Biosynthesis of Vitamin K (Menaquinone) in Bacteria · BIOSYNTHESIS OF VITAMIN KIN BACTERIA 243 is of unique interest that bacteria again came into the picture and, as will be seen,

MICROBIOLOGICAL REVIEWS, Sept. 1982, p. 241-280 Vol. 46, No. 30146-0749/82/030241-40$02.00/0Copyright C 1982, American Society for Microbiology

Biosynthesis of Vitamin K (Menaquinone) in BacteriaRONALD BENTLEY* AND R. MEGANATHANt

Department ofBiological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

INTRODUCTION .......................................................... 241DISCOVERY OF VITAMIN K IN BACTERIA ........... ........................... 243BIOSYNTHESIS OF MENAQUINONES .............. .............................. 245

Geeneral Informaton ............................................................ 246Role of Shikimate .......................................................... 247A Possible Role for 1-Naphthol?.................................................. 251Origin of the "Three Carbon" Unit............................................... 252First Aromatic Intermediate, o-Succinylbenzoate ................................... 2541,4-Dihydroxy-2-Naphthoate, a Naphthalenoid Intermediate.......................... 254Role of Naphthalene Compounds Other than 1-Naphthol............................. 255

INDIVIDUAL REACTIONS IN MENAQUINONE BIOSYNTHESIS ...... .............. 256Formation of o-Succinylbenzoate.................................................. 256Formation of succinic semialdehyde-thiamine pyrophosphate complex ..... .......... 257Subsequent reactions.......................................................... 257

Formation of 1,4-Dlhydroxy-2-Naphthoate ............ ............................. 258Structure of o-Succinylbenzyl Coenzyme A Intermediate ........ ..................... 261Prenylation Reaction.......................................................... 262Methylation of Demethylmenaquinone............................................. 263Formation of Reduced Isoprenyl Units ............................................ 263

GENETICS OF MENAQUINONE BIOSYNTHESIS ......... ......................... 263men Mutants of Escherichia coli .................................................. 263men Mutants of Bacillus subtdis.................................................. 264men Mutants of Staphylococcus aureus .............. .............................. 265

FACTORS INLUENCING MENAQUINONE BIOSYNTHESIS........................ 265Aerobic Versus Anaerobic Growth................................................ 265Other Factors Influendng Menaquinone Biosynthesis................................ 267

VITAMIN K-REQUIRING BACTERIA ............... .............................. 268Mycobacterium paratuberculosis................................................... 268Bacteroides melaninogenicus...................................................... 270Lactobacilus bfidus var. pennsylvanicus ............. .............................. 272Other Microorpnisms........................................................... 272Are the Vitamin K-Like Growth Factors Secreted by Bacteria ActuaHly Menaquinones?.. 272Are Growth Factors with Vitamin K Activity Converted to Menaquinones? ..... ....... 273

VITAMIN K BIOSYNTHESIS BY INTESTINAL BACTERIA ....... .................. 273LITERATURE CITED............................................................ 274

INTRODUCTIONAls der geistige Vater der Vitaminlehre ist

wohl Gowland Hopkins zu betrachten ....F. Rohmann, 1916 (183)

Research work on a disease (Johne's Disease)which affects any of the larger domesticatedanimals is necessarily very costly .... On thisaccount our experiments, though covering afairly wide field, have not been so numerous insome cases as we should have wished. In view ofthe importance of this disease to agriculturists,the question is one which should be investigatedwith public money. In our work on this disease,however, we have received no assistance fromthe Board of Agriculture or from the Develop-ment Fund Commissioners, even though appli-

t Present address: Department of Biological Sciences,Northern Illinois University, DeKalb, IL 60115.

cations for a grant have been made after theessential part ofour work-the cultivation of thebacillus-had been verified by the Danish Gov-ernment bacteriologists.F. W. Twort and G. L. Y. Ingram, 1913 (225)

In his Nobel Prize Lecture in 1929, Hopkinspointed out that "it is abundantly clear thatbefore the last century closed there was alreadyample evidence available to show that the needsof nutrition could not be adequately defined interms of calories, proteins and salts alone.....It is sure that until the period 1911-1912, theearlier suggestions in the literature pointing tothe existence of vitamins lay buried" (107). Atthe time of Hopkins' Nobel Lecture, almost allof the work on vitamins had concerned animaland human nutrition. Fat- and water-solublevitamins had been distinguished, but the firstchemical structure, that of vitamin A, was not

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242 BENTLEY AND MEGANATHAN

determined until 1931. Hopkins was apparentlyunaware that, at the same time as his own workon vitamin requirements in animals, a growthfactor requirement for "Johne's bacillus" (My-cobacterium paratuberculosis) was establishedby his fellow countrymen, Twort and Ingram(222, 224).

In June of 1910, these workers had considereda possible vaccine for Johne's disease-a chron-ic, specific enteritis principally affecting cattle.Whereas a specific bacillus (Johne's bacillus)was present in the intestinal mucous membraneand mesenteric glands of infected animals, allattempts to cultivate the organism on ordinary,artificial laboratory media had failed. They con-sidered that these failures "must be due, eitherto some substance in the medium acting as apoison, or to the absence of some material orfoodstuff necessary for its vitality and growth"(222, 224). Since related bacilli grew in ordinarylaboratory media, the poison alternative seemedhighly improbable; they were, therefore, forced"to conclude that the failure to grow the bacillusmust be due to the absence of some necessaryfoodstuff." Further, the close relationships be-tween the tubercle bacillus and Johne's bacillus(both live in the bodies of bovines, for instance)suggested that the former could grow in ordinarymedium because it could elaborate a certainrequired substance, the "Essential Substance."When dead human tubercle bacilli were incorpo-rated into an egg medium, Johne's bacillus wasgrown in culture for the first time. This resultwas announced by Twort in a preliminary fash-ion in 1910 (222) and more fully in 1911 (224).The timothy grass bacillus (Mycobacteriumphlei) was also found to be an excellent sourceof Essential Substance. Extraction of Mycobac-terium phlei cells with ethanol yielded a yellow-ish mass which supported growth; on furtherextraction with chloroform, the best stimulationwas obtained with the material insoluble in chlo-roform. As Hanks has noted, this work providesthe first discovery of a biological growth factor,a vitamin, for a microorganism (96). However,"der geistige Vater der Vitaminlehre" did notrefer to Twort and Ingram's work in either hisclassical paper (106) or his Nobel Prize Lecture(107) and appears to have shared the feeling thatbacterial nutrition and animal nutrition had noconnection (69).The nature of Essential Substance remained

unexplored for 30 years. In the meantime, vita-min K had been characterized and shown to be amethyl naphthoquinone (see below). Woolleyand McCarter (231) were aware of the report (6)that phthiocol, 2-methyl-3-hydroxy-1,4-naph-thoquinone, isolated from Mycobacterium tu-berculosis possessed vitamin K activity. SinceEssential Substance was present in this orga-

nism and was to some extent soluble in fatsolvents as well as in water, phthiocol, 2-methyl-naphthoquinone, and a potent concentrate ofvitamin K were tested as growth factors forJohne's bacillus. All three materials were shownto stimulate growth, but extracts of Mycobacte-rium phlei were somewhat more effective thanany of the quinones. Whether, in fact, vitamin Kcan actually be considered to be required bystrains of Mycobacterium paratuberculosis willbe examined in a later section (Vitamin K-Requiring Bacteria). Writing in 1949, 1 yearbefore his death and after the destruction of hisinstitute in the London "blitz," Twort withobvious pride, but frustrated by the inability toobtain financial support, referred to his earlywork as follows: "The name Vitamin at thattime had not been coined, although my 'Essen-tial Substance' has since been named 'VitaminK' " (223).

It would be of interest to speculate on theconsequences of a serious and determined effortto characterize Twort's Essential Substance atan earlier date. However, the work "lay bur-ied," and its significance was not appreciateduntil Knight called it to attention in 1936 (125).Remarkably, the Twort and Ingram paper of1912 also contains a reference to the possibilityof an ultramicroscopic virus working in symbio-sis with Johne's bacillus. Although experimentsto test this possibility were negative, later workled to another of Twort's major contributions tomicrobiology, the Twort-d'Herelle phenomenonof transmissible lysis (70).Had the yellowish mass extracted from Myco-

bacterium phlei been examined at some timebefore say 1935, the identification of a naphtho-quinone and vitamin K might have beenachieved at an earlier date. In fact, possibleconnections between animal and bacterial me-tabolism remained unappreciated for manyyears after Twort and Ingram's work. It was notuntil 1934 that Fildes could write, "It is notimpossible that substances shown by the bacte-rial chemist to be necessary for the propergrowth of bacteria may subsequently be found tobe necessary for the growth of animals" (69).Instead, vitamin K was discovered by the classi-cal approach of animal nutrition. In 1929, HenrikDam began nutritional studies with chickens,thus "lighting a candle which pushed back thedarkness and revealed a new vitamin factorwhich is now recognized to be of vital impor-tance to the health of mankind" (97). The his-tory of the work of Almquist and Dam and theircolleagues is, for the most part, well known (4,59, 155), and some new and interesting reminis-cences have appeared recently (5, 120, 172).Only those portions of the work of immediateinterest to microbiologists will be noted here. It

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BIOSYNTHESIS OF VITAMIN K IN BACTERIA 243

is of unique interest that bacteria again cameinto the picture and, as will be seen, in a noveland unexpected manner.To conclude this general introduction, it

should be noted that available review articles inthe general area of vitamin K have been listed bySuttie (207). A major function for vitamin K inmammalian metabolism has now been clarified:it is a required cofactor for the carboxylation (byC02) of protein-bound glutamate residues toform y-carboxyglutamates (205). The functionsof vitamin K in bacteria have been discussed inseveral review articles (88, 89, 118, 129, 208,214). The vitamin has a major role as an electroncarrier. The clinical uses of vitamin K are wellknown (148).Many other 1,4-naphthoquinone derivatives

are found in nature, particularly in plants andfungi, and have also been exploited by humansin many ways. For example, the plant metabo-lite lawsone (2-hydroxy-1,4-naphthoquinone) isthe material responsible for the yellow-to-orangedyeing properties of henna; this material hasbeen used by men and women for at least 2,000years-reputedly, for instance, by Cleopatraand Mohammed (213). In more recent times,some naturally occurring naphthoquinones, orclosely related materials, have been used orconsidered for use as antibiotics, e.g., the axen-omycins (22), frenolicin (63), kalafungin (173),the nanaomycins (173), the naphthocyclinones(235), and the ansamycins (178).

DISCOVERY OF VITAMIN K IN BACTERIAThe discovery of vitamin K biosynthesis in

bacteria came about from detailed studies of thenutrition of chickens. In 1931 McFarlane et al.(156, 157) investigated the fat-soluble vitaminrequirements of the chick. The basic rationsincluded either fish meal (from white nonoilyfish) or meat meal (from which the fat waspartially extracted). When either meal was firstextracted with ether, the animals suffered poorgrowth and, if injured, bled to death. The bleed-ing condition was most pronounced when ether-extracted fish meal (rather than meat) was usedand was similar to that reported earlier by Dam.Dam's diets, however, were based on casein asthe protein source (56, 57). Somewhat later,Holst and Halbrook described a "scurvy-likedisease" in chicks, also using a fish meal ration(104). The feeding of 5 g of cabbage per birdduring weeks 5 and 6 of deficiency gave acomplete recovery.At about this same time, Cook and Scott

stated that the hemorrhagic condition in chick-ens "can be ascribed to the fish meals used inthat they contained objectionable materials and/or lacked some accessory factor" (50). Reminis-cent of the earlier work by McFarlane et al. (156,

157), these workers encountered no problemswith a diet of "commercial meat scrap." Theobjectionable materials were said to be nitroge-nous bases (51), and feeding a number of suchcompounds did produce hemorrhagic symptoms(e.g., mono-, di-, and trimethylamine, diethyl-and dipropylamine, ergot, nicotine). Further,methylamine was detected in fish meal. Theseworkers unfortunately failed to appreciate thesignificance of one of their observations: whenfish meal was washed with water and allowed todry at 65°C, the syndrome was much reduced.

Since fish meals were used for animal feeding,the problem of "toxic fish meal" versus "non-toxic meat meal" became a cause celebre. Atthat time, Almquist was working on problems ofprotein quality in animal protein concentratesfor the feeding industry. He has recollected that"meat scraps were made mostly from the offalfrom meat packing, which would include theviscera and incidental manure, condemned liv-ers, dead animals picked up from the hinterland,and what have you. The starting material oftencould be pretty 'ripe'. . . . It occurred to me thatpossibly the opportunity for bacterial action orother spoilage on the raw materials going intothese animal protein concentrates might havesomething to do with the problem. So I moist-ened some good fish meal with water and let itstand in a warm cabinet. It stunk up the place"(H. J. Almquist, letter to R. Bentley, dated 26March 1982 [a recollection of events that goback nearly half a century]). The water-moist-ened fish meal was examined by a graduatestudent, Halbrook (92), and found to prevent thehemorrhagic symptoms in chicks (Table 1). Wa-ter extraction followed by drying gave a similarresult. If the water-extracted fish meal wastreated with alcohol before drying, hemorrhagicsymptoms were present (see Table 1). Halbrookconcluded that the protective action of water-extracted fish meal "can only be explained bybacterial action, especially since mere moisten-ing of the fish meal had a similar effect, whereaswater extraction followed by moistening withethyl alcohol to prevent bacterial action failed toprevent the> symptoms from occurring."

Halbrook's thesis also records that untreatedrice bran had little effect in preventing the occur-rence of hemorrhagic symptoms, but did sowhen it was extracted with water and driedslowly under conditions conducive to bacterialaction. With reference to either fish meal or ricebran, he concluded that "either a deficiencyfactor is synthesized by bacterial action or atoxic factor is destroyed." A fact arguing againstthe "toxicity theory" was his finding that theallegedly toxic trimethylamine hydrochloride(51) had no effect when added to water-extractedfish meal.

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TABLE 1. Effect of various treatments of fish mealon occurrence of hemorrhages in chicksa

Fish meal % of chicks with No.treatment hemorrhagic of

symptomsb expt

Normal 38.5-100 (77.2)C 6Ether 83.0-86.0 (85.0) 2

extractedWater 0.0-9.2 (2.3) 4

extractedWater 0.0 1

moistenedWater 83.3 1

extracted,alcoholmoistened

a The fish meal was derived from Pacific Coastsardines, using equal parts of whole fish and heads andviscera, by a superheated steam-drying process. Thesedata are abstracted from Table V of Halbrook's thesis(92).

b Each experiment involved 12 to 15 chicks. Differ-ent batches offish meal were used in each experiment.

c Average is given in parentheses.

Some confusion had been caused in the earlywork since not all batches of fish meal allowedthe development of the hemorrhagic syndrome.Again quoting Almquist's recollection, "Some-times fish meals were made from offal from thecanning operations, or from fish which had gone'soft' and unfit for canning because of delaybetween catching and canning times. Sometimesthey were almost entirely made from .the cut-tings from the canning operation." These mealswere less likely to cause the bleeding problem,again supporting a role for bacterial action.Jukes has also suggested that the better micro-biological state of some fish meal batches result-ed from the cold temperatures in the sardinefishing grounds off the coast of California due tothe Alaskan current. Sardine fishing was done atnight, and under optimal conditions the fishwere quickly transported, still cold, for immedi-ate processing at the factory (120).Almquist and Stokstad cited Halbrook's the-

sis work in a paper published in 1935 (9) andreported further experiments with fish mealwhich had been moistened and allowed to putre-fy. By this time they were able to rationalize thenontoxic quality of meat meal by pointing outthat it is "well known that many animal proteinconcentrates offered for poultry feeding are notprotected from the action of microorganisms inthe raw state and during manufacture." It wasclear that "antihaemorrhagic power cannot beattributed specifically to any feed ingredientunless the possibility of action upon it by micro-organisms has been guarded against" (10).

Unfortunately for Almquist, the "deficiency-toxicity" dispute had become heated at theUniversity of California since commercial inter-ests were at stake for the fish meal producers (nopun intended). A manuscript by Almquist andStokstad was, for a time, actually embargoed bythe administration of the University of Califor-nia (4, 120); when submitted to Science it wasrejected because of the previous claim that tox-icity resulted from the presence of nitrogenousbases (51). After these delays, the paper wasaccepted by Nature (10), appearing in the 6 Julyissue of 1935 (no receipt date given). Almquistthereby lost priority to a paper in the samejournal by Dam (58) which had been received on19 March and appeared in the 27 April issue. Inthis paper, Dam suggested the name vitamin Kfor the antihemorrhagic factor and showed it tobe present in hog liver fat, hemp seed, certainvegetables, and to a lesser extent in cereals.Vitamin K was derived from the spelling ofcoagulation in German and in the Scandinavianlanguages (59) and was also the first letter of thealphabet not then assigned to another vitamin.Dam was evidently of the opinion that Almquistshould have shared the Nobel Prize for thediscovery of vitamin K (4, 120). That he did notmust presumably be attributed to the delaycaused by the deficiency-toxicity dispute.

Strangely enough, a "replay" of toxicity ver-sus deficiency occurred some years later whenyoung rats, fed irradiated beef, were found todevelop hemorrhages. As Matschiner has noted,several laboratories believed that a toxic princi-ple was responsible rather than a nutritionaldeficiency of vitamin K (152). In fact, noncon-taminated ground beef contains about 0.07 ,ig ofvitamin K per g, and this amount is sufficient toprotect rats against hemorrhage.For some time, "putrefied fish meal" was a

major source of what became known as vitaminK2. In 1938, for example, Osterberg describedthe process in some detail (175). He wished toobtain material for a clinical trial of vitamin K injaundice. Some 15 pounds (ca. 6.8 kg) of com-mercial fish meal (from tuna) was ether extract-ed (a staggering volume of ether must have beenrequired) and, after drying, was moistened andallowed to putrefy for 1 week in a warm andmoist atmosphere. After drying and extractionwith petroleum ether, 15 ml of impure oil wasobtained; the data suggest a vitamin K contentof perhaps 15%.

Almquist and Stokstad considerably extendedtheir observations. They showed, for example,that the vitamin could be biosynthesized, pre-sumably by bacterial action, within the intestinaltract of chicks (11). This followed from the factthat droppings from chickens on a vitamin K-free diet could be extracted to yield material that

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was adequate as a source of the antihemorrhagicvitamin. When droppings were collected in 1%phenol solution to inhibit further bacterial ac-tion, the potency of the extract was lower.Extreme care was necessary in these and othernutritional experiments. To prevent bacterialsynthesis it was necessary, for instance, to re-move feed which the chicks had carried to thewater troughs.The "fish meal organism," probably Bacillus

cereus, was isolated and grown on substratessuch as beef broth, fish meal broth, proteose-peptone broth, and nutrient agar; in each case itfunctioned as a rich source of vitamin K (8).Various pure bacteria were also grown on nutri-ent agar, and vitamin K was found, for example,in Bacillus cereus, B. mycoides, B. subtilis,Chromobacterium prodigiosus (= Serratiamarcescens), Escherichia coli, Mycobacteriumtuberculosis, Sarcina lutea, and Staphylococcusaureus. No activity was observed with extractsfrom yeast or Pseudomonas aeruginosa (nowknown to contain only ubiquinones). It wasevident that the factor, vitamin K, was a productof the metabolism of many bacteria.

Bacteria continued to play an important rolein the vitamin K story. Almquist became awareof the isolation of the naphthoquinone phthiocol(2-methyl-3-hydroxy-1,4-naphthoquinone) fromMycobacterium tuberculosis by Anderson andNewman (12). Since he had evidence that hisvitamin K preparations were quinones and sinceMycobacterium tuberculosis was a good sourceof vitamin K, he obtained a phthiocol sample. Itwas shown to have definite vitamin K activityand became the "first identified form of vitaminK" (6). With evidence accumulating for a phytylgroup in vitamin K1, Almquist and Klose con-densed phytol and 2-methylnaphthoquinone toachieve a synthesis of vitamin Kl. This form ofthe vitamin, present in alfalfa and other greenplants, is therefore 2-methyl-3-phytyl-1,4-naph-thoquinone. Their paper (7), received by theJournal ofthe American Chemical Society on 21July 1939, appeared at the same time as similarwork from the laboratories of Fieser (67, re-ceived 12 August 1939) and Doisy (30, received21 August 1939; a more detailed paper [144]carries the date 5 September 1939).

It was from bacterially putrefied fish meal thatthe first crystalline antihemorrhagic vitamin wasprepared in Doisy's laboratory (excluding phthi-ocol from consideration). A page fromR. W. McKee's notebook, dated 10 November1938, has been published (160). The momentouscrystallization of the purified vitamin occurred"a few days later." Since this crystalline bacte-rial material (161) was clearly different from thehighly purified, but noncrystalline, materialfrom alfalfa, the two were distinguished as vita-

min K1 (from green plants; now phylloquinone= leaf quinone) and vitamin K2 (from bacteria;now menaquinone = methyl naphthoquinone).

It is perhaps striking that another 10 yearswere to elapse before the isolation of a mena-quinone from a pure bacterial culture (as op-posed to putrefied fish meal) was undertaken. In1948 Tishler and Sampson isolated and crystal-lized a menaquinone from B. brevis (221). Thesaga of the antihemorrhagic vitamin present inbacteria has a final irony. Although Doisy andhis colleagues (31, 161) characterized the materi-al from the putrefied fish meal as 2-methyl-3-farnesylfarnesyl-1,4-naphthoquinone (i.e., MK-6 in present nomenclature), it was later shownthat the major component is, in fact, MK-7; MK-6 is present but only as a minor component(114). It is now known that the natural mena-quinones have all trans configurations for theappropriate side chain double bonds; the doublebond of phylloquinone was also shown to be 2'-trans and the chiral centers at 7' and 11' wereshown to be R (23). In the hydrogenated mena-quinone from Mycobacterium phlei MK-9 (II-H2), the configuration at the 7' position is also R(19).

It is now abundantly clear that bacteria con-tain both normal and modified menaquinonetypes; in addition, cyanobacteria contain phyllo-quinone rather than menaquinones. A compre-hensive account of the various forms of vitaminK present in bacteria has been given by Collinsand Jones (48). Common side chain variations inthe menaquinones are hydrogenation of one ormore of the isoprenoid double bonds and theintroduction of oxygen atoms. Of particular im-portance for our present purposes is the occur-rence ofdemethylmenaquinones (DMK). As willbecome apparent, the DMK are precursors tothe menaquinones themselves.

In this review, we have made no attempt to becompletely consistent with respect to the no-menclature of the various materials. The termvitamin K has been used in the initial introduc-tory material and in those situations where phys-iological activities are discussed or where aninclusive term for materials with antihemorrhag-ic activity is needed. In other, more specificcases, the International Union of Pure and Ap-plied Chemistry-International Union of Bio-chemistry nomenclature has been used (112).

BIOSYNTHESIS OF MENAQUINONES

An introduction to the discovery of the mena-quinone biosynthetic pathway will be given firstalong with a general description of the overallprocess. Subsequently, the work leading to theidentification of intermediates will be reviewed

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in detail, and the individual reactions of thepathway will be considered in terms of mecha-nism and enzymology, as far as is possible.Genetic considerations will then be coveredseparately.

General InformationThe work described in the preceding section

clearly showed that bacteria have a high abilityfor menaquinone biosynthesis. However, before1964, the biosynthetic question had not receivedany experimental attention. Fieser et al. (68) hadsuggested that phylloquinone might be derivedby condensation of phytol with 2-methyl-1,4-naphthoquinone, and they had also attempted tomake a connection between the two farnesylresidues (two C15) found in the menaquinonefrom putrefied fish meal (as noted earlier, only aminor component) and the known presence ofsqualene, C30H50, in fish oils. In 1964, Martiusand Leuzinger (150) found that phylloquinoneand 2-methyl-1,4-naphthoquinone could be con-verted to menaquinone by the action of thevitamin K-requiring Bacteroides melaninogeni-cus (then termed Fusiformis nigrescens). Fur-thermore, it was shown that 1,4-naphthoquinoneitself was converted to menaquinone by Bacter-oides melaninogenicus and that the requiredmethyl group was contributed by methionine(206). The role of methionine as the methylgroup donor has been confirmed for the biosyn-thesis of MK-9 (II-H2) by Mycobacterium phleiand Mycobacterium smegmatis (81, 117) and forMK-8 in E. coli (53, 64, 115). Schiefer andMartius also observed the conversion of 2-meth-yl-1 ,4-naphthoquinone to menaquinone, usinganimal mitochondrial preparations (194). Theisoprenoid side chain was contributed by pyro-phosphate esters of polyisoprenoid alcohols.This work pointed to a mevalonoid origin for thesecond side chain of vitamin K. In 1967, Threl-fall et al. were able to show the utilization ofmevalonate for the biosynthesis of phylloqui-none (219), and in 1969 Hammond and Whiteextended these observations to a bacterial sys-tem (93). It is now generally agreed that theprimary precursors of the two side chains ofmenaquinones are methionine and mevalonate.

Studies of the origin of the naphthoquinonenucleus proceeded more slowly than those of theother isoprenoid quinones and the related cy-clized forms such as vitamin E. In part, this slowdevelopment stemmed from the low levels ofmenaquinones and biosynthetic enzymes pre-sent in bacteria, and in part it was from the factthat the biosynthetic pathway has turned out tobe unique and to involve unprecedented reac-tions. Most of the presently available infor-mation concerning vitamin K biosynthesis ingeneral has, hbwever, been obtained from ex-

periments with bacteria. The work reviewedhere will be concerned almost exclusively withmenaquinone biosynthesis in a rather smallnumber of bacteria (generally E. coli, Mycobac-terium phlei, and Bacillus subtilis). The onlyother possible experimental organisms availablefor a study of vitamin K biosynthesis are greenplants and cyanobacteria. Although. phylloqui-none biosynthesis seems to be generally thesame as that of menaquinones, the exact path-way in plants remains unclear at the presenttime, and virtually no work with enzyme sys-tems has been carried out.

In 1964, Cox and Gibson observed the conver-sion of [G-14C]shikimate into both ubiquinoneand menaquinone by E. coli, thus providing thefirst evidence for a role for the shikimate path-way (52). The incorporation (1) value was notgiven; the dilution (D) can be calculated to be 9.4for menaquinone and 7.9 for ubiquinone; I andDhave the usual meanings (41). Chemical degrada-tion of two labeled samples of E. coli menaqui-none (MK-8) showed that essentially all of theradioactivity was retained in the phthalic anhy-dride. Hence it was concluded that "the ben-zene ring of the naphthaquinone (sic) portion ofvitamin K2 (MK-8) arises from shikimate in E.coli." These authors also suggested that shiki-mate was first converted to chorismate. Soonafterwards, more complete chemical degrada-tions of menaquinone derived from radioactiveshikimate established that all seven carbon at-oms of this precursor were incorporated into themenaquinone molecule (43). The remainingthree atoms of the naphthoquinone nucleus weresubsequently found to be derived from 2-keto-glutarate; both carboxyl groups of this precursorwere removed at some stage (40, 180, 181).The work just summarized established that

the immediate precursors of the menaquinoneswere as follows: shikimate (chorismate) plusnoncarboxyl carbon atoms of 2-ketoglutarateforming the naphthoquinone nucleus, with themethyl and isoprenoid side chains obtained,respectively, from S-adenosylmethionine and anisoprenoid alcohol pyrophosphate ester. Twoimportant aromatic intermediates were subse-quently characterized. They are the benzenoidderivative o-succinylbenzoate (OSB; 60) and thenaphthalenoid compound 1,4-dihydroxy-2-naphthoate (DHNA; 182). The broad outlines ofthe biosynthetic pathway to menaquinones aresummarized in Fig. 1. Evidence has also beenobtained for the participation of at least twoother intermediates; each possibility will be dis-cussed in detail below.The branch of the shikimate pathway through

OSB is also responsible for the biosynthesis ofphylloquinone (215), some simpler plant naph-thoquinones such as lawsone and juglone (60),

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RbOj COOH COOH

HO Co21-

. COOH PyruvateChorismate

Shikimate

OSB

CH2RI- HOOC-8-

OH

HH COCOOH

CO2OH

DHNA

R"= Prenyl

MK 'FIG. 1. Major intermediates in menaquinone biosynthesis. The ketoglutarate unit is not identified due to

space limitations. The abbreviations used here and elsewhere are as follows: OSB = o-succinylbenzoate (4-[2'-carboxyphenyl]-4-oxobutyrate); DHNA = 1,4-dihydroxy-2-naphthoate; DMK = demethylmenaquinone; MK =menaquinone.

and (with the addition of further carbon atomsderived from mevalonate) of some plant anthra-quinones such as those of Rubia peregrina (60).OSB is also a precursor to the orchid alkaloidshihunine (134) in Dendrobium pierardii and D.lohohense. Shikimate pathway branches lead toother plant naphthoquinones by way of differentintermediates, as indicated:

reduced naphthalene material geosmin, pro-duced by various streptomycetes, is apparentlya degraded sesquiterpene derived from mevalon-ate (26).

Role of ShikimateAfter their early observation (52) that shiki-

mate was a menaquinone precursor, Cox and

t,4-hydroxybenzoate - .alkannin (195)OSB +- shikimate homogentisate - chimaphilin (33)

"'3-amino-5-hydroxybenzoate -rifamycins (122)

Furthermore, it has always appeared likely thatthe naphthoquinone (and related) ring systemsof rifamycins and similar antibiotics were de-rived from the shikimate pathway (24). Thisexpectation has been upheld by the discovery of3-anino-5-hydroxybenzoate as a rifamycin pre-cursor (122).Most of the 1,4-naphthoquinones found in

nature, however, are produced by plants andfungi, and the majority of these are derived by"'polyketide" pathways (24); some bacterialnaphthoquinones are also produced in this way(e.g., 5,8-dihydroxy-2,7-dimethoxy-1,4-naph-thoquinone produced by a Streptomyces strain[158]). One final pathway for naphthoquinonebiosynthesis must be noted. In a few cases, plantnaphthoquinones are derived entirely from mev-alonate. In bacteria, however, this precursor isused sparingly; few, if any, bacteria producesterols. However, as noted, the isoprenoid sidechain of menaquinones derives from this materi-al as do the long-chain alcohols such as bacto-prenol. Although not a naphthoquinone, the

Gibson in 1966 (53) converted the labeled mena-quinone to 1,4-diacetoxy-2-methylnaphthalene-3-acetic acid by the procedure used earlier forubiquinone (27). This material was more vigor-ously oxidized with KMnO4 to form phthalicanhydride (Fig. 2); in two experiments this mate-rial contained 92 or 95% of the activity present inMK-8.

It had become apparent at about this time thatpurification of radioactive menaquinone samplesby the usual chromatographic procedures wasunreliable: contamination of such samples byother lipids was observed. Esters of fatty andaromatic acids were particularly troublesome(17, 42, 82). In addition, Cox and Gibson hadused materials of relatively low specific activity(the maximum activity in their phthalic anhy-dride sample was ca. 8 cpm/mg), and theirchemical degradation did not reveal whether allof the shikimate carbons were incorporated intothe naphthoquinone nucleus. Campbell et al.(43, 44), therefore, carefully purified menaqui-none samples by use of the (lipophilic) Sephadex

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8OH

COOH

9

2 C02

HOOCII,COOHHOOC,N,CH-3

FIG. 2. Utilization of all seven carbon atoms of shikimate for menaquinone biosynthesis. Radioactivity isindicated as *, and the same symbol within a six-membered ring implies that all carbon atoms are labeled. Thesequence is as follows; a, bacterial biosynthesis; b, reductive acetylation of the menaquinone; c, 03/KMnO4 (53);d, Os04/HI04, then KMnO4 (43); e, KMnO4 in acetone, only phthalate being isolated (53); f, H202 (43); g,Schmidt degradation.

LH-20 and Sephadex LH-50 (170). The sampleswere converted to the diacetate of the mena-quinol for verification of radiochemical purity.

In this work, the incorporation from shikimateto menaquinone ranged from 0.1 to 1.6% with E.coli, and the dilution values were 290 to 5.3.With Mycobacterium phlei and Streptomycesalbus the incorporations were lower (0.02 and0.007%, respectively) and the dilutions werehigher (3,300 and 16,000, respectively). A chem-ical degradation was devised so that all carbonatoms of the naphthoquinone nucleus could berecovered. By treatment with Os04-HI04 andthen KMnO4, the atoms of the nucleus (alongwith two carbons from the polyisoprenoid sidechain) were obtained as 1,4-diacetoxy-2-methyl-3-naphthalene acetic acid. Further degradationof the latter with H202 yielded phthalic acid,acetic acid, and malonic acid (see Fig. 2).Schmidt degradation of the phthalate yielded thecarboxyl carbons as CO2. When [G-14C]shiki-mate was used as precursor, the quinone carbonatoms, C-1 plus C-4, contained 12 to 16% of thetotal radioactivity of the menaquinone (experi-ments using E. coli, Streptomyces albus, andMycobacterium phlei). The "generally labeled"shikimate ([G-14C]shikimate) used in these ex-periments was a commercial preparation ob-tained by exposing Ginkgo biloba seedlings to14CO2. Chemical degradations established that,on average, the COOH group of the shikimatecontained 15.6% of the total radioactivity.Hence, it was clear that, in these organisms, allseven carbon atoms of shikimate were incorpo-rated into the menaquinone molecule and thecarboxyl carbon of shikimate provided one (orboth) of the carbonyl functions of the menaqui-none (43, 44).

At the same time, Leistner et al. (135) alsoexamined the conversion of shikimate intomenaquinone in the following organisms: Bacil-lus megaterium, Bacillus subtilis, E. coli, "Mi-crococcus lysodeickticus," Proteus vulgaris,and Sarcina lutea. The highest incorporations(1.1 and 2.7%) were obtained with Bacillusmegaterium. Radioactive menaquinone samplesfrom the latter organism were directly oxidizedwith KMnO4 to phthalic acid; this acid was thendecarboxylated. It was again observed that thephthalate had all of the menaquinone radioactiv-ity. The two carboxyl groups of phthalate con-tained a total of 13.9% of the menaquinoneactivity, in agreement with the work just cited(43, 44). Thus, it was clear that the ring ofshikimate was incorporated intact into ring A ofa variety of bacterial menaquinones; the shi-kimate carboxyl was also utilized, becomingeither one (or possibly both) of the quinonecarbonyl groups. The correct situation is shownin Fig. 2, and evidence in support of it will bediscussed.

In ingenious experiments, Leduc et al. investi-gated which of the shikimate atoms provide thetwo atoms at the A/B ring junction (133). At-tempts to resolve this question by the use of[1,6-14C2shikimate were frustrated by difficul-ties in purifying degradation products; however,the use of [3-3H]shikimate and degradation to amixture of 3- and 4-nitrophthalates provided asolution. The question is complicated by possi-ble symmetry in a biosynthetic intermediate (asnoted below, this is not the case) and by actualsymmetry in a degradation product (phthalate).The experiments can be best understood withreference to Fig. 3. Following isolation of la-beled MK-9 (I-H2) after administration of [3-

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BIOSYNTHESIS OF VITAMIN K IN BACTERIA

HO..

Pathway

B

FIG. 3. Origin of the atoms at the menaquinone ringjunction. R = prenyl; X = COOH. The numbers identifyatoms from shikimate at all times; 3H radioactivity is indicated as *, and if a 1:1 dilution has arisen, it is indicatedas O). Two possible pathways, A and B, are considered. For each of the pathways, there are two possibleincorporation modes without a symmetrical biosynthetic intermediate and a third in which randomization couldhave taken place with a symmetrical intermediate. After conversion of the labeled menaquinones to phthalates,nitration gave a mixture of the 3-nitro and 4-nitro derivatives; the "top" nitrophthalate in the figure is the 3-nitroderivative; the "bottom" nitrophthalate is the 4-nitro derivative. The nitrophthalates were separated beforedetermination of radioactivity.

3HJshikimate to Mycobacterium phlei, the sam-ple was oxidized with KMnO4 to phthalate. Thelatter was nitrated (HNO3-H2SO4) to a mixtureof 3- and 4-nitrophthalates; the acids were sepa-rated by thin-layer chromatography on cellu-

lose. If the phthalate carries 3H at positions 4and 5, the 3-nitro derivative carries 3H in twopositions (4 and 5) and the 4-nitro carries it onlyin one (position 5). Thus, for pathwayA the ratioof radioactivity, 3-nitro/4-nitro = 2. For path-

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0H x

02N x


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04

tCH~~~H3CH3

I

HO )jCOOH

HOHOH

o#<,~CH3~

CH3

4c

+

A B

FIG. 4. Utilization of shikimate carboxyl group inmenaquinone biosynthesis. The reactions are as fol-lows: a, biosynthetic conversion of [7-14C]shikimate toMK-9 (II-H2) by Mycobacterium phlei, followed bychemical conversion to 2-methyl-3-ethyl-1,4-naphtho-quinone; b, reaction with 03; c, hydrolysis and esteri-fication of the anhydride mixture followed by separa-tion of the two diketoesters by gas chromatography; d,formation of quinoxalines, followed by ester hydroly-sis and decarboxylation of the resulting acids. Com-pound A formed by this sequence contained only 3.6%of the original radioactivity of the menaquinone, andcompound B contained 101%.

way B the situation is reversed. The 3-nitroderivative contains 3H at position 6; the 4-nitrocontains it at positions 3 and 6. Hence, theradioactivity ratio in this case is 3-nitro/4-nitro =0.5. Since the experimentally determined valuewas 0.5, pathway B operates. In other words,the atoms of the A/B ring junction are providedby C-1 and C-2 of shikimate. Not answered bythis work, however, was the question, Does theshikimate carboxyl give rite to C-1, to C-4, or toboth C-1 and CA of the naphthoquinone nucle-us?That the carboxyl group of shikimate provided

C-4 was shown by Baldwin et al. (21). In thiswork, shikimate was labeled in the carboxylgroup. A chemical degradation yielding C-1 andC-4 of the naphthoquinone ring, as separate

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chemical entities, was devised as shown in Fig.4. The end products of the degradation were twoquinoxaline derivatives, one containing C-1 butnot C4 (compound A, Fig. 4) and the othercontaining C-4 but not C-1 (compound B, Fig. 4).It was found that only compound B contained14C (101% of the MK-9 [II-H2] activity). Hence,the origin of the C-4 carbonyl in the shikimatecarboxyl was demonstrated. This work alsodemonstrated that there are no symmetrical in-termediates in the menaquinone biosyntheticpathway.

It has also been shown (193) that in menaqui-none biosynthesis from shikimate the pro-R hy-drogen at position 6 is eliminated (Fig. 5). Therequired precursors, (6S)-[7-14C, 6- H]- and(6R)-[7-14C, 6-3H]shikimate, were obtained en-zymatically from (E)- and (Z)-[3-3HJphospho-enolpyruvate (71, 174). The organism used wasBacillus megaterium 248, a shikimic acid auxo-troph. Very high incorporations were obtainedwith this mutant (15 to 17%). The incorporationswere determined for both 3H and 14C, and theratios of activity are shown in Table 2. Hence,3H retention from the 6S material was 84.0o,and that from the 6R material was only 18.6%.These experiments are consistent with a role forchorismate (see below) since the same pro-6Rhydrogen of shikimate is eliminated during cho-rismate biosynthesis.As a result of their initial work, Cox and

Gibson had suggested chorismate as the"branch point" for menaquinone formation.This suggestion was, in part, based on the obser-vation that addition of 3,4-dihydroxybenzalde-hyde or adrenaline to E. coli cultures diminishedthe incorporation of radioactivity from shiki-mate into menaquinone (the following mate-rials were without effect on the incorporation:catechol, phenylpyruvate, 4-hydroxyphenyl-pyruvate, 2,3-dihydroxybenzoate, 4-hydroxy-benzoate, 3,4-dihydroxybenzoate, menadione).3,4-Dihydroxybenzaldehyde (or some relatedpyrocatechol derivative) was known to be re-quired for growth of certain aromatic auxo-trophs of E. coli and was described as the "sixthfactor" (61, 62). Cox and Gibson also found that

0~q Hs0

A H 0HO... OOHR

HO COH 0

FIG. 5. Stereospecificity of the conversion of shi-kimate to menaquinone. R = Prenyl. The precursorshown is (6S)-[7-14C, 6-3H]shikimate.


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an auxotrophic strain of "Aerobacter aero-genes" (strain 170-44) did not form detectablequantities of menaquinone when grown on medi-um containing phenylalanine, tyrosine, trypto-phan, and 4-aminobenzoate. Similar results withthis strain and with E. coli 159-4, which isblocked after shikimate, were obtained by Dan-sette and Azerad (60) and Leduc et al. (133).When 3,4-dihydroxybenzaldehyde was presentat a final concentration of 10 ,uM, menaquinonewas produced (ubiquinone was produced underall growth conditions). This "A. aerogenes"strain is unable to convert 5-enolpyruvoyl-shikimate 3-phosphate into chorismate. Theseresults strongly implicated chorismate as thecommon branch point. The authors stated, fur-thermore, that there was little reason "to sup-pose that 3,4-dihydroxybenzaldehyde itself is onthe direct route of biosynthesis of vitamin K." Aclearly defined role for chorismate as a substratefor the enzymatic synthesis of OSB has nowbeen demonstrated (162) and is discussed below.The possible role of 3,4-dihydroxybenzalde-

hyde has remained enigmatic. Radioactive sam-ples of 3,4-dihydroxybenzaldehyde and the cor-responding acid were not incorporated intomenaquinones by cultures of E. coli, Mycobac-terium phlei, Bacillus megaterium, Proteus vul-garis, and "A. aerogenes" (43, 44, 83, 135).

Guerin et al. (83) were unable to stimulate "A.aerogenes" 170-44 to produce menaquinone onaddition of 3,4-dihydroxybenzaldehyde (con-trary to Cox and Gibson); furthermore, Leistneret al. (135) reported only a 40o suppression ofthe incorporation of activity from labeled shiki-mate into menaquinone with E. coli in the pres-ence of 3,4-dihydroxybenzaldehyde, whereasCox and Gibson found a larger value (althoughno numerical value was determined by theseauthors, the figure reproduced in their papersuggests a suppression of at least 85%). Otherworkers found a slight increase in shikimateincorporation when 3,4-dihydroxybenzaldehydewas added. It is now the general consensus thatneither 3,4-dihydroxybenzaldehyde nor the cor-responding acid plays any role in menaquinonebiosynthesis.

A Possible Role for 1-Naphthol?In 1967 Sandermann and Simatupang had iso-

lated 2,2-dimethylnaphthochromane from teakwood and had suggested on comparative phyto-chemical grounds that 1-naphthol would be anearly precursor of the chromane, plant naphtho-quinones, and materials such as tectol (188).This possibility was tested for menaquinonebiosynthesis in the same year by Leistner et al.(135). Using Bacillus megaterium, 1-[1-14C]-naphthol was reported to be incorporated to theextent of 1.5% (purification by thin-layer and

TABLE 2. Stereospecificity of the shikimate --

menaquinone conversion

3H/14C ratioCompound (6S)-[7-14C,6_3H] (6R)-[7-14C,6-3H]

shikimate expt shikimate expt

Precursor shikimate 8.70 6.92Menaquinone 7.30 1.29

reversed-phase thin-layer chromatography toconstant specific activity). On oxidation tophthalate all of the menaquinone activity wasretained (102%), and all of the radioactivity waslocated in the carboxyl groups. As a result, thefollowing partial pathway was suggested: 1-naphthol -- 1,4 - naphthoquinol-- 1,4 - naphtho-quinone-*menadione--*MK. Similar results with1-[1-14C]-naphthol administration (I = 0.3%) toStaphylococcus aureus were reported by Ham-mond and White (93, 94). In their work, thelabeled samples of menaquinone were also de-graded with permanganate to phthalate andphthalic anhydride. The phthalate derivativescontained about 87% of the menaquinone 14C.However, despite these chemical degrada-

tions, it now appears most probable that themenaquinone samples in this work were contam-inated with impurities that were difficult to re-move. Using more rigorous purifications, otherworkers have failed to show conversion of 1-naphthol to menaquinone in Mycobacteriumphlei, "Micrococcus lysodeikticus," Bacillusmegaterium (several strains, including that usedby Zenk and his colleagues), Proteus vulgaris,and Proteus mirabilis (21, 35, 44, 64, 83).A problem with 1-naphthol is its surprising

instability in growth medium. On standing 1-[1-14C]-naphthol in sterile growth medium in theabsence of bacteria at 37°C for 44 h, only 0.06%of original radioactivity was reisolated as 1-naphthol (21; H. Rapoport, personal communi-cation). The medium used was as follows: glu-cose, 3%; Casamino Acids, 1.3%; potassiumfumarate, 0.1%; Tween 80, 0.2%; K2HPO4,0.1%; MgSO4 * 7H20, 0.003%; FeSO4 * 7H20,0.002%; pH adjusted to 7.0 with KOH. In dis-tilled water under the same conditions, 96% ofradioactivity was recovered as 1-naphthol. Notonly does this instability present a problem inbiosynthetic experiments with 1-naphthol, butthere is, in addition, the possible degradation of1-naphthol by bacterial action. For example, asoil Pseudomonas grown on 1-naphthol as solecarbon source produced 3,4-dihydro-3,4-dihy-droxy-1(2H)-naphthalenone as an early interme-diate (227). Other workers found 4-hydroxy-3,4-dihydro-1(2H)-naphthalenone as a bacterialdegradation product of 1-naphthol (34).When the origin of the oxygen atoms in mena-

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quinone was studied, further evidence eliminat-ing 1-naphthol was obtained (203). For thiswork, Mycobacterium phlei was grown either inordinary water with an 1802 atmosphere or inH2180 and ordinary oxygen. Special isolationtechniques were used to prevent (nonenzymatic)exchange of the quinone oxygens with water.Menaquinone, MK-9 (II-H1), obtained bygrowth in the presence of ' 02 contained noexcess of 180 above the natural abundance. Onthe other hand, in a medium containing H218Othe quinone oxygen atoms were shown to con-tain 80 (in all cases samples were converted bypyrolysis to carbon monoxide for mass spectro-metric analyses). If 1-naphthol were a biosyn-thetic intermediate to menaquinone, introduc-tion of a second oxygen function would benecessary. Presumably this would require an"aromatic hydroxylase" enzyme. Since mostsuch enzymes utilize molecular oxygen, incor-poration of 180 (from 1802) into menaquinonewould be expected. No such incorporation wasobserved, so these experiments also cast consid-erable doubt on the proposed role for 1-naph-thol.

Origin of the "Three Carbon" UnitSince all seven carbon atoms of shikimate

were incorporated into the naphthoquinone nu-cleus, a search was undertaken for the source ofthe remaining three carbons (44, 121). Likelycandidates, such as pyruvate, glycerol, anddiethyl malonate, were not well converted toMK-8 by E. coli or to MK-9 (II-H2) by Myco-bacterium phlei. (I values, 0.0001 to 0.003%).Hammond and White also reported a low incor-poration (I = 0.035%) of activity from [2-4C]glycerol into the menaquinones of Staphylo-coccus aureus (93). [1-14C]- and D-[U-14C]ribosewere also not utilized effectively (I values, 0.003to 0.017%). When the three-carbon amino acidsalanine and serine were examined, the I valueswere only slightly higher (0.005 to 0.01%) but,surprisingly, very low dilution values were ob-tained (1.1 to 1.4), suggesting a rather directutilization.

Radioactive acetates were reasonably wellutilized by M4ycobacterium phlei (up to I =0.22% for [21 Clacetate); as expected, most ofthe incorporated 14C was associated with theisoprenyl side chain (about 80%7o). Surprisingly,chemical degradation showed significant activityin ring A. Since the aromatic portion of tyrosinewas also found to be radioactive, it was clearthat this incorporation occurred by leakage ofacetate radioactivity into the shikimate pool(44). The carboxyl carbon of acetate labeled C-1or C-4 (or both) of ring B to some extent,whereas the methyl carbon tended to label C-2

or C-3 (or both) of ring B. Acetate utilization byE. coli was much lower than with Mycobacte-rium phlei (0.002% with [1-]4C]acetate). WhenD-[l- 4Cglucose was used for growth of E. coli,higher incorporations were obtained (0.006 to0.04%). Most of the incorporated 14C (about809%) was associated with the isoprenyl sidechain; there was again a tendency to find some14C at C-2 or C-3 (or both) of ring B, presumablyas a result of the formation of [2-14C]acetate (oracetyl coenzyme A [CoA]).These experiments with acetate, coupled with

the observations that three carbon amino acidswere incorporated (albeit poorly) but with lowdilution suggested further testing of intermedi-ates of the citric acid cycle. The first test of thehypothesis used a system for which the chemicaldegradations were simpler than those for mena-quinones. Various plants produce lawsone (2-hydroxy-1,4-naphthoquinone), and it wasknown that shikimate utilization was involved(236); furthermore, in juglone (5-hydroxy-1,4-naphthoquinone) biosynthesis in Juglans regia,C-2 or C-3 (or both) of ring B were formed fromthe methylene carbon of malonate or the methylcarbon of acetate (136; compare the bacterialresults just described). Campbell, therefore, ad-ministered [1-14C]- and [U-14C]alanine, [U-14C]aspartate, and [2-14C]glutamate to excisedshoots of Impatiens balsamina plants, assumingthey would be converted, respectively, to pyru-vate, oxaloacetate, and 2-ketoglutarate (40). Ofthese precursors, the first three showed I valuesof 0.03 to 0.33%; the incorporation with [2-4C]glutamate was substantially higher, namely,1.36%. The chemical degradation used was oxi-dation to phthalate and C02, with further decar-boxylation of phthalate. It was found that [1-14C]-alanine gave a rather random distribution ofradioactivity. With [U-14C]-alanine, there wassubstantial labeling at C-2 or C-3 or both (73% oftotal lawsone activity); and with [U-14C]aspar-tate, at C-1 or C-4 or both (46%), as well as C-2and C-3 (35%). In the experiment with [2-14C]glutamate, a very specific incorporation oc-curred: 99% of the lawsone activity was at C-1or C-4 or both.Campbell concluded from this very significant

observation that the three central carbon atomsof ketoglutarate constituted the "missing threecarbon unit" (Fig. 6). He suggested that the unitactually attacking shikimate was the thiaminpyrophosphate (TPP) adduct of succinic semial-dehyde. This anion could be formed by the first(decarboxylase) enzyme of the ketoglutarate de-hydrogenase complex, and the addition wasmechanistically a Michael addition; the requiredanion could also have been formed by a separatedecarboxylase. It was clear that in the formationof the naphthoquinone system, both carboxyls

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Ala CH3COSCoA + HOOCCH2COCOOH Asp

HO COOH coo COOH

HO%V

4: . -

OH OH TPP COOH

Shikimate I

0 *0 0HO *COOH *OH

HO and/or OHOH 0 0

LawsoneFIG. 6. Amino acid utilization in naphthoquinone biosynthesis. The amino acids were administered to

Impatiens balsamina plants and lawsone was isolated. The labeling in the amino acids was uniform for Ala ("C= U), uniform for Asp ("C = 0), and at the 2 position for Glu ("C = A). It was assumed that 2-ketoglutaratewas formed by transamination from Glu or by tricarboxylic acid cycle reactions from acetyl CoA andoxaloacetate as shown. This drawing is based on the original (40) and shows a trihydroxydecalindione carboxylicacid as a possible precursor; this precursor is no longer considered to be involved.

of the glutamate-ketoglutarate precursor had tobe removed.A role for glutamate-ketoglutarate was quick-

ly confirmed in bacterial systems. Glutamatewas incorporated into MK by E. coli, Mycobac-tenum phlei, Corynebacterium diphtheriae, andStreptomyces albus with good I values (0.002 to0.02%) and little dilution (44, 181). In E. coli,which also contains ubiquinone, the ratio ofactivity MK/Q was high (20:1, 35:1) in twoexperiments (cf., for example, ratios of about1.1:1 for acetate feeding). This was in line withthe known biosynthesis of ubiquinone via p-hydroxybenzoate, a route giving no place toglutamate or ketoglutarate. In chemical degrada-tions, the use of [U-14C]glutamate gave approxi-mately equal labeling in C-1 (or C4), C-2, and C-3. With [2-14C]glutamate, the menaquinonesamples from E. coli and Corynebacteriumdiphtheriae were shown to be labeled at C-1 orCA or both, but there was no label in C-2 and C-3. Hence, there was a specific utilization ofglutamate C-2 for one of the quinone carbonylpositions. In a feeding of 2-[U_14C]ketoglutarateto E. coli, Robins and Bentley found an incorpo-ration (I = 0.011%) comparable to that for [U-

14C]glutamate (I = 0.015%) (180). The ratio ofactivity MK/Q was 24:1, a value in line with thatfound in the glutamate experiments. Chemicaldegradation established that the 2-[U-14C]keto-glutarate contributed activity essentially equallyto C-1 or C4 or both, C-2, and C-3 (16.3, 17.0,and 14.6%, respectively).As a result of this work, the origin of all of the

carbon atoms of menaquinones became known(Fig. 7).

Mevalonate0 I

PrenylShikimate-

I

CH3to S-Adenosyl-

2 CO2y methionine

2-KetoglutarateFIG. 7. Primary biosynthetic precursors of mena-

quinones.

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First Aromatic Intermediate, o-Succinylbenzoate

The discovery of a new pathway from choris-mate was foreseen by Young et al. in 1969 (233).In isotope competition experiments, neither 3-(1'-carboxyvinyloxy)benzoate (a chemical pre-

cursor to 3-hydroxybenzoate) nor 3,4-dihydro-3,4-dihydroxybenzoate (known to be producedenzymatically from chorismate) reduced 14Cincorporation from shikimate into MK-8 of E.coli. They concluded that if "none of the knowncompounds derived from chorismic acid areconcerned in vitamin K biosynthesis, it is proba-ble that one more metabolic conversion of chor-ismic acid is yet to be found" (233).

Important evidence for a new intermediate,the benzenoid compound OSB, was quicklyobtained by Dansette and Azerad (60). (Interest-ingly, this important compound had been firstprepared in 1884 [184]). Earlier, however, theyhad suggested a possible role for carboxyphenyl-pyruvate (133). These earlier speculations wereinfluenced by the finding that C-2 of malonate oracetate was incorporated into C-2 or C-3 or bothof the naphthoquinone ring of juglone in theplant Juglans regia (136). It was suggested thatthe carboxyphenylpyruvate was derived fromisochorismate; hence, all nine carbon atoms ofchorismate would have been retained in thenaphthoquinone nucleus, the remaining one be-ing the central methylene of malonate. Howev-er, o-[2-14C]carboxyphenylacetate, a potentialfurther intermediate from carboxyphenylpyru-vate, either as the free acid or ester, was notincorporated into menaquinone by Mycobacte-rium phlei or "A. aerogenes" 170-44 (133).With the knowledge that three carbons of the

naphthoquinone were obtained from ketogluta-rate, and reasoning that a condensation withchorismate might give an aromatic compounddirectly, they then synthesized OSB (60). Thecompound supported growth and allowed mena-quinone formation in E. coli 159-4 (a mutantblocked after shikimate) and with "A. aero-genes" 170-44 (blocked after 5-enolpyruvylshi-kimate 3-phosphate). Furthermore, [14C]OSBwas incorporated into MK-9 (II-H2) in Mycobac-terium phlei with a low dilution (I = 70%; D =1.85). Much smaller I values were obtained in"A. aerogenes" 62-1 and E. coli K-12 (0.18 and2.25%, respectively). In these organisms, D val-ues were measured for both MK-8 and DMK-8.With "A. aerogenes," the two D values were0.14 and 0.38, and in E. coli they were 8 and18.5. Thus, despite the relatively low incorpo-ration in these bacteria compared with Myco-bacterium phlei, the utilization of OSB proceed-ed with good specificity (Fig. 8). Also ofimportance, in the last two organisms, was thelack of 14C in ubiquinone.

MICROBIOL. REV.

The MK-9 (11-H2) from Mycobacterium phleiwas degraded to phthalate with KMnO4; decar-boxylation of the phthalate showed that all of thelabel of the menaquinone was localized in C-1 orC-4 or both (see Fig. 8). [2',4-14C21OSB was alsofound to be an excellent precursor of MIK inBacteroides melaninogenicus (I = 0.9%; D =1.8) (182). A somewhat higher incorporation of[2,3-14C2]OSB into menaquinone of E. coli K-12was reported later; in this work I = 1.8% and D= 6.3 (44). Campbell et al. also showed that [1-14CJOSB was not incorporated under the sameconditions. Hence, the "aliphatic" carboxyl ofOSB is clearly lost during MK biosynthesis (seeFig. 8).

It was not until 1981 that a direct study ofOSBbiosynthesis was carried out (164). For thiswork, glutamate samples specifically labeledwith 14C at C-5 and C-1, or uniformly labeled,were administered to E. coli AN209, a mutantshown to accumulate OSB (see below). Theisolated OSB was converted to a dimethyl deriv-ative for examination by radiogas chromatogra-phy. When the precursor glutamate contained4C at position 5, or was uniformly labeled, thedimethyl OSB was radioactive. From [1-14C]glu-tamate, however, the dimethyl OSB was without14C. Hence, in OSB biosynthesis, C-1 of gluta-mate is lost and C-2 to C-5 are retained.

1,4-Dihydroxy-2-Naphthoate, a NaphthalenoidIntermediate

In 1969, Campbell suggested a Claisen-typereaction as the mechanism for formation of thenaphthalenoid nucleus. The reaction involvedthe carboxyl group introduced by shikimate(chorismate) and the C-4 methylene of ketoglu-tarate (40). A trihydroxy-carboxy-decalindionewas written as a possible intermediate. Later,Campbell et al. and Robins et al. suggested arole for 1,4-dihydroxy-2-naphthoate possiblyformed from the decalin derivative (44, 181).Dansette and Azerad (60) realized that the dihy-droxynaphthoate could be directly formed by aClaisen-type process on OSB (Fig. 9). Theyadministered [14C]OSB and unlabeled DHNA tothe plant Impatiens balsamina, but found nochange in incorporation compared with controls.The first evidence implicating DHNA as a

menaquinone intermediate was obtained in 1973(182). The growth ofa vitamin K-requiring strainof Bacteroides melaninogenicus was stronglystimulated by DHNA; this material was effectiveat 10-5 M and was comparable to phylloquinoneor MK-9. Among the nonquinonoid naphthalenecompounds, it is the most effective growth stim-ulator for this organism. Furthermore, [2,3-14C2]DHNA was administered to E. coli, usinganaerobic growth conditions to minimize oxida-


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0

QCH30

&CO2

COOH

COOH

MK

FIG. 8. Utilization of OSB for menaquinone biosynthesis. R = Prenyl. The results of two separateexperiments have been combined. The reactions are: a, biosynthetic utilization; b, degradation to phthalateeither directly with KMnO4 or as shown in Fig. 2.

tive degradation (25). A high incorporation intoMK-8 was obtained (I = 3.3%) coupled with alow D value (1.4). (By contrast, in a controlexperiment under the same conditions, 1,4-[1,4-14C2]naphthoquinone was less efficiently incor-porated and with higher dilutions: I = 0.3%; D =14.5.) The MK-8 derived from the [2,3-14C2]DHNA was degraded to phthalate and mal-onate. The phthalate was without radioactivity,and the malonate contained 50% of the activityof the naphthylacetate (see Fig. 2 for reactions).Hence, the conversion apparently was a directone, without degradation. The carboxyl groupis, of course, removed at some further stage (seeFig. 1).

It is of interest that DHNA is degraded bycell-free extracts of a nonfluorescent pseudomo-nad (grown on m-cresol) to pyruvate and phthal-ate (108). This reaction was postulated to in-volve a keto derivative of OSB. In phenan-threne-grown Aeromonas sp. S45P1, the related

Chorismate,Ketoglutarate

1-hydroxy-2-naphthoate is degraded to 2-car-boxybenzaldehyde and hence to phthalate (123).

Role of Naphthalene Compounds Other than 1-Naphthol

In addition to the somewhat contradictoryresults obtained in the incorporation experi-ments with radioactive 1-naphthol, equally con-tradictory results have been obtained with someother naphthalene compounds. Some of the ob-servations are summarized in Table 3. In fourorganisms, there is apparently a utilization oflabeled menadione for MK biosynthesis; theseobservations indicate that the final stage in men-aquinone biosynthesis is prenylation rather thanmethylation. In the case of Staphylococcus aur-eus, this possibility gains some credence fromthe fact that menadione (i.e., MK-0) can actuallybe shown to be present along with other, moreusual isoprenylogs (93).To conclude that menadione is, in some cases,

Ao ,COOH

OHDHNA

OSBFIG. 9. Formation of 1,4-dihydroxy-2-naphthoate. The (no longer accepted) trihydroxydecalindione carbox-

ylic acid was proposed by Campbell et al. (44) and Robins et al. (181), and OSB was proposed by Dansette andAzerad (60).

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TABLE 3. Incorporation of naphthalenoid materials into bacterial menaquinonesPrecursor Organism Incorporation Reference

[methyl-14C]menadione "Aerobacter aerogenes" 170-44 + 83[methyl-14C]menadione Bacillus megaterium + 217[methyl-t4Cjmenadione Bacteroides melaninogenicusa + 150[methyl-14C]menadione Staphylococcus aureus + 93, 217[methyl-14C]menadione Escherichia coli - 44, 217[methyl-14C]menadione Micrococcus luteus - 220[methyl-14C]menadione Mycobacterium phlei - 44, 2171,4-[1,4-14C]naphthoquinone Mycobacterium phlei - 441,4-[1-14C]naphthoquinone "Aerobacter aerogenes" 170-44 + 831,4-[1,4,5,8-14C]naphthoquinone "Aerobacter aerogenes" 170-44 + 831,4-[5,8(?)-3H]naphthoquinone Bacteroides melaninogenicus + 150[methyl-3H,1',2'-14C]phylloquinone Bacteroides melaninogenicus + 150

a [methyl-3H]menadione was also examined in this organism.

a precursor to menaquinones, it would be neces-sary to carry out further experiments. Thesewould involve rigorous purification of samplesderived from tracer experiments with labeledmenadione. The problem is illustrated by workon the administration of [methyl-14C]menadioneto E. coli and Mycobacterium phlei. Althoughthe initial extracts containing the MK fractionswere strongly radioactive, on gel filtration chro-matography followed by derivatization, the ra-dioactivity was lost (44). Unfortunately, rigor-ous purification procedures such as these havegenerally not been used, so the precise signifi-cance of a positive incorporation result is diffi-cult to assess.The incorporations observed in some cases

with labeled samples of naphthoquinone are alsodifficult to evaluate. Since the evidence stronglyindicates that a symmetrical intermediate is notinvolved in menaquinone biosynthesis, it ap-pears that some kind of aberrant pathway mustbe involved, if indeed the results are not due tothe presence of radioactive contaminants. Twoorganisms, "A. aerogenes" 170-44 and Bacte-roides melaninogenicus, seem to be particularlyactive in converting naphthalenoid compoundsto menaquinones. The Bacteroides melanino-genicus strain examined by Martius and Leu-zinger (150) was actually the Lev strain andrequired a supplement of vitamin K for growth.A particularly striking result was the conversionof phylloquinone, labeled in the methyl groupwith 3H and in the phytyl side chain with "C to amenaquinone containing only 3H. This observa-tion implies that the phytyl side chain was

removed and replaced with the typical mena-quinoid side chain. This result may again besuspect. The purification used was a Craig coun-tercurrent distribution, and on a single separa-tion the MK fractions appeared to be labeledwith both isotopes. On repetition of the counter-

current distribution, it appeared that the 3Hactivity showed two maxima; the second wasvery small and contained a reduced level of 14C.However, the experimental evidence (150 [Fig.3b]) is less than convincing, although it wasclaimed that this small peak was MK-9. A muchlarger peak containing large amounts of both 14Cand 3H was also present. Martius and his col-leagues also observed that radioactive phyllo-quinone was converted to MK4 in animalswhen administered orally, but not when admin-istered by injection (29). This again suggestedthat the phylloquinone side chain was removedby intestinal bacteria. The methodology reliedon the Craig countercurrent distribution tech-nique and is not above suspicion.Whatever the merits of these incorporation

experiments, the genetic and enzymologicalstudies in organisms such as Bacillus subtilis andE. coli, and the enzymological work in Micro-coccus luteus and Mycobacterium phlei, provideno indication for a role for menadione or naphth-alenoid derivatives other than DHNA in mena-quinone biosynthesis.A further complication in work on the possible

significance of naphthalenoid compounds is theknown toxicity of many of these materials forbacteria (14). In particular, substituted naphtho-quinones are quite inhibitory to the growth ofbacteria (131). For 50%o inhibition of growth ofE. coli and Staphylococcus aureus, the concen-trations ranged from 4.76 x 10-5 to 36.0 x io-0M and 0.09 x 10-5 to 9.0 x 1i-0 M, respectively(61 compounds were examined in this work). Inthe more sensitive Staphylococcus aureus, men-adione had a 50%o growth inhibition concentra-tion of 0.55 x 10' M, and phylloquinone hadone of 11.1 x 1i-0 M. Even in a bacterium witha specific growth requirement, toxicity may oc-cur beyond a critical concentration (139). InBacillus cereus, the antibacterial action of mena-

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dione results from a specific inhibition of RNAsynthesis in growing cells (124).

INDIVIDUAL REACTIONS INMENAQUINONE BIOSYNTHESIS

Formation of o-Succinylbenzoate

The formation of OSB from chorismate and 2-ketoglutarate (glutamate) represents an unusualsynthesis of an aromatic compound from a cy-clohexadiene structure. These reactions providethe largest unknown area of the entire biosyn-thetic pathway, and it is only recently that a cell-free synthesis of OSB has been demonstrated byMeganathan (162). Improved techniques for theisolation, purification, and identification of smallquantities of OSB were devised (164); a majorcomponent was the use of radiogas chromatog-raphy to examine OSB as its dimethyl ester. Acell-free extract of E. coli, prepared by use of aFrench press, was incubated with 2-[U-14C]ke-toglutarate and chorismate in the presence ofthiamine pyrophosphate (TPP). In the absenceof chorismate, no OSB was produced, and in theabsence of TPP there was a decreased synthesisof OSB. The organism originally used was an E.coli mutant, AN154, blocked in all of the aro-matic pathways with the exception of that formenaquinone (see Genetics of MenaquinoneBiosynthesis). The occurrence of "OSB syn-thase" activity has, however, now been demon-strated in wild-type strains (R. Meganathan andR. Bentley, unpublished data).The chemistry of this reaction is clearly com-

plex; the fundamental addition of the succinylside chain has for long been considered to re-quire the succinic semialdehyde anion complexofTPP (40). If this is indeed the case, as many asfive separate stages might be needed for theoverall process: (i) formation of the succinicsemialdehyde-TPP complex from ,2-ketoglutar-ate; (ii) addition of the succinic semialdehyde-TPP complex to chorismate; (iii) regeneration ofTPP; (iv) removal of the pyruvoyl group orig-inally associated with chorismate; and (v) re-moval of the hydroxyl group originally associat-ed with chorismate.Formation of succinic semialdehyde-thiamine

pyrophosphate complex. A succinic semialde-hyde-TPP anion is presumably formed duringthe action of the 2-ketoglutarate dehydrogenasecomplex by the first (decarboxylase) enzyme

(Fig. 10). Although this first enzyme of theketoglutarate dehydrogenase complex remainsas a prime candidate, an alternate and separatedecarboxylase cannot be ruled out at this time.After dialysis, the E. coli OSB synthase prepara-

tions were stimulated by the addition of TPP,and these same extracts showed stimulation by

TPP when 2-ketoglutarate decarboxylase activi-ty was assayed by ferricyanide reduction (91;modified to read absorbance at 420 nm). Whenextracts were prepared from a 2-ketoglutaratedecarboxylase-negative mutant of E. coli, therewas a decreased incorporation of 14C from 2-[U-14C]ketoglutarate into OSB. This observation isconsistent with the known leakiness of thisgroup of mutants and their ability to grow anaer-obically on lactatefumarate media.Subsequent reactions. Figure 11A shows a

possible reaction sequence in which the orderingof the stages is as follows: anion addition, re-moval of TPP, removal of the pyruvoyl group,and removal of the hydroxyl. An analogy for theproposed biosynthetic process has been provid-ed recently by a stereoselective synthesis ofdecalin derivatives (113; Fig. 12).

It is not easy to predict how many enzymesare needed for the various stages. The maximumis probably four, in addition to the decarboxyl-ase. This number could be reduced if more thanone stage was catalyzed by a single enzyme or ifsome of the reactions were concerted. For ex-ample, the anion addition (step 1, Fig. 11A)and removal of TPP (step 2, Fig. llA) couldperhaps require only one enzyme. An attractivepossibility for anion addition concerted withremoval of the chorismate OH group is shown asstep 1 in Fig. llB. If the enzyme also catalyzedTPP removal (step 2, Fig. llB), only two en-zymes, in addition to the decarboxylase, wouldbe required. A concerted mechanism for theaddition of glutamate to chorismate during theaction of anthranilate synthetase was proposedearlier (204, 212); in this case, addition takesplace at position 6 of chorismate, and directevidence for a postulated intermediate has, ap-parently, not been obtained.Some evidence for two enzymes comes from

the fact that there are two groups of E. colimutants, menC and menD, which require OSBfor growth and are, therefore, blocked in OSBbiosynthesis (55, 84, 85). This suggests the pos-sible occurrence of a definite intermediate in theformation of OSB from chorismate and 2-keto-glutarate. To investigate this possibility, cell-free extracts were prepared from menC andmenD mutants. After incubation of these ex-tracts separately with 2-[U-14C]ketoglutarateand chorismate followed by protein denatur-ation, ethyl acetate extraction was performed inthe hope of isolating any such intermediate. Thetwo materials obtained after removal of ethylacetate were then further incubated with anextract from the other mutant. Evidence wasobtained that the menC mutant did, in fact, forman intermediate and that this intermediate wasconverted to OSB by the extract from the menD

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R2 R3 R2 R3

R 5s

H R4icooe9TPP L0 H

2 R3

RlIKS

R-OOH

FIG. 10. Formation of the succinic semialdehyde-TPP anion. R1 = Pyrimidine component of TPP; R2 = CH3;R3 = CH2CH20P2063-; R4 = CH2CH2COOH. The suggested anion is shown as the final structure; it is onepossible resonance structure.

mutant (R. Meganathan and R. Bentley, Abstr.Annu. Meet. Am. Soc. Microbiol. 1982, K138,p. 159). It appears from these preliminary ex-periments that the menD gene codes for anenzyme which is responsible for the formation ofan intermediate, "X," and that the enzymecoded for by the menC gene converts this inter-mediate to OSB:

three possible structures for the intermediate areindicated in Fig. 11.

Formation of 1,4-Dihydroxy-2-NaphthoateThe enzymatic conversion OSB -+ DHNA

was first demonstrated in E. coli (37) and Myco-bacterium phlei (159) extracts. It was of consid-erable interest that the conversion showed an

menD menCChorismate + succinic semialdehyde-TPP -- X -- OSB

t~-+ Co22-ketoglutarate + TPP toQ|^ltO mt

The chemical nature of X remains to be deter-mined; assuming that it does not contain TPP,

HOOC>,,, COOHHf R StIL2 HO I

I 1 ~~2TPP

A

adsoiuLe aepenUence on Ine presence oLi -oAand ATP. However, initial attempts to purify theenzyme(s) were not successful. It was subse-

--3 - TPP

tep 3

CH3COCOOH

BHOOC . O/3OOHsH 0e-OH

I 1p 2

YCCOOH

6 0

CH3COCOOHSte 3

HOOC 0 COOH HOOC O'" OOH"If HR Stop 24- (

4D Th0-TP&P7TPPFIG. 11. Reaction mechanisms for OSB biosynthesis. R = CH2CH2COOH. Compounds are identified as

follows: 1, chorismate; 2, succinic semialdehyde-TPP anion; 6, OSB. The postulated intermediate, X, could havestructure 4, 5, or 8, assuming it does not contain TPP.

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HFIG. 12. Chemical analogy for the biosynthetic reaction. The chemical process was carried out with KF in

dimethyl sulfoxide.

quently found that a cell-free enzyme extractfrom Mycobacterium phlei could be treated withprotamine sulfate in the presence of 20% di-methyl sulfoxide so that one protein was precip-itated and a second remained in solution; neitherprotein alone formed DHNA, but a combinationof the two did so (163). These facts, combinedwith the following, provided strong evidencethat a CoA derivative of OSB was involved asan intermediate in the overall conversion. (i) Bythe use of [14C]ATP, it could be shown thatAMP and pyrophosphate were formed during

COOHarO~COHC

ATP, CoAo E-I

OSB OSE

0OCoA

Ho J* Non-enzymaticI0

the reaction, which is typical of those ligasesforming CoA esters. (ii) When the protein re-maining in solution (on treatment with prot-amine sulfate as just described) was incubatedwith [2-14C]OSB, ATP, and CoA, the spirodilac-tone derivative of OSB was produced (but noDHNA). This result could be explained by for-mation of the (unstable) OSB-CoA derivative,followed by a spontaneous, nonenzymatic lac-tonization. These processes are summarized inFig. 13.The two enzymes have been termed OSB-

COSCoACOOH

0 E-IEB-CoA DHNA

CoA-SH

OSB-CoA

0

0SPIRODILACTONE

FIG. 13. Formation of OSB-CoA and its conversion to DHNA by an enzyme and to a spirodilactonenonenzymatically. The enzymes involved are: E-I = OSB-CoA synthetase; E-II = DHNA synthase.

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CoA synthetase (E-I) and DHNA synthase (E-II); the separability of the Mycobacterium phleienzymes has received an independent confirma-tion (98). Further evidence for their existencecame from enzymological studies on mutants ofBacillus subtilis and E. coli (165; D. J. Shaw,J. R. Guest, R. Meganathan, and R. Bentley,unpublished data).

Extracts from a (wild-type) men' Bacillussubtilis catalyzed DHNA formation at a some-what higher pH, 7.5 to 8.5, than did Mycobacte-rium phlei extracts (pH 6.9). Extracts of mutantstrains RB413 (men-325) and RB415 (men-329)produced DHNA in combination with extractsfrom either RB388 (men-310) or RB397 (men-312); no DHNA was formed by any extractseparately. Complementation analysis withpreparations of OSB-CoA synthetase andDHNA synthase from Mycobacterium phleishowed that RB388 (men-310) and RB397 (men-312) lacked OSB-CoA synthetase but did pos-sess DHNA synthase; the reverse situation heldwith RB413 (men-325) and RB415 (men-329).Mutants defective in the structural gene forOSB-CoA synthetase are now termed menE,and those defective in the structural gene forDHNA synthase are termed menB. E. coli mu-tants deficient in one or the other enzyme havesimilarly been identified by the same methods.

In this work, fresh cells were used and werelysed with lysozyme. In addition to unchangedOSB and DHNA, the thin-layer chromatogramsshowed a third spot which was identified as thespirodilactone of OSB. We believed that forma-tion of spirodilactone resulted from a low levelof DHNA synthase in the extracts; when aDHNA synthase preparation from Mycobacte-rium phlei was added to the incubation mixtures,spirodilactone formation was suppressed andDHNA production increased. No difficulty wasexperienced in showing DHNA formation fromOSB with extracts prepared from 5-year-oldspray-dried cells of Micrococcus luteus. Thereis, therefore, no ready explanation for the failureof Hutson and Threlfall to show DHNA forma-tion in extracts from Micrococcus luteus.

In earlier work with E. coli, we had sometimesencountered some formation of the spirodilac-tone (37); it appears that bacterial extracts con-tain different levels ofDHNA synthase and withthose that we have examined the situation is asfollows: Mycobacterium phlei extracts nevershow formation of spirodilactone; E. coli ex-tracts sometimes show formation of spirodilac-tone; Micrococcus luteus and Bacillus subtilisextracts always show formation of spirodilac-tone. The OSB-CoA derivative is rather unsta-ble and, unless a high level of DHNA synthase

OsOSB menEA tht'OSB-CoA men th DHNAOBOSB-CoA synthetase DHNA synthase

It was also observed that when extracts frommenB mutants of Bacillus subtilis and E. coliwere incubated with [14C]OSB, ATP, and CoA,radioactive spirodilactone was the only productformed. Further, the spirodilactone formationcould be suppressed by adding DHNA synthasefrom Mycobacterium phlei. This result againsupported the formation of an unstable OSB-CoA compound which decomposed to spirodi-lactone by elimination of CoA-SH (see Fig. 13).Other workers were unable to demonstrate

the conversion of OSB to DHNA by using cell-free extracts prepared from spray-dried cells ofMicrococcus luteus (111); similar negative re-sults were also obtained in a limited number ofexperiments with E. coli extracts. With theMicrococcus luteus extracts, they routinely ob-served the formation of OSB spirodilactone onincubation of OSB with CoA and ATP. Micro-coccus luteus contains high levels of menaquin-one (about five times those in E. colt), and thesesurprising results raised the possibility of analternate pathway for menaquinone biosynthe-sis. In our hands, incubation of cell-free extractsof Micrococcus luteus under the same condi-tions did lead to the formation of DHNA (166).

activity is present, spirodilactone formation oc-curs with elimination of CoA-SH (see Fig. 13).

It is not known at present whether thesediffering responses result from different levels ofDHNA synthase relative to OSB-CoA synthe-tase in different organisms or whether it reflectspossible loss of DHNA synthase activity onextraction from cells. So far, most attention hasbeen given to the two enzymes present in Myco-bacterium phlei, Micrococcus luteus, and Bacil-lus subtilis (163, 165, 166). In general, DHNAsynthase appears to be less stable than the OSB-CoA synthetase. For example, in Mycobacte-rium phlei preparations, the OSB-CoA synthe-tase shows a definite resistance to low pH andenzymatic activity can be recovered after expo-sure of the preparations to 0.1 N HCI for 5 min.Under these conditions, the Mycobacteriumphlei DHNA synthase is completely inactivated(163).The OSB-CoA synthetase from Mycobacte-

rium phlei has been purified approximately1,200-fold (Table 4); on acrylamide gel electro-phoresis, however, there is present one majorand two or three minor bands (R. Meganathan,C. Dippold, and R. Bentley, unpublished data).

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TABLE 4. Purification of OSB-CoA synthetase

Procedure Fraction Vol U Total Protein Activity Yield Purifi-Procedureno. (MI) (m)'a (m/l (U/mg of (% cationprotein) (fold)

Extract 37.1 24.5 909.0 30.00 1.23 100Protamine Sb 59.4 12.7 754.4 2.90 4.30 83 3.6

sulfateprecipitation

Acid 49.8 11.8 587.6 0.69 17.10 65 13.9treatmentand dialysis

(NH4)2SO4 1.5 367.6 551.4 3.80 96.70 61 78.6precipitationand dialysis

Affi-Gel Blue 12-14 3.0 156.9 470.7 0.15 1,046.00 52 850.0Column

Matrex Gel 12-14 3.0 125.1 375.3 0.08 1,564.00 41 1,271.0Green Aa Unit = nanomoles of DHNA produced per 30 min.b S, Supernatant.

In this purification the use of relatively nonspe-cific affinity columns was particularly useful;possibly construction of OSB-containing affinitycolumns might lead to a complete purification.

In E. coli these two enzymes do not behave asjust described. Attempts to use the dimethylsulfoxide-protamine sulfate method for the sepa-ration of the E. coli enzymes have led to loss ofall DHNA synthase activity, although the OSB-CoA synthetase activity was retained. A similarresult was obtained with ion-exchange chroma-tography. Furthermore, the E. coli OSB-CoAsynthetase is inactivated by acidic conditionsunder which the Mycobacterium phlei, Bacillussubtilis, and Micrococcus luteus enzymes retainactivity.

Structure of o-Succinylbenzyl-Coenzyme A Inter-mediate

In 1981, Heide and Leistner achieved theisolation of the putative OSB-CoA derivative(98). A preparation of OSB-CoA synthetasefrom Mycobacterium phlei was prepared by theprotamine sulfate precipitation methods just de-scribed. After incubation of [4'-14C]OSB, ATP,CoA, and Mg2+ with the enzyme preparation,separation was attained by paper chromatogra-phy (Whatman 3 MM paper; butanol-acetic acid-water, 5:2:3). The CoA derivative (Rf = 0.48)was eluted with 3 M formic acid and was furtherpurified on a Hg-Sepharose column which re-tained residual CoA-SH. Formation of the 14C-labeled CoA derivative was only observed in thepresence of enzyme and ATP. Use of 3H-labeledCoA-SH also gave a radioactive product; withboth labels, it was possible to show that the3H/14C ratio was that expected from a mono-CoA derivative rather than a di-CoA ester. The

OSB-CoA derivative was active as a substratewith DHNA synthase, as expected.The OSB-CoA ester was relatively unstable,

as had been concluded before its isolation. Itwas most stable at acid pH and was converted toOSB spirodilactone plus CoA-SH under neutralconditions and to OSB plus CoA-SH underalkaline conditions.

Subsequently, Leistner and his colleagueshave provided evidence that the CoA moiety islocated on the aromatic carboxyl group of OSB(128). In this work, paper chromatography wasreplaced by thin-layer chromatography on cellu-lose for the isolation of the ester. The nonesteri-fied carboxyl was reacted with diazomethane,and the resulting diester (CoA, CH3) was hydro-lyzed under mild conditions to cleave the thioes-ter bond. The 14C-labeled product was com-pared with reference samples of "aliphatic" and"aromatic" ester by thin-layer chromatographyon silica gel. (Reference samples were obtainedby partial hydrolysis of dimethyl OSB and wereidentified on the basis of Rf values and 1H-nuclear magnetic resonance and mass spectra.In particular, the base peak at mlz 149 wasobtained by the fragmentation shown in Fig. 14.)This work provided convincing evidence thatthe aromatic carboxyl carries the CoA unit, ashad been originally suggested (37, 159, 163). Thecorrect location of the CoA unit is, in fact,shown in Fig. 13 and 14.

Similar results have been obtained in ourlaboratory (R. Meganathan, G. Emmons, L. A.Ernst, I. M. Campbell, and R. Bentley, unpub-lished data). [14C]OSB was incubated with puri-fied preparations of OSB-CoA synthetase ob-tained from Mycobacterium phlei; productswere separated by thin-layer chromatography oncellulose plates (n-butanol-acetic acid-water,

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5:2:3). The slowest-moving peak (Rf = 0.51) wasremoved, dissolved in methanol, and treatedwith diazomethane. The labile thioester bondwas subsequently hydrolyzed at pH 8.0 to yielda monomethyl OSB. This product was subjectedto the action of diazoethane, and the mixedmethyl ethyl ester of OSB was examined byradiogas chromatography and mass spectrome-try. For identification purposes, dimethyl anddiethyl derivatives of OSB were prepared byalkylation, respectively, with diazomethane anddiazoethane; mixed esters were obtained byexchange reactions. Study of the mass spectra ofthese known compounds indicated the charac-teristic fragment ion shown in Fig. 14. Themixed methyl ethyl ester obtained from enzy-matically synthesized OSB-CoA was shown tohave the ethyl group on the aromatic carboxyl,indicating location of the CoA at that position.

Prenylation ReactionIn the presence offarnesyl pyrophosphate and

Mg2+, cell-free extracts of E. coli were shown toconvert DHNA to MK-3 or DMK-3 or both (25).In more detailed investigations, Shineberg andYoung obtained a membrane-bound enzyme,1,4-dihydroxy-2-naphthoate octaprenyltransfer-ase, from E. coli; this enzyme was active witheither synthetic solanesyl pyrophosphate or(natural) octaprenyl pyrophosphate, but solane-syl monophosphate was not a substrate (201).The enzyme showed a lipid and Mg2+ require-ment. The overall conversion, DHNA -* DMK,actually requires three stages: removal of theDHNA carboxyl as C02, the attachment of theisoprenoid residue, and a quinol -- quinoneoxidation. The demethylmenaquinol is a likelyintermediate, and possibly its conversion toDMK is a spontaneous process (Fig. 15). Thequestion of whether more than one enzyme isinvolved has not been answered unequivocally;the available evidence, however, suggests oneenzyme, possibly with a concerted mechanism.Evidence for a single enzyme is that the decar-boxylation product, 1,4-naphthoquinol, cannotexist as even a transient intermediate since this

11OS<COSCoA

Q+/CCOOH0

OSB-CoA

COSCoA b

I - COOCH3

0

would lead to a symmetrical situation. Further-more, as will be described below, menA mutantsof E. coli accumulate DHNA and not 1,4-naphthoquinol or 1,4-naphthoquinone (234).The 1,4-dihydroxy-2-naphthoate octaprenyl-

transferase has some features in common with 4-hydroxybenzoate octaprenyltransferase in-volved in ubiquinone biosynthesis (both aremembrane bound and require Mg2+). In addi-tion, both of these enzymes appear to use acommon pool of membrane-bound octaprenylpyrophosphate as the prenyl donor (127, 234).Genetic evidence, however, indicates that theyare quite distinct. The side chain lengths of themenaquinones are probably determined by theavailability of the isoprenyl pyrophosphate sub-strate(s) within the membranes. In addition tothe situation just described for E. coli, there isan association between the polyprenyl pyro-phosphate synthetase isolated from Bacillussubtilis, which produces all trans-heptaprenylpyrophosphate (211), and the fact that this orga-nism produces exclusively MK-7. It is of interestin this connection that an enzyme reactingDHNA with phytyl pyrophosphate has been de-tected in spinach chloroplasts (197); this enzymeis involved in the biosynthesis of phylloquinone.A similar enzyme is presumably present in thecyanobacteria, which also biosynthesize phyllo-quinone.

Saito and Ogura have also investigated aprenyl transferase enzyme in the membranefraction of Micrococcus luteus (185). This en-zyme was found to be relatively nonspecific withrespect to the prenyl unit; both all trans-famesylpyrophosphate and all trans-geranylgeranyl py-rophosphate were effectively converted toDMK. The all trans forms of octaprenyl pyro-phosphate and farnesyl and geranyl phosphatewere less effective. Inactive compounds weredimethylallyl pyrophosphate, trans-farnesol,and trans-octaprenol. It appears from these re-sults that a trans configuration in the 2 doublebond is important. With regard to the prenylacceptor, a greater specificity was observed. Asexpected, DHNA was very active, and the fol-

oOOR-. C OOCH3

0

2

>sCOOR

c

3FIG. 14. Structure ofOSB-CoA. Two sets of experiments are covered. In the work of Leistner and colleagues

(128) the OSB-CoA derivative was converted with CH2N2 (a) to the methyl ester, 1; the acid, 2, R = H, wasobtained on mild alkaline hydrolysis (b). On mass spectrometry (c), this material provided the ion, 3, R = H, m/z= 149. In unpublished work from our laboratory, the same methyl ester, 1, was hydrolyzed (b) at pH 8; theproduct was realkylated with diazoethane to 2, R = C2H5. On mass spectrometry (c), the ion, 3, R = C2H5, m/z =177, was obtained.

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JV "", -< -h DMK

R -P207 P2037-OH 27OH

DHNAFIG. 15. Prenylation of 1,4-dihydroxy-2-naphthoate. R = Prenyl.

lowing were much less effective as acceptors: 2-carboxy-4-hydroxy-a-tetralone, 1,4-dihydroxy-3-methyl-2-naphthoate, and 1-hydroxy-2-naphthoate. A number of substituted benzoicand naphthoic acids and naphthalenes were in-active.

Methylation of DemethylmenaquinoneA cell-free extract of Mycobacterium phlei

was shown to incorporate radioactivity fromL-[14CH3]methionine into menaquinones; ATPand MgCI2 were required, probably to facilitatethe formation of S-adenosylmethionine (18, 45).Similarly, the conversion of DMK-3 to MK-3was demonstrated in E. coli extracts by usingS-[14CH3]adenosyl-L-methionine (37). Thestructural requirements of the methylase systemwere investigated by Samuel and Azerad (187).DMK-1 was not a substrate, and a side chaincontaining two or more prenyl units was re-quired. DMK-3 and DMK-4 showed maximalactivity, which decreased progressively up toabout DMK-9. Saturation of one or more of theisoprene units, other than the first, had littleeffect on the methylation rate; both DMK-3 (II-H2) and demethylphylloquinone were good sub-strates. Saturation of the first double bond (adja-cent to the nucleus) resulted in loss of activity.The trans configuration was necessary in thefirst double bond. Structural changes in thenaphthoquinone ring, by partial saturation (5,8-dihydro-2-phytyl-1,4-naphthoquinone) or by re-placement (2,3-dimethylbenzoquinone), resultedin loss of activity.The methylase enzyme (S-adenosylmethio-

nine:2-demethylmenaquinone methyltransfer-ase) is localized in the particulate membranefraction of Mycobacterium phlei. Lipids couldbe removed.from the preparations by treatmentwith acetone to yield a powder, stable for somemonths. However, attempts at further solubili-zation and purification were not satisfactory,and reproducible results could not be obtained.A change in substrate specificity occurred in thepresence of a phospholipid fraction; under theseconditions, DMK-2 was the preferred substrate.

Investigations with [methyl-2H3]methionine

have shown that in formation ofMK-9 (I-H2) byMycobacterium smegmatis all three 2H atoms ofthe methyl group were transferred ("mecan-isme-CD3") (117). A similar result has beenobtained for MK-8 produced by E. coli strain518 (115) and for the formation of a hydroxylatedphylloquinone by the alga Euglena gracilis(218). In contrast, only two hydrogens are trans-ferred, for example, in the biosynthesis of the"extra" methyl group of C28 sterols and in theformation of tuberculostearic acid by Mycobac-terium smegmatis (132).

Formation of Reduced Isoprenyl Units

Although the formation of menaquinones withone or more isoprene units in the reduced condi-tion is common (48), little is known of thebiosynthesis of these materials. The reduction ofMK-9 to MK-9 (II-H2) has been demonstrated incell-free extracts of Mycobacterium phlei (18).The reduction required NADH or NADPH aselectron donor, but nothing further is knownconcerning this enzyme.

GENETICS OF MENAQUINONEBIOSYNTHESIS

men Mutants of Escherichia coliThe most complete genetic information relat-

ing to menaquinone biosynthesis has been ob-tained with mutant strains of E. coli. Five sepa-rate genes have been identified, and for three ofthese the association with a particular biosyn-thetic enzyme has been established. The othertwo genes are concerned in the first committedstep of menaquinone biosynthesis where theprearomatic compound chorismate is convertedto the fully aromatic OSB by a complex se-quence of reactions. The precise role of thesetwo genes is not yet clear. The enzymologicalconsequences of the mutations were discussedabove (Individual Reactions in MenaquinoneBiosynthesis); hence, no further descriptions ofenzymology will be given here. The presentunderstanding of the relationship of the mengenes to the biosynthetic pathway is summa-rized below for convenience.

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menD menC showed a nutritional requirement for OSB (orChorismate - "X"(?)---- )OSB , DHNA); as noted earlier, these genes may be

concerned with the synthesis and transformationof an intermediate between chorismate and

OSB synthase OSB.

menE menB menA)-OSB-Col..A. )-DHNA )DMK

OSB-CoAsynthetase

DHNA synthase

The first E. coli mutant, deficient in mena-quinone biosynthesis, was isolated during asearch for ubiquinone-deficient mutants; strainswere selected which were unable to grow onmalate as the source of carbon and energy aftermutagenesis by N-methyl-N'-nitro-N-nitroso-guanidine (54). Subsequently, this mutation wasshown to be cotransducible with metB and argEat frequencies of 30 and 50%, respectively;hence the gene, designated menA, was tentative-ly located at min 78 on the E. coli chromosome(169). It should be noted that E. coli mutantsdeficient in genes for both ubiquinone and mena-quinone (i.e., ubiA menA) have been construct-ed by transduction techniques (228). In growthstudies on glucose media the generation timeincreased in ubi strains and was very prolongedin the double quinone mutant. The followinggeneration times were determined: AN387 (ubi+men') and AN386 (ubi+ men), both 60 min;AN385 (ubi men'), 180 min; AN384 (ubi men),420 min. Clearly, menaquinone has a role inaerobic metabolism when ubiquinone is absent.In addition to these strains, another doublequinone mutant, AN187 (ubiD menA), was alsoconstructed by transduction techniques (169).

In 1975, Young isolated menaquinone-defi-cient E. coli mutants by treatment of strainAB3311 with N-methyl-N'-nitro-N-nitrosoguan-idine (201, 232). The mutants selected werethose unable to grow on succinate as a solecarbon source, but able to grow on glucose; theselection procedure was a fortuitous one. Theorganisms of one group were menA mutants andwere found to be unable to attach the isoprenylside chain; hence, such mutants accumulatedDHNA. A second group of mutants was orig-inally termed menB and was found to accumu-late OSB (201, 232). These mutants, blocked inthe conversion of OSB to DHNA, were latershown to consist of two groups, now termedmenE and menB (D. J. Shaw, personal commu-nication).Guest identified two further groups of mu-

tants, menC and menD, which were selected fortheir inability to use fumarate as a terminalelectron acceptor (84, 85). These mutants

DHNA octa-prenyl transferase

The menB, -C, and -D genes form a cluster at48.5 min on the E. coli linkage map; the menAgene (originally placed at 78 min) is actuallylocated at 88 min according to the recalibratedlinkage map (20). A transducing phage carryingsome of the men genes has been isolated from apool of lambda phages that were constructedfrom R - HindIII digests of E. coli DNA and thecorresponding insertion vector (86). This phage,XG68 [XmenCB(D)], was used in attempts tocomplement lesions in menB, -C, and -D; com-plementation did occur with menB and menCmutants but the menD mutants were transducedat low frequencies or not at all. Thus, thetransducing phage contains functional menC andmenB genes, but only part of the menD gene.Restriction analyses established the presence ofa bacterial DNA fragment (11.5 kilobases) linkedby an R - Hindlll target to the right arm of the Agenome but fused to the left arm of the vector(86). Studies with this phage have led to areinterpretation of the mapping data; the geneorder is now believed to be as follows: nalA-....menC....menB....menD ....purF. The exactlocation of the menE gene has not been deter-mined. The single menE mutant that is available,AN213, has been found to be leaky, allowingsufficient menaquinone to be formed for growthto occur anaerobically on lactate-fumarate me-dia (D. J. Shaw, personal communication).

men Mutants of Bacillus subtlisMutants of B. subtilis, an organism that does

not contain ubiquinone, were obtained by nitro-soguanidine treatment and were selected bysimultaneous resistance to two aminoglycosideantibiotics by Taber and his colleagues (209).The use of aminoglycoside antibiotic resistancefor the selection of men mutants derives fromthe role of quinones in transport of these antibi-otics into the bacterial cell (36, 210). The extentof aminoglycoside entry is related to the pres-ence and efficiency of electron transport and thegeneration of an electrochemical proton gradi-ent. Hence, those organisms resistant to amino-glycoside antibiotic action are likely to be men

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mutants. Taber has noted that the MK concen-tration must be very low before aminoglycosideresistance is expressed (208).The B. subtilis mutants, obtained as just de-

scribed, were divided into two groups: group I,whose nutritional requirement was satisfied ei-ther by OSB or DHNA; and group II, compris-ing those capable of growing only in the pres-ence of DHNA. The group I mutants could notbe separated either biochemically or geneticallyinto menC and menD. However, group II mu-tants could be divided into two groups on thebasis of syntrophy experiments, fine-structuremapping, and in vitro complementation by cell-free extracts (209). The term menE for mutantslacking OSB-CoA synthetase was first applied toB. subtilis strains, and menB was used for thosedeficient in DHNA synthase (165). The menC,menD, menE, and menB genes were localizedbetween bioB and ald on the B. subtilis genomeby genetic mapping with bacteriophage PBS1(209). By three-point transductional crosses withbioB and by cotransductional frequencies withksgB, the men genes were found to be groupedin the probable order, bioB....menE....menC,-D....menB....ksgB. It will be noted that thisordering is different from that proposed for theE. coli genes.

men Mutants of Staphylococcus aureusMenadione-requiring, small-colony mutants

of S. aureus (another organism without ubiqui-none) were obtained in 1968 by selection withneomycin (192). In the absence of menadione,growth was poor and there were reduced bio-chemical activity and respiration, as well asdeficient pigment formation. Menadione con-centrations of 0.1 to 1.0 Lg per ml of mediumgave rise to fully restored growth and respira-tion; higher concentrations were inhibitory.Some of the mutants were also stimulated byshikimate and formation of menaquinones wasdemonstrated. Hence, these strains (thentermed meq) were truly vitamin K-deficient mu-tants. It was later shown that some of themutants were deficient at the level of the com-mon aromatic pathway, others prior to naphtho-quinone ring formation, and others at the level ofsynthesis of the isoprenoid side chain (190, 191).Despite the potential usefulness of these mu-tants, little further work with them has beenreported. It has been shown that menaquinonedeficiency is accompanied in every case by animpaired nitrate respiration. Reinitiation ofMKbiosynthesis (e.g., by addition of shikimate)restores electron transport. These results clearlyimplicate menaquinone in nitrate respiration(189).A mutant of S. aureus, auxotrophic for mena-

dione and glycerol, was obtained from a glycerol

auxotroph by treatment with kanamycin andselection on nutrient agar with and withoutmenadione (80). This organism makes the nor-mal array and distribution of isoprenologs fromthe menadione provided. The enzymatic lesion,prior to menadione, has not been determinedand it is not clear whether menadione is a normalbiosynthetic intermediate in this organism (seealso Individual Reactions in Menaquinone Bio-synthesis).

Menadione-requiring strains of S. aureus,similar to those of Sasarman et al., have beenisolated clinically (1). In one study of eightstrains isolated from patients, four required thia-mine and four required menadione. One of themenadione-requiring strains was isolated from apatient receiving warfarin ("anti-vitamin K")after surgery. The menadione-requiring strainswere resistant to aminoglycoside antibiotics.

FACTORS INFLUENCING MENAQUINONEBIOSYNTHESIS

Aerobic Versus Anaerobic GrowthMany bacteria biosynthesize both ubiqui-

nones and menaquinones (48). In these orga-nisms, the interrelationships between theamounts of the various quinones are complexand are influenced by aerobic or anaerobicgrowth. For instance, among those bacteria ableto use fumarate as a terminal electron acceptor,the major quinone patterns appear to be MKalone, DMK alone, MK plus Q, or DMK plus Q(129). A complete account of these effects,which would of necessity have to consider thefunctions of the quinone components, is beyondthe scope of this review. Only those aspectsrelating directly to biosynthesis will be consid-ered here.

Despite some early confficting reports (32,137, 177), it is now generally recognized thatmenaquinone biosynthesis in facultative anaer-obes is increased by anaerobiosis. At the sametime, ubiquinone biosynthesis is diminished, andthere are also changes in the various cyto-chromes. In E. coli there is considerable varia-tion in the observed effects, depending to someextent on the cultural conditions and on theparticular strain which is examined. An extremecase was encountered with E. coli B/r, whereubiquinone biosynthesis was reduced to a verylow level indeed under anaerobic conditions(Table 5). A smaller change in ubiquinone con-tent was noted in E. coli AN98 (argE metE). Inthis strain the aerobic cultures contained 252nmol of Q-8 per g (wet weight), and the anaero-bic cultures contained 140 nmol of Q-8 per g (wetweight) (169): at the same time, the menaqui-none content increased from 104 to 246 nmol ofMK-8 per g (wet weight).

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TABLE 5. Levels of ubiquinone (Q) andmenaquinone (MK) in E. coli B/r under aerobic and

anaerobic conditions (177)

Conditions nmol/g, dry wt

Q MK

Stationary culture, 190 150aerobica

Vigorous aeration, 570 26log phaseb

Vigorous aeration, 330 24resting phasec

Anaerobicd 3 250

a Average from two experiments.b Average from four experiments.c Average from three experiments.d Single experiment.

Since some organisms contain all three qui-none types, Q, DMK, and MK, complete infor-mation requires analysis for all three. It shouldbe noted that there are significant differences inthe standard redox potentials of the twonaphthoquinones (101, 102). For MK, AE'o =

-74 mV; for DMK, AE'o = +36 mV. Thus, incontrast to Q (AE'o = +113 mV) and MK, DMKcan act equally well in succinate respiration andin fumarate reduction; MK can function in fuma-rate reduction but not succinate respiration(228).Work with Haemophilus influenzae RAMC 18

Bensted will illustrate these points (102). Thisorganism normally biosynthesizes DMK only.The quinone content of disrupted bacteria wasdepleted by pentane extraction, and reincorpo-ration of Q, MK, or DMK was studied. Forelectron transport from NADH to fumarate, MKand DMK were very effective and more activethan Q by a factor of 4 to 5. For electrontransport from succinate to 02, Q and DMKwere effective, and MK had only a slight effect.It appears that electron transport depends notonly on the redox potential but also on thechemical structure of the quinone.

In an early approach to the study of all qui-none components, three representative orga-nisms were examined. Anaerobiosis was foundto influence the content ofMK and DMK differ-ently. As anticipated, anaerobic growth dimin-ished ubiquinone formation from 1.5- to 3-fold inEscherichia freundii, Proteus mirabilis, andAeromonas punctata (230). The combinedamounts of MK plus DMK increased anaerobi-cally by 1.45-fold in E. freundii and 1.58-fold inProteus mirabilis; there was no change, howev-er, with Aeromonas punctata. For the individualnaphthoquinone components, the amounts ofMK increased under anaerobic conditions,whereas the amounts ofDMK decreased and, in

the case of Proteus mirabilis, DMK was notfound at all. The changes in the ratio DMK/MKfor aerobic versus anaerobic growth were, re-spectively, 2.0 to 0.4 (E. freundii), 0.8 to 0.0(Proteus mirabilis), and 13 to 3.3 (Aeromonaspunctata).A series of E. coli mutants carrying two

possible combinations of genes for ubiquinoneand menaquinone biosynthesis (ubi+, ubi, men',men) has also been examined with respect tochanges in the levels of Q, MK, and DMK forthe change from aerobic to anaerobic conditions(228). The change in Q concentration for anaero-biosis was to about 20% or less of the aerobicvalues; the combined MK plus DMK increased2.7-fold (ubi+ men') or 1.85-fold (ubi men')under anaerobic growth conditions. In terms ofthe individual naphthoquinone components,DMK concentrations increased somewhat, andthe MK concentrations increased considerably(Table 6). In these mutants, the changes inDMK/MK ratio were from 7.6 to 1.0 (aerobicanaerobic, ubi+) and from 3.5 to 1.7 (aerobicanaerobic, ubi). These changes are differentfrom that observed in E. freundii.The following organisms also reduce their Q

content in favor of increased DMK contentwhen cultivated anaerobically in the presence offumarate: Haemophilus parainfluenzae HIM412-6 and NCTC 4101, H. haemoglobinophilus,H. parasuis, and H. paragallinarum (103).These are examples of organisms which do notcontain MK components.Some strains of E. coli K-12 actually synthe-

size relatively high levels of Q under anaerobicconditions, in the presence of fumarate. Theselevels are in the range of 50 to 70% of thoseobtained under aerobic conditions (2). The lev-els are higher than those for cells grown anaero-bically with nitrate as electron acceptor (228). Itis worth mentioning that under anaerobic condi-tions the hydroxylations required in Q biosyn-thesis are carried out by three "alternative"hydroxylation reactions, not involving molecu-lar oxygen (rather than by the aerobic monooxy-genases). The aerobic monooxygenases areprobably flavin enzymes rather than cytochromeP-450 type enzymes (127). The oxygen-depen-dent synthesis of Q-8 in E. coli appears to have astandby position in the anaerobic cell and can beactivated quickly if oxygen becomes available(126). It is of interest to recall that in menaqui-none biosynthesis the quinone oxygen atoms arenot derived from molecular oxygen (203); this is,of course, consistent with the known biosynthet-ic pathway for MK. These oxygen atoms derivefrom the carboxyl group originally associatedwith shikimate and the carbonyl group originallypresent in 2-ketoglutarate. Despite the generalstructural similarities, the quinone functions in

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the naphthoquinones are biosynthetically verydifferent from those in the benzoquinones.The increased biosynthesis of MK in E. coli

under anaerobic conditions is apparently relatedto its role as an obligatory hydrogen carrier forthe oxidation of dihydroorotate coupled to fuma-rate reduction (169). It has, apparently, not beendetermined whether DMK can function in thissystem. In any event, this system is probablyinhibited by molecular oxygen. It has also beensuggested that the low levels ofQ with anaerobicgrowth are an expression of a regulatory mecha-nism (169) rather than an interference with abiosynthetic reaction (229).

In line with the general effects of anaerobiosiswhich have just been summarized is an effect ofKCN. Aerobic growth of E. coli with an oxidiz-able substrate (e.g., succinate) in the presence ofKCN leads to an approximately ninefold in-crease in menaquinone content; the ubiquinonecontent is essentially unchanged (16). At thesame time, the cytochromes synthesized aretypically the same as those in anaerobicallygrown cells.An increased formation of menaquinones un-

der aerobic conditions occurred when Staphylo-coccus aureus cultures were shifted from anaer-obic to aerobic growth (74). This organism is, ofcourse, one which does not contain ubiquinoneand so menaquinone is used for electron trans-port to oxygen. The increase was about 1.6-fold.The various isoprenylogs were affected differ-ently; during the shift, the amount of MK-9increased and that of MK-7 decreased. Ex-pressed as percentage of the total MK content,the changes for anaerobic to aerobic were asfollows: MK-7, 26.0 to 18.9; MK-8, 66.0 to 62.5;MK-9, 8.0 to 18.6. The reason for these changesis not clear.

Other Factors IDfluencing MenaquinoneBiosynthesis

Little attention has been paid to the influenceof general cultural conditions on the biosynthe-sis of menaquinones. Conclusions from one

study (28) with a strain of Serratia marcescensare as follows. (i) The greatest amount of Q-8and MK-8 was present during stationary-phasedevelopment (2 to 3 days) on an optimal medi-um. (ii) The amount of MK-8 was little influ-enced by changes in glucose concentration from10 to 80 g/liter. (iii) The best growth and yield ofMK-8 were obtained in the presence of 20 to 50 gof glutamate per liter. (iv) There was littlechange in the ratio of MK-8/Q-8 as a function ofmedium composition and growth phase.During exponential growth of Staphylococcus

aureus, the proportions of the various mena-quinone isoprenologs changed, although the to-tal amount remained essentially constant (2.0 +

0.1 ,umol/g, dry weight). MK-8 increased from36 to 70% of the total during seven to eightdoublings, and in the same period the propor-tions of MK-0, MK-1, MK-5, MK-6, and MK-7decreased (93).When Staphylococcus aureus is grown at

25°C, the cells contain more total menaquinonethan at 20°C (28% more) or 37°C (16% more).There are also differences in the amounts of thevarious menaquinone isoprenologs as growthtemperature changes (119). During a shift downfrom 37 to 25°C, total menaquinones increasedby about 20% and the proportions of very short-chain components decreased, whereas MK-8increased. No explanation was offered for thesechanges.

Since diphenylamine was known to suppressthe formation of carotenoids and ubiquinone invarious bacteria, Salton and Schmitt examinedthe effect of this material on the menaquinonecontent of membranes isolated from "Micrococ-cus lysodeikticus," Sarcina lutea, and Bacillusmegaterium (186). At diphenylamine concentra-tions of 50 gu.g/ml of medium, the menaquinonecontent was reduced to 77% of the normal valuewith "Micrococcus lysodeikticus" and to 84%with Sarcina lutea. These levels of diphenyl-amine reduced the carotenoid content to 10o orless of the normal. With the nonpigmented Ba-cillus megaterium, diphenylamine concentra-

TABLE 6. Levels of demethylmenaquinone (DMK) and menaquinone (MK) in E. freundii and E. coli strainsunder aerobic and anaerobic conditions

nmol/g, wet wtaStrain Aerobic Anaerobic

DMK MK DMKIMK DMK MK DMK/MKE. freundii 160 80 2.0 90 260 0.4E. coli AN387 (ubi+ men+)b 38 5 7.6 58 59 1.0E. coli AN385 (ubi men') 120 34 3.5 179 106 1.7E. coli AN386 (ubi+ menA) <2 <2 <2 <2E. coli AN384 (ubi menA) <2 <2 <2 <2

a The amounts are expressed as nanomoles per gram, dry weight, for E. freundii (230).b Data for all E. coli mutants are from reference 228. These results are for anaerobic growth in the presence of

nitrate.

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tions of 12.5 and 25 pg/ml of medium reducedthe content of MK-7 to 9%o of the normal, a

much smaller effect. Clearly, the biosynthesis ofmenaquinones was less sensitive to the effect ofdiphenylamine than was that of the carotenoids.In similar work with Staphylococcus aureus

under both aerobic and anaerobic conditions,diphenylamine inhibited menaquinone biosyn-thesis by about 50% (95). The diphenylamineconcentrations in this work did not influence thegrowth rate; cyclic carotenoid biosynthesis was

inhibited by either 25 to 35% (anaerobically) or

60 to 90% (aerobically). The mechanism bywhich diphenylamine exerts these effects is ap-

parently not known.Some observations have been made on the

effect of 5-aminolevulinate, a precursor to heme,on quinone levels in E. coli strains (90). A 5-aminolevulinate-deficient mutant of E. coli wasobtained and was grown both aerobically andanaerobically in the presence or absence of 5-aminolevulinate. Under aerobic conditions theformation of menaquinone was decreased by thepresence of 5-aminolevulinate, but there was nosuch action under anaerobic conditions (Table7). The precise significance of this effect is notclear.The lack of action of 5-aminolevulinate under

anaerobic conditions has been confirmed in theAN359 strain ofE. coli, which carries a mutationin the hemA gene (13). Under anaerobic condi-tions, in the absence of 5-aminolevulinate,growth was about one-third of the rate withexcess 5-aminolevulinate present. Cytochromescould not be detected in the membranes of thosecells grown without 5-aminolevulinate, but thelevels of naphthoquinones were normal. It maybe noted that other workers have implicated amenaquinone requirement in the anaerobic bio-synthesis of heme, specifically for the oxidationof protoporphyrinogen (116).

VITAMIN K-REQUIRING BACTERIAIn 1945, a comprehensive review of the

growth requirements of bacteria for vitaminscontained the statement ". . . most of the fat-soluble vitamins are not known to have anypotency for bacteria" (176). In a listing of 130bacteria, the only reference to vitamin K wasveiled: Mycobacterium paratuberculosis waslisted as requiring "anti-hemorrhagic com-pounds." Clearly, the ability to synthesize de-methylmenaquinones and menaquinones waswidespread among bacteria. Since that time,however, several instances of other vitamin K-requiring bacteria have been discovered. Thebest documented cases are discussed here. Oth-er examples of a requirement for menaquinone,or for a biosynthetic precursor thereof, are pro-vided by the men mutants of E. coli, Bacillus

TABLE 7. Influence of 5-aminolevulinate on E. colimutants deficient in heme biosynthesis

5-Amino- nmol/g, wet wtStrain levulinate Q MK DMK

1, aerobic - 86 30 _b1, aerobic + 84 31, anaerobic - 36 341, anaerobic + 38 27AN359, - 73 33 50

anaerobicAN359, + 78 31 52

anaerobic

a Strain 1 is a 5-aminolevulinate synthetase-defi-cient mutant (90); AN359 has a mutation in the hemAgene.

b , Not determined.

subtilis, and Staphylococcus aureus (see Genet-ics of Menaquinone Biosynthesis). On the prac-tical level, it is worth noting that vitamin K, asmenadione, is added to the brain heart infusionbroth (supplemented) medium used for culturingmany anaerobes. This addition is recommended,for instance, for isolation of anaerobes fromvarious clinical specimens (99).

Mycobacterium paratuberculosisIn their early work on the cultivation of M.

paratuberculosis (see Introduction), Twort andIngram recognized clearly that not all bacteriaand not all strains of the same bacterium weresources of Essential Substance (224, 225). Pro-duction of the growth factor by M. phlei wasfound to be dependent on culture media compo-nents; thus, glycerol-containing media weregood substrates for Essential Substance produc-tion. Since bioassay required about 2 months forthe growth ofM. paratuberculosis, progress wasnecessarily slow.When Woolley and McCarter reported in 1940

that M. paratuberculosis could be grown onmedia containing phthiocol, 2-methylnaphtho-quinone, or a vitamin K concentrate, it appearedthat this organism did have a vitamin K require-ment. However, they also realized that none ofthese materials was as effective as an extract ofM. phlei (231). In an examination of the samequestion, Glavind and Dam showed that a con-centrated bovine tuberculin preparation, whichwas known to have a definite vitamin K activity,stimulated growth of a M. paratuberculosisstrain about threefold (78). These results (Table8) were obtained before 1941, using a strainisolated in Denmark. A strain obtained by Damin 1946 from W. A. Hagan, Ithaca, N.Y. (on thesuggestion of D. W. Woolley and J. R.McCarter) showed less stimulation. This strainwas clearly different from that used earlier, sinceit gave about 10 times as much growth under

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similar conditions. In the early experiments, 2-methylnaphthoquinone, or the correspondinghydroquinone disulfate, gave at most only veryslight growth stimulation and amounts of 250 ,ugper ml of medium were actually inhibitory. Withphthiocol, a somewhat more consistent stimula-tion (average yield, 97 versus 81 mg for controls)was seen. In all of these experiments, there wasa considerable variation (Table 8). These au-thors concluded that the tuberculin preparationcontained a growth-stimulating factor differentfrom vitamin K. They also stated that "thestrains used in the two laboratories (i.e., Wool-ley and McCarter vs. Glavind and Dam) mighthave been different with respect to their vitaminK requirements."A new growth factor for M. paratuberculosis

was investigated by Francis et al. beginning in1949 (72, 73, 202). These authors used strain 129of "Myco. johnei" (sic) obtained from R. E.Glover. This strain could not be easily adaptedto growth in the absence of growth factor. FromM. phlei they isolated a crystalline aluminumcomplex of a material termed mycobactin (now,mycobactin P). Optimal growth ofM. paratuber-culosis required 40 to 80 gLg of mycobactin perml of medium. This was the first of severalmycobactins, now recognized as iron-chelatinggrowth factors for mycobacteria (202). Franciset al. observed no effect of phthiocol, 2-methyl-naphthoquinone, and vitamin K in their assaywith M. paratuberculosis. They concluded thatmost, if not all, of the activity of simple extractsof M. phlei in promoting growth of M. para-tuberculosis could be accounted for by myco-bactin P (it is present to the extent of about 1%of dry weight of M. phlei).The work on the chemistry and microbiology

of the mycobactins is certainly elegant and volu-minous. The various members of the group aregenerally recognized as growth factors for my-cobacteria, including M. paratuberculosis. Anargument favoring the identity ofTwort's Essen-tial Substance with mycobactin concerns thereported solubility characteristics. The beststimulation of the growth ofM. paratuberculosiswas reported by Twort and Ingram (224, 225) tobe obtained with material insoluble in chloro-form; there was evidence, however, that it wasto some extent soluble in lipid solvents. Thesecharacteristics do not agree well with the excel-lent lipid solubility of vitamin K and the generaldifficulty of obtaining it in a water-soluble form;for instance, a form of vitamin K, used clinicallyis actually an aqueous colloidal preparation(AquaMEPHYTON).As a result of the discovery of the mycobac-

tins, any possible role of vitamin K has, since1949, been minimized. For example, an exten-sive discussion ofM. paratuberculosis and other

TABLE 8. Stimulation of growth of Mycobacteriumparatuberculosis by tuberculin

mg of sediment/200 ml of growth medium

cubleirnEp Danish strain, U.S. strain,addition no. 14 14

(ml) Individual Avg Individual Avgflasks flasks v

0 1 59, 65, 75, 112 78 843, 856 8500 2 68,73, 90, 91 810.02 1 194, 235, 251 226 1,586, 1,627 1,6070.02 2 199, 206 203

host-dependent microbes does not contain anyreference to vitamin K (%). Nevertheless, forthe specific case of M. paratuberculosis, it isperhaps not heretical to suggest that the pictureis not totally clear. For example, in 1970, aninvestigator well aware of the role of mycobactinshowed that "water soluble vitamin K" (thesodium dibenzoylsulfonate of 2-methylnaphtho-hydroquinone) could be substituted for M. phleiextract in the isolation and repeated subcultureof M. paratuberculosis (47). Coletsos noted thatthe culturing of M. paratuberculosis, despitemycobactin, is burdened with a considerablenumber of failures, so that stocks of this orga-nism are extremely rare. In his work, threestrains (one isolated from a cow; two isolatedfrom sheep) were maintained for 15 years onmedia containing the menadione derivative.The problem of the actual growth require-

ment(s) is made more complex by the "circum-vention of the mycobactin requirement of Myco-bacterium paratuberculosis" (167). Forinstance, during the first transfer of strain 68from the mycobactin-containing Trypticase(BBL Microbiology Systems)-glycerol mediumonto Watson-Reid medium (pH 5.5) a mycobac-tin requirement was observed. Subsequently, 18transfers were made without mycobactin. Fur-thermore, autoclaved Watson-Reid mediumcontained an unidentified growth factor, notpresent in filtered medium. (This work did notconsider a possible vitamin K requirement.)

Although an evaluation of all of the nutritionaland other factors for growth of M. paratubercu-losis is beyond the scope of this review, itappears to us that the existence of vitamin K-requiring strains is still possible. It would not bebeyond the bounds of probability to suggest thatthere may even be strains requiring both vitaminK and mycobactin for optimal growth. (Com-pare the double requirement for vitamin K andheme of Bacteroides melaninogenicus, dis-cussed below.) Ironically, the crude extracts ofM. phlei, used for a long time for the growth ofM. paratuberculosis, probably contained signifi-cant amounts of both materials. In other bacte-ria, both vitamin K-requiring and vitamin K-

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independent strains are well known (see below).There is clearly much variation in M. paratuber-culosis, and it is known that phage-inducedchanges in mycobacteria may be severe. Forinstance, lysogenization of M. phlei F89 withmycobacteriophage B2hF89 gave a strain withacquired properties characteristic for M. smeg-matis (75).

It is known that within the M. avium group ofmycobacteria there are various degrees of myco-bactin dependence, as is the case with M. para-tuberculosis (151). In particular, with M. avium,mycobactin dependence occurs when media areinoculated with small numbers of viable units;large inocula "presumably contain sufficientmycobactin associated with the organisms toenable growth to occur."

Possibly the discovery of mycobactin has ledto the selection of mycobactin-requiring strains.It would be of considerable interest to isolate M.paratuberculosis from an animal with Johne'sdisease and to examine in detail the effects ofvitamin K and mycobactin, separately and incombination. With the exception of the work ofColetsos in 1970, any possible effect of vitaminK on freshly isolated strains of M. paratubercu-losis has apparently not been investigated. Inany event, such fresh isolations are now uncom-mon. Between the years 1973 to 1979 the CentralLaboratory of Veterinary Research (Alfort,France) isolated some 332 cultures of mycobac-teria from 590 pathological specimens of differ-ent origin. Of these, only two strains wereidentified as M. paratuberculosis (216).

Since vitamin K is present to a large extent inthe diet of bovines and is also biosynthesized byother intestinal bacteria, it may well have beenused as a growth factor by M. paratuberculosis.It may be more than coincidence that it is theintestinal mucous membrane which is uniquelythe site of the lesions produced by this organism(47). It is also a remarkable coincidence thatboth vitamin K and some of the mycobactins arederived from the shikimate pathway (110, 149).Those mycobactins containing 6-methylsalicylicacid rather than salicylic acid, however, derivethe aromatic acid by way of polyketide path-ways (109).

Thus, at least this one strain of M. paratubercu-losis is able to biosynthesize menaquinones.However, mycobactin biosynthesis by anystrain of this organism has, apparently, not beendemonstrated.

Bacteroides melaninogenicusA vitamin K requirement for some strains of

B. melaninogenicus has been discovered. Thechain of circumstances leading to this is striking-ly similar to that which provided evidence for agrowth factor requirement for Mycobacteriumparatuberculosis. In 1928 Burdon (38) investigat-ed "the nonspore-bearing, black pigment pro-ducing, anaerobic microorganism, "Bacteriummelaninogenicum" (sic), which had been de-scribed and named in a brief note a few yearsearlier (171). Burdon believed it was probablethat the earlier workers had not obtained purecultures. Growth of such pure cultures wasfound to be "uncertain, slow, and usually mea-ger." However, the organism exhibited "to amarked degree the habit of growing in veryintimate mixture with other bacteria ...." Inmixed cultures, under anaerobic conditions,growth on blood agar was found to be rapid andluxuriant. A "melanin-like" pigment wasformed, and the red color of the hemoglobincontaining agar eventually was completely lost.The bacterium was widely distributed on normalmucous membranes and skin in humans, partic-ularly in the mouth and on the external genitalia.Although Burdon had noted the fact of good

growth in mixed cultures, he was apparently notaware of the work of Twort and Ingram, nor didhe postulate the formation of any special sub-stance(s) by the contaminants. He spoke of asymbiosis between it and other organisms suchas Streptococcus viridans (39).

It required another 15 years before the pig-ment was recognized as a heme compound, notmelanin (198). The name, Fusiformis nigrescens,was then proposed for the organism, and subse-quent revisions now have led to the presentlyused B. melaninogenicus.

In 1954, a gram-negative, nonmotile anaerobewas isolated (along with other organisms) from

Shikimate -chorismate v OSB -. MKSa iso-chorismate -- salicylate -- mycobactin

One strain of M. paratuberculosis has beenshown to contain menaquinone; MK-9 (I-H2)was the major component with smaller amountsofMK-9 and MK-8 (49). The distribution patternwas similar to that in other mycobacteria. (Itwas not stated whether the particular strainexamined was mycobactin dependent, and ap-parently mycobactin was not used for growth.)

the mouth of a patient with a clinical diagnosis ofhypertrophic gingivitis (146). The organism,K110, produced a black pigment and was as-signed to the "ill-defined species Bacteroidesnigrescens." This organism normally grew inassociation with a streptococcus, JS9, fromwhich it was separated with difficulty. However,"growth occurred in Difco thioglycollate broth

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only when 20%o of a sterile Seitz filtrate of 5-daybroth culture of JS9 was added." It was laterfound that filtrate of a hemolytic Micrococcusaureus was more effective than filtrate of JS9. Alittle later, Macdonald et al. (147) noted that"cross-streaking strain K110 against all of thestrains disclosed that in the presence of strainsJR3, JR4, JR5 or JS9, strain K110 grew well. Itwas found to grow poorly in the presence ofstrain JB3B and not at all with any of the otherstrains." (Strains JR4, JR5, and JS9 were appar-ently streptococci, JR3 was an aerobic gram-positive, nonmotile rod, and JB3B was also agram-positive nonmotile rod.) It was clear fromthis work that K110 required a growth factor,which could be obtained in a water-soluble formfrom various bacteria.

In 1958, Lev also isolated an organism, appar-ently "Fusiformis nigrescens," from the rumencontents of cows (138). It was first isolatedassociated with an anaerogenic strain of Pro-teus. On attempting to grow it in pure culture,the organism died out after giving rise to atypicalforms. Mindful of the work with Mycobacteriumparatuberculosis and Mycobacterium phlei andthe work of Woolley and McCarter with vitaminK, Lev added a suspension of menadione inwater to a blood agar plate. Good growth of ""F.nigrescens" was obtained and serial subculturewas possible. Menadione was also able to reviveatypically growing cultures.

Similarly, Gibbons and Macdonald found thathuman strains of B. melaninogenicus, whichgrew as satellites adjacent to Staphylococcusaureus, presumably by deriving a growth factor,could also be grown and maintained by additionof menadione and, indeed, a number of othernaphthoquinones and naphthalene derivatives(77). Benzenoid and anthraquinonoid materialswere ineffective. In this work, 12 of 14 strains ofB. melaninogenicus isolated from the humanmouth were found to require hemin for growth.Half of the isolates, in addition, required thegrowth factor from Staphylococcus aureus or avitamin K replacement. Clearly, there are threegroups of B. melaninogenicus strains. (i) thosegrowing on Trypticase soy broth plus 0.05%sodium thioglycolate (basal); (ii) those requiringbasal medium plus heme; (iii) those requiringbasal medium plus heme plus vitamin K.A further nutritional complication is that for a

rumen strain of B. melaninogenicus succinatecan replace the heme requirement in the pres-ence of vitamin K, and, in addition, succinatecan to some extent replace the vitamin K re-quirement in the presence of heme (141). Also,succinate increased the growth rate when addedto a blood- and vitamin K-supplemented culture.The precise role of succinate is not entirelyclear. Labeled succinate is, however, incorpo-

rated into ceramide phosphorylethanolamine,ceramide phosphoglycerol, and other phospho-lipids. In vitamin K-depleted cultures, additionof vitamin K increases this incorporation. It is ofinterest that in another Bacteroides, B. rumini-cola, an unusual reductive carboxylation of suc-cinate to 2-ketoglutarate has been reported (3).In growing cultures of this organism, addition of[1,4-14Cj2succinate leads to 4C in amino acidsas well (particularly glutamate, arginine, proline,aspartate, threonine, and alanine). A possibleconnection between succinate, 2-ketoglutarate,and menaquinone biosynthesis cannot be ruledout.

In a study of a large number of oral isolates(human, canine) of B. melaninogenicus and B.asaccharolyticus, the strains were found to fallinto two classes: asaccharolytic organisms pro-duced butyric acid whereas saccharolytic (B.melaninogenicus) organisms actually producedsuccinic acid (154). Of 177 strains of B. melan-inogenicus isolated from humans, about 11%required vitamin K; on the other hand, of 160strains isolated from dogs, 68% required vitaminK. There was no correlation between vitamin Kdependency and the source of the human iso-lates. B. asaccharolyticus, which is found in theoral cavity, also shows a vitamin K requirementwith some strains. Of 23 human isolates, about21% were vitamin K dependent. Of 30 canineisolates, 90%o were vitamin K dependent (154).

Before 1973, a number of synthetic com-pounds had been examined as replacements forthe growth factor or vitamin K requirement ofB.melaninogenicus (77, 139, 145, 206). Althoughseveral naphthalene and naphthoquinone com-pounds had this ability, benzene derivativeswhich were examined did not (benzoquinone,hydroquinone, pyrogallic acid, phthalic acid,and y-phenylbutyric acid) (145). However, in1973 Robins et al. were able to show that threemonocyclic compounds replaced the vitamin Krequirement of Lev's strain of B. melaninogeni-cus (182). These three compounds, shikimate,chorismate, and OSB, were all materials identi-fied as menaquinone precursors; another precur-sor, DHNA, behaved similarly. Benzoic acid,phthalic acid, and ubiquinone could not replacevitamin K. (Phthalic acid had been examinedearlier by Macdonald. We regret that we omittedto reference that work [145] when our paperappeared.)

In general, the genus Bacteroides appears tobe well endowed with a biosynthetic capacity formenaquinones: 36 of 37 strains have been shownto contain no ubiquinones but a variety of mena-quinones from MK-5 to MK-14 (199). "Bacte-roides melaninogenicus subsp. levii" (140) wasstated to contain neither menaquinones nor ubi-quinones; contrary results had, however, been

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reported earlier (179). Although identified assubspecies levii, the strain used by Shah andCollins (140) was not obtained from Lev and wasisolated from a cattle horn abscess rather thanfrom bovine rumen contents (as was Lev'sstrain). It was not stated whether this strainshowed a vitamin K requirement, although men-adione was apparently added to the growthmedium. Robins et al. (182) also found MK-9and MK-10 in Lev's strain in a ratio of 2:1. Itappears that in this strain the defect arisesbefore shikimate; aromatic amino acids are pre-sumably available from the blood generally pres-ent in the medium.

Lactobacillus bifidus var. pennsylvanicusA batch of commercial lactulose (Bifiterlose;

4 - 0 - a - D - galactopyranosyl -D - fructofuranose)was found to enhance the growth of L. bifidusvar. pennsylvanicus in the presence of humanmilk (or materials such as N-acetyl-D-glucosam-ine and NH3). By extraction of 1.8 kg of lactu-lose with methanol, followed by further extrac-tion from water with ether, a brown syrup wasobtained (2.97 g). By chromatography on a cellu-lose column and vacuum sublimation, a paleyellow sublimate (25.4 mg) was obtained. Afterfurther crystallization, this material was identi-fied as menadione. Synthetic menadione wasalso shown to function in the same way; it wasdescribed as a "supplementary factor," re-quired in addition to that for N-acetyl-D-gluco-samine-containing saccharides (79). Why theparticular batch of carbohydrate contained men-adione is not known; lactulose is obtained byepimerizing lactose, and contamination of thelatter also seems unlikely. A few other naphtho-quinones, including phylloquinone, were alsoshown to function in the same way as mena-dione; benzoquinone and hydroquinone wereinactive.

Other MicroorganismsAn oxygen-dependent and menadione-requir-

ing variant of Haemophilus parainfluenzae wasobtained from cultures grown on heated bloodagar (143). Of two colony types present, onlyone showed the requirement. The response oc-curred only over a limited range (0.5 to 2.5,ug/ml) and was limited to menadione, mena-dione bisulfite, 2-methyl-4-amino-1-naphthol,and 2,3-dimethyl-1,4-naphthoquinone. The re-sponse occurred on blood-Lemco broth but noton blood or heated blood agar plates. Strains ofHaemophilus parainfluenzae generally containeither ubiquinone and demethylmenaquinone oronly demethylmenaquinone (102, 103).An investigation of the nutritional require-

ments of a number of anaerobic coryneforms ledto the identification of four strains of Propioni-bacterium acnes type II with nutritional require-

ments for heme and vitamin K (cf. Bacteroidesmelaninogenicus). Ten other water-soluble vita-mins were also present in the defined medium. Itwas suggested that the heme and vitamin K wererequired for synthesis of cytochromes (65).A brief note from the 15th Joint Leprosy

Research Conference indicated that vitamin K3(0.005 ,ug/ml) and vitamin B12 (0.16 ,ug/ml) sig-nificantly enhanced growth of Mycobacteriumlepraemurium and suggests that these materialsmay have given possible multiplication of M.leprae (168).

In 1942, an apparent influence of vitamin K onthe growth of intestinal bacteria, particularly E.coli, was reported (1%). It was found that inyoung chicks E. coli grew rather slowly. Ifchicks were maintained for 26 days on a dietproducing vitamin K deficiency, the intestinal E.coli largely disappeared. Vitamin K administra-tion restored the coli count by up to 70% after 8to 18 days. There were morphological differ-ences when the isolated intestinal bacteria weregrown on media with and without vitamin K. Itwould be of interest to see this work confirmed;it has received little attention since most work-ers do not regard E. coli as requiring vitamin Kfor growth.

In Aspergillus niger, menadione was said toexert a slight growth stimulation at low concen-tration but rapidly inhibited growth as the con-centration was increased (226). The result issurprising since fungi do not contain menaquin-ones.

Are the Vitamin K-Like Growth FactorsSecreted by Bacteria Actually Menaquinones?It is common to ascribe response of a "vita-

min K-dependent" organism, such as Bacte-roides melaninogenicus, to another organism byassuming the excretion of naphthoquinones. Toquote one example, it has been stated that"naphthoquinones were detected in Bacteroidesfragilis using a naphthoquinone-dependentstrain of Bacteroides melaninogenicum" (36).Indeed, the stimulation of growth of vitamin K-dependent bacteria by the presence of otherbacteria has been used or suggested for use as anassay for vitamin K. It appears that rarely, ifever, in such work has a distinction been madeamong several possibilities. The test organismcould: (i) secrete into the agar a menaquinone(or menaquinol) in the typically lipophilic form;(ii) secrete into the agar a menaquinone (ormenaquinol) in a modified, watersoluble form;(iii) secrete into the agar a biosynthetic precur-sor of menaquinone, likely as a water-solublematerial.

(i) Since menaquinone is typically localized inthe cytoplasmic membrane (130), it seems un-likely that it would be easily excreted by bacteri-

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al colonies into agar in its lipophilic form. Al-though bacteriolytic enzymes are present, forexample, in Staphylococcus aureus (15), thepossible role of such enzymes in the release ofmenaquinone is not known.

(ii) Lev and Milford have provided evidencefor the existence of water-soluble factors withvitamin K activity in pig liver and in Bacteroidesmelaninogenicus (142). The pig liver extractsappeared to contain a protein with vitamin Kbound to it in some manner; microbiologicalactivity was associated entirely with this form(and no ether-extractable vitamin K was pres-ent). Similar, water-soluble forms, presumablyprotein bound, were obtained from Bacteroidesmelaninogenicus. When this organism wasgrown in the presence of vitamin Kl, a lipidsoluble form was also present. Such protein-bound forms of vitamin K might be excreted bybacterial colonies into agar.

(iii) Whereas it could be argued that the mostlikely material to be excreted into agar would bea water-soluble menaquinone precursor (e.g.,OSB, DHNA) this possibility has generally beenoverlooked in work dealing with vitamin Kassay. However, the "cross feeding" of specificmen mutants by other mutants or by wild-typeE. coli and Bacillus subtilis is well known (85,209).

It is not impossible that Twort and Ingram'sEssential Substance could have contained a vita-min K precursor, and some of the growth factorsfor Bacteroides melaninogenicus have proper-ties more closely resembling an aromatic acidthan a menaquinone. For example, the proper-ties of the growth factor produced by Staphylo-coccus aureus were summarized as follows(145): (i) withstood 15 min at 100°C, but 15 minat 121°C gave 95% loss of activity; (ii) notinactivated by vacuum drying, ethylene oxide,or UV light; (iii) ether extractable at pH 1.5 withretention of activity; (iv) absorbed by charcoaland both anion- and cation-exchange resins; (v)gummy residue obtained on evaporation wassoluble in NaOH or ethanol, and redissolvedresidue was active; (vi) extracts (ether?) of alka-line preparation at pH 11.5 were inactive. Theseproperties, in particular, the stability to UVradiation, do not agree with those of a mena-quinone. In this case, therefore, a good argu-ment can be made that the growth factor isactually a water-soluble precursor of menaqui-none.

Are Growth Factors with Vitamin K ActivityConverted to Menaquinones?

In many of the studies of the vitamin Krequirements of bacteria, the growth supplementused has been the readily available menadioneor phylloquinone rather than the menaquinone

which would be expected as the normal bacterialcomponent. There has been little study of themechanisms by which materials with vitamin Kactivity are actually taken up by growing bacte-rial cells and are then subjected or not to furthertransformations. Unfortunately, the results thathave been obtained are somewhat contradic-tory. In the early work of Martius with Bacte-roides melaninogenicus (obtained from Lev)(see Biosynthesis of Menaquinones) conversionof labeled menadione and phylloquinone to men-aquinone was said to have been achieved (150).Whether or not the purifications obtained in thiswork were adequate is now difficult to decide.When the same organism was grown by otherswith phylloquinone as the supplement, a sub-stantial amount of phylloquinone itself was actu-ally recovered from the cells. Only a trace ofother quinones was present; this material wasshown by mass spectrometric analysis to be amixture of MK-9 and MK-10 (D. J. Robins andR. Bentley, unpublished data). Other growthfactors for this organism (shikimic acid, OSB,DHNA, 1,4-naphthoquinone) gave mixtures ofMK-9 and MK-10, and an abnormal mass spec-trometric pattern was obtained in one experi-ment with 6-methyl-1,4-naphthoquinone as thegrowth factor, as well as a much lower yield of"quinone." As noted earlier, other workershave stated that "neither menaquinones or ubi-quinones were detected in 'Bacteroides melan-inogenicus subsp. levii (JP2)' " (140).

In studies of a men mutant of Staphylococcusaureus, menadione was used as the growth fac-tor (191). Isolation of quinonoid material fromthe cells "revealed a spectrum (UV) differingfrom that of vitamin K2 (30)," i.e., MK-6. It wasconcluded, therefore, that such cells did notcontain a normal menaquinone component. TheUV spectrum was derived from material recov-ered from thin-layer chromatography, and it ispossible that the abnormal spectrum resultedfrom the presence of contaminants; a rigorouslypurified compound was probably not examined.In any event, there has been no further charac-terization of this material. In work with a Staph-ylococcus aureus strain which produced mena-quinones, Hammond and White observed thatradioactive menaquinones, from MK-2 to MK-9,were not taken up by exponentially growingcultures (93). The samples were added to culturemedia in dimethyl sulfoxide solution, and essen-tially 100%o of added activity was recovered inthe culture media. With MK-0 (menadione),about 0.5% of the added material was incorpo-rated by the bacterial cells.

VITAMIN K BIOSYNTHESIS BYINTESTINAL BACTERIA

Beginning with the pioneering work of Alm-quist and Stokstad (9), the biosynthesis of vita-

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min K by intestinal bacteria has come to berecognized as an important component in animaland human nutrition. With the exception of thechick, it is, in fact, not easy to induce vitamin Kdeficiency in experimental animals. With thedevelopment of powerful antibiotics, vitamin K-responsive hypoprothrombinemia became a sig-nificant clinical problem (210), and the antibiot-ic-associated defect in vitamin K biosynthesiscan actually prove to be lethal (105). To cite onlyone recent example, gastrointestinal bleedingwas encountered in patients being treated withcefamandole (105).Some work on the role of intestinal bacteria

has been carried out with gnotobiotic rats (46).When these animals are fed a vitamin K-freediet, they rapidly develop a hemorrhagic condi-tion which can be reversed by associating themwith bacteria isolated from conventional animals(87). A variety of bacteria isolated from the oralcavity or feces of rats was tested singly and incombination. Bacterial suspensions weresprayed into the containers for the gnotobiotes(control, vitamin K-deficient, germfree apimalsshowed no response to uninoculated media).The following bacteria gave no significant rever-sal: rat oral strains of Lactobacillus acidophilusand a diphtheroid organism; rat enteric strains ofa sporeformer and two Bacteroides strains.However, an E. coli strain from rat feces and anunclassified sarcina-like micrococcus did re-verse the vitamin K deficiency symptoms. Thiswas the first demonstration that an experimen-tally induced vitamin deficiency could be re-versed by colonization of the host animal by asingle strain of bacteria.Evidence for a role for bacterial biosynthesis

in bovines comes from a study of the menaqui-none composition in liver. Bovine liver containsMK-10, -11 and -12; these three menaquinonesand MK-13 as well have also been isolated frombovine rumen contents (153). It appears likelythat the liver menaquinones are bacterial inorigin and are deposited after intestinal absorp-tion. It has been postulated that the vitamin Kcontent of liver is determined, in fact, by nutri-tional sources and is not dependent on metabolicevents. In support of this statement is the factthat the liver of the horse, a herbivore which isnot a ruminant, contains only phylloquinone (152).The lower part of the intestinal tract, where

the bacterial density is highest, seems the likelysite for vitamin K absorption. In rats, it has beenshown that the large bowel can absorb bacterial-ly synthesized menaquinone (although it has norole in the absorption of lipids). The absorptionrate appears to be more than adequate to pro-vide the animal with the daily requirement (100).A more complete discussion of this question isgiven by Suttie (207).

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Knowledge of the physiology of vitamin K inhumans is far from complete: it was notedrecently that "the fundamental question of therelative importance of the diet or intestinal mi-cro-flora in providing man's requirements forvitamin K still remains to be answered" (200).

Since Bacteroides species are among the mostnumerous of the bacteria inhabiting the humanintestinal tract and since strains such as Bacte-roides fragilis do synthesize vitamin K, it wassuggested that Bacteroides fragilis types weremore significant in providing the human vitaminK requirement than were E. coli strains (76).Other workers have investigated human bac-

terial strains and have shown that vitamin K is" produced by some strains ofBacteroides fragi-lis, bifidobacteria, clostridia and Streptococcusfaecalis." Although this conclusion is reason-able, it should be noted that the method usedwas "a plate test, with Bacteroides melanino-genicus as the indicator organism" (66). Possi-ble problems in this regard have been discussedearlier. It seems clear that the major bacterialpopulation contributing vitamin K to humannutrition remains to be identified with certainty.

ACKNOWLEDGMENTS

We are very grateful to H. J. Almquist for his recollectionsof the early days of vitamin K research, to E. L. R. Stokstadfor supplying a copy of the thesis of R. Halbrook, and to H.Rapoport for clarifying aspects of his work on 1-naphtholdegradation. This review has also benefited from our manydiscussions with I. M. Campbell, J. R. Guest, D. J. Shaw, andH. Taber. We are particularly grateful to Drynda L. Johnstonand the staff of Langley Library for their cheerful willingnessin meeting countless requests for obscure books, journals, andthe like.The work carried out in our laboratory has been supported,

in part, by Public Health Service research grant GM 20053from the National Institutes of Health. This support is grate-fully acknowledged.

LITERATURE CITED

1. Acar, J. F., F. W. Goldstein, and P. LaGrange. 1978.Human infections caused by thiamine- or menadione-requiring Staphylococcus aureus. J. Clin. Microbiol.8:142-147.

2. Alexander, K., and I. G. Young. 1978. Alternative hy-droxylases for the aerobic and anaerobic biosynthesis ofubiquinone in Escherichia coli. Biochemistry 17:4750-4755.

3. Allison, M. J., and I. M. Robinson. 1970. Biosynthesis ofa-ketoglutarate by the reductive carboxylation of succi-nate in Bacteroides ruminicola. J. Bacteriol. 104:50-56.

4. Almquist, H. J. 1975. The early history of vitamin K.Am. J. Clin. Nutr. 28:656-659.

5. Almquist, H. J. 1979. Vitamin K: discovery, identifica-tion, synthesis, functions. Fed. Proc. 38:2687-2689.

6. Almquist, H. J., and A. A. Klose. 1939. The anti-hemor-rhagic activity of pure synthetic phthiocol. J. Am. Chem.Soc. 61:1611.

7. Almquist, H. J., and A. A. Klose. 1939. Synthetic andnatural antihemorrhagic compounds. J. Am. Chem. Soc.61:2557-2558.

8. Almquist, H. J., C. F. Pentler, and E. Mecchi. 1938.Synthesis of the antihemorrhagic vitamin by bacteria.


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Biosynthesis of Vitamin K (Menaquinone) in Bacteria · BIOSYNTHESIS OF VITAMIN KIN BACTERIA 243 is of unique interest that bacteria again came into the picture and, as will be seen,