Vol. 158, No. 2JOURNAL OF BACTERIOLOGY, May 1984, p. 474-4800021-9193/84/050474-07$02.00/0Copyright © 1984, American Society for Microbiology

Pleomorphism of Fusobacteria Isolated from the Cockroach HindgutM. A. FOGLESONG,t D. L. CRUDEN, AND A. J. MARKOVETZ*

Department of Microbiology, University of Iowa, Iowa City, Iowa 52242

Received 5 July 1983/Accepted 31 January 1984

Fusobacteria are commonly isolated from the hindgut of the cockroach Eublaberus posticus. Elevenstrains isolated from E. posticus by us were keyed to four species, Fusobacterium necrophorum, F. varium,F. gonidiaformans, and F. prausnitzii, using current taxonomic criteria. With the exception of F.gonidiaformis, all species showed rods with swollen centers and large bodies. The pleomorphism of F.varium was examined by phase microscopy and scanning and transmission electron microscopy. Thepleomorphic process begins with a gradual swelling at the center of the rod until a large round body isformed. Some of these round bodies then fragment, giving rise to rod-shaped cells. When 10% yeast extractwas added to growth media, pleomorphism was not induced. A dialyzable factor was found to account forthis observation. Fermentation of [1-14C]glutamic acid gives rise to butyrate labeled in the carboxyl carbon,indicating that butyrate is formed by the hydroxyglutarate pathway which may be characteristic for thegenus Fusobacterium.

Several investigators have noted or studied the pleomor-phism of anaerobic gram-negative rods. In addition to spin-dle shapes with pointed ends and long and filamentousforms, "bizarre forms," rods with "bulbous swellings," and"round bodies" have been described. These pleomorphicorganisms originally were identified as "Bacteroides var-

ius," "Bacteroides funduliformis," "Sphaerophorus necro-

phorus," or "Fusobacterium ridiculosum." However, be-cause both nomenclature and criteria for identification anddifferentiation of anaerobic gram-negative rods havechanged considerably since many of these reports were

published, it is difficult to be certain how these organismswould be classified if the same strains were seen today. Inthe following section, we have tried to update both nomen-clature and identification, when possible. Synonyms are

given in parentheses after the present classification.In 1933, Eggerth and Gagnon (13) reported that broth

cultures of Fusobacterium varium ("B. varius") sometimescontained vacuolated or coccoid forms. Organisms thoughtto be Fusobacterium necrophorum ("B. funduliformis," "S.necrophorus") were described as having bulbous enlarge-ments that formed round bodies. These segmented to yieldnew rod-shaped cells (11, 22). The DNA base composition ofthe rods and the large body forms was the same (12). Cellscould be maintained in the rod stage, large body stage, or

intermediate stage by proper selection of conditions incontinuous culture (V. R. Dowell and E. 0. Hill, Bacteriol.Proc. 1964, G84, p. 29). The organism studied by Dowell isnow considered an atypical strain of Fusobacterium morti-ferum (V. R. Dowell, personal communication). Wahren andHolme (23) also commented about the marked morphologi-cal changes during the growth of F. necrophorum ("S.necrophorus"). Pearson and Balish (19) isolated from thehuman intestinal tract a bacillus that formed blebs and largespheroids. By biochemical tests the isolate most clearlyresembled F. mortiferum ("F. ridiculosum"). In an ultra-structural study of F. varium ("Sphaerophorus varius") andthe infective process of an associated phage, swollen cellswere seen, but these were interpreted as cells that were

ready to lyse and release phage (4). No swollen rods or otherlarge bodies were mentioned.

* Corresponding author.t Present address: Eli Lilly and Co., Indianapolis, IN 46206.

474

The initial report from this laboratory on the intestinalanaerobic microbiota from the cockroach reported on theisolation of a Fusobacterium sp. (M. A. Foglesong and A. J.Markovetz, Abstr. Annu. Meet. Am. Soc. Microbiol. 1973,G111, p. 44) that showed pleomorphic forms, i.e., swollenrods and large bodies (M. A. Foglesong and A. J. Marko-vetz, Abstr. Annu. Meet. Am. Soc. Microbiol. 1974, G104,p. 37). The present study reports on the identification ofseveral members of the genus Fusobacterium isolated fromthe hindgut of the cockroach Eublaberus posticus and on thepleomorphic development via swollen rods and large bodiesof one species, F. varium. Also, the fate of [1_14C]glutamicacid metabolized by F. varium is described.

MATERIALS AND METHODS

Isolation and nutrition. Specimens of E. posticus weredissected in an anaerobic chamber or under a stream of N2 ina partially covered dissection dish. The hindgut was quicklyremoved, homogenized with a tissue grinder in PYG medium(15), and streaked in roll tubes under N2 by a modifiedHungate technique (5) or on plates of PYG in the anaerobicchamber under an atmosphere of 85% N2-10% H2-5% CO2.Colonies were purified by repeated restreaking. Stocks weremaintained on PYG medium with transfers at 3-day intervalsor were stored frozen at -70°C.

Isolates were characterized with media and tests specifiedfor fusobacteria in the Virginia Polytechnic Institute (VPI)Anaerobe Laboratory Manual (15). Tubes of Scott anaerobicmedia (Scott Laboratories, Inc., Fiskeville, R.I.) were usedto determine fermentation of carbohydrates. Fermentationproducts were analyzed by flame ionization gas chromatog-raphy of ether extracts of acidified PYG culture fluid forvolatile fatty acids and chloroform extracts of methyl estersof nonvolatile fatty acids prepared as described in the VPIAnaerobe Laboratory Manual (15). Analyses were per-formed on a gas chromatograph (model 2700; Varian Instru-ments, Palo Alto, Calif.) equipped with a flame ionizationdetector with a 50-m capillary column of Carbowax 20M ondeactivated silica (VFA) (Hewlett-Packard, Avondale, Pa.).Volatile fatty acid analyses were run isothermally at 60°C,whereas methyl ester analyses were run for an initial periodat 60°C followed by temperature programming at 4°C/min toa final temperature of 110°C.

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Gaseous end products were analyzed by use of a Smithfermentation tube and a Fisher-Hamilton gas partitioner.Gaseous samples obtained from the headspace of culturegrown in 25-ml Erlenmeyer flasks fitted with gas-tight serumstoppers were injected into the gas partitioner which wasequipped with a 53-cm, 28-200-mesh silica gel column and a1.9-m, 42-60-mesh 13x molecular sieve (Fisher ScientificCo., Pittsburgh, Pa.).Growth curves were performed in PYG medium contain-

ing various amounts of yeast extract (1, 3, 5, 7, or 10%). Astandard inoculum of 15 drops of a PYG (1% yeast extract)-grown culture with an optical density at 660 nm of 0.1 wasused for nutritional experiments.

Electron microscopy. Fusobacterium cells were fixed fortransmission electron microscopy by the method of Kellen-berger et al. (17), embedded in Epon, and sectioned with adiamond knife. Sections were poststained with uranyl ace-tate and Reynolds lead citrate stain (21).For scanning electron microscopy, cells were fixed as

above, critical point dried, mounted on stubs, sputter coatedwith gold and palladium, and viewed with a Kent CambridgeStereoscan S4 scanning electron microscope.

Slide cultures. Morphological development of F. variumwas observed by preparing slide cultures. The developmentof the organisms was examined and photographed with aLeitz phase microscope with a photographic attachment. Toprepare slide cultures, depression slides were coated with asmall amount of PYG semisolid agar (0.7%), and the slideswere inoculated in an anaerobic chamber with F. varium inthe rod stage of development. A cover slip was placed overthe agar and sealed with paraffin wax. In later experiments, asmall PYG agar block (10 mm square by 2 mm thick) wasinoculated, placed in a depression slide, fitted with a coverslip, and sealed with paraffin. To enable the gas produced bythe organisms during growth to escape, a 21-gauge needlewas inserted through the paraffin directly under the coverslip. All steps were performed in an anaerobic chamber.Large body viability. Viability of large bodies of F. varium

was tested by plate count dilution of liquid PYG (3% yeastextract)-grown cultures. Cell counts were determined with aPetroff-Hauser counting chamber, and appropriate dilutionsof the cultures were plated on PYG (1% yeast extract)medium.

Butyric acid analysis. A 10-ml culture of F. varium in basalPYG (3% yeast extract) medium containing 3.5 x 106 dpm ofDL-[1-14C]glutamic acid (Amersham-Searle Corp.) wasgrown to an absorbance of 0.76. To extract the fatty acid endproducts from the culture fluid, the medium was adjustedback to pH 7 with 10 N NaOH, and the cells and proteinswere precipitated with zinc sulfate. The precipitate wasremoved by centrifugation and washed twice with distilledwater. The supernatant fluid and precipitate washings werecombined, acidified to pH 2 with HCI, and continuouslyextracted with diethyl ether. After 48 h of extraction, theether was separated from the aqueous phase and dried withNa2SO4, and the volume was reduced to 4 ml withoutheating under a stream of N2. The resulting [l4C]butyric acidwas collected by preparative gas-liquid chromatography on astainless steel column (1/8 in. by 8 ft [0.31 by 243.8 cm])packed with 10% SP-1200 plus 1% phosphoric acid on Gas-Chrom G AW DMSC. The gas chromatograph was fittedwith a stream splitter that allowed an approximate split ratioof 10 parts to the collector and 1 part to the detector. Butyricacid (200 LI) was collected from the column into glasscapillary tubes (310 by 1 mm) packed in dry ice and elutedfrom the capillary tubes with ether.

[14C]butyric acid was chemically decarboxylated by theSchmidt degradation. The procedure was a modification ofthe one outlined by Phares (20). [14C]butyric acid in etherwas placed in the reaction flasks, and enough 5% NaOH wasadded to give two distinct phases. The mixture was shakenthoroughly to ensure conversion of butyric acid to thesodium salt. The mixture was evaporated to dryness under avacuum with heating. After the reaction flask was cooled to4°C, 0.3 ml of H2SO4, prepared by combining one part offuming H2SO4-20% excess S03 with three parts of concen-trated H2SO4, was carefully added. The [14C]sodium buty-rate was completely dissolved by warming and shaking, andafter cooling, 50 mg of NaN3 was added. The flask waswarmed and shaken until the NaN3 was almost completelydissolved, and the flask was attached to a trap containing 5ml of hyamine hydroxide. The clamp leading to the sweeptube was closed, a vacuum was applied to the trap, and theentire apparatus was placed in a water bath at 30°C. Thetemperature was raised to 70°C over a 30-min period, andafter 30 min at 70°C, the clamp leading to the sweep tube wasopened, and the entire system was swept with C02-free airfor 15 min. Radioactive CO2 was counted by dispensing theentire 5 ml of hyamine hydroxide into 20 10-ml vials of Insta-Gel (Packard Instruments, Downers Grove, Ill.).To recover the three-carbon compound (propyl amine)

remaining after decarboxylation, the hyamine hydroxide inthe trap was replaced with 5 ml of 0.2 N H2SO4, 5 N NaOHwas added to adjust the pH of the reaction mixture to 12, andthe system was swept with C02-free air for 25 min at 80°C.We then added 5 ml of 5% KMnO4 and 0.5 ml of 0.5 N NaOHto the H2SO4 in the trap, and the trapping vessel was tightlystoppered and the mixture was heated for 20 min at 80°C togenerate propionate. Recovery of the resulting fatty acid wasachieved by acidifying the mixture to pH 2 with H2SO4 andextracting it three times with diethyl ether. The ether wasdried with Na2SO4, and the fatty acid was analyzed andcollected by gas-liquid chromatography and checked forradioactivity.

RESULTSOrganisms. Eleven strains of bacteria isolated from the

hindgut of E. posticus on a variety of media over a period ofseveral years were identified as Fusobacteril/m spp. Allwere obligately anaerobic, gram-negative, nonmotile, non-sporeforming rods which produced butyrate and smalleramounts of acetate, propionate, and succinate as fermenta-tion products. Table 1 shows the characteristics used todistinguish among the strains. Most produced large amountsof H2. None of the strains produced acid from lactose,maltose, starch, or sucrose. None hydrolyzed starch orgelatin or reduced nitrate. Seven of the strains were identi-fied as F. necrophorum, two as F. varirmn, and one each asF. gonidiaformans and F. praiusnitzii. The two indole-negative strains of F. necrophorum were placed in this groupbecause of their strong production of propionate from lactateand lack of fermentation of mannose. Of the 11 strains, 10(all except F. gonidiaformans) were pleomorphic, producinglarge bodies. One of the strains of F. i'arium was studiedfurther. This strain was previously incorrectly classified byus as F. symbiosum (14).

[14C]butyrate formation from glutamate. Butyric acid(52,000 dpm) recovered from culture fluid was collected inether from the gas chromatograph and decarboxylated by theSchmidt reaction. Evolved CO, was checked for radioactiv-ity. Decarboxylation of butyrate by this procedure producedpropylamine, which was left in the reaction mixture. Propio-

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476 FOGLESONG, CRUDEN, AND MARKOVETZ

TABLE 1. Fusobacteria isolated from the hindgut of E. posticusa

Strain (no.) E E

F. varium (2) + la, lw la, lw w 1-,1+ +F. necrophorum (7) + 5w, 2- 3w, 4- - 5+, 2- + +F. gonidiaformans (1) - - - - + +F. prausnitzii (1) + w - - - - - +

a a, Strong acid; w, weak acid.

nate was generated by the oxidation of propylamine withKMnO4 in the presence of NaOH. After completion of theSchmidt degradation, 13,000 dpm remained as unreactedbutyric acid and 13,000 dpm of "4CO2 was formed. Thepropionate recovered after decarboxylation contained only450 dpm, so it appeared that C-1 of butyrate accounted forthe bulk of the radioactivity in butyrate. These resultsindicate that F. varium forms butyrate from glutamate by thepathway used by Peptococcus asaccharolyticus ("Pepto-coccus aerogenes") whereby C-1 of glutamate becomes thecarboxyl carbon of butyrate (6).

Observations by phase and electron microscopy. Examina-tion of cultures of F. varium grown in PYG broth revealednumerous pleomorphic forms, i.e., rods, rods with swollencenters, and large bodies. The development of a bacteriumcould be followed by anaerobic slide culture, using phasemicroscopy. A bacterium initiated growth in the form of arod. After ca. 6 h, a small dense region appeared in thecenter of the organism. Three hours later the dense areadeveloped into a swollen region which continued to swell asthe bacillary arms degenerated resulting in the formation of alarge body. This developmental process was also monitoredwith electron microscopy. Cells were grown in PYG broth,removed at intervals corresponding to the different formsobserved, and examined by scanning electron microscopy.Figure 1A shows the organism as it appeared in the rod formbefore the development of swollen regions. The rods wereregular in shape and ranged from 0.9 to 1.0 p.m in width and 3to 4 p.m in length. The swollen region occurred predominant-ly in the central portion of the bacterium, and this can beseen at various stages of development in Fig. 1B. Thebacillary arms still measured ca. 1 p.m in diameter with theswollen regions measuring from 2.0 to 2.5 p.m in width.Continued swelling of the centers, in conjunction withgradual shrinkage of the bacillary arms, culminated in theproduction of large bodies (Fig. 1C). At this stage, the largebodies measured ca. 4 p.m in diameter. The bacillary armswere 1 by 4 p.m and no longer possessed a regular appear-ance. Further development of the large bodies showed acontinuing shrinkage of the arms (Fig. 1D).

In an attempt to ascertain any discernible differences infine structure during pleomorphic development, thin sec-tions were observed by transmission electron microscopy.Figure 2A shows an organism as it appears before initiationof the swelling process. The distribution of chromatin mate-rial (light areas from which ribosomes have been excludedprobably contained DNA) indicates that the rod-shaped cellwas preparing to divide. Further development showed in-creased swelling (Fig. 2B), and the cell wall was stillcontinuous with the bacillary arms. Figure 2C shows a crosssection through a large body. The cell wall was intact aroundthe entire organism, and no gross internal abnormalities

were apparent. The inner cytoplasmic membrane was repre-sented by the heavily staining dark line surrounding the largebody. The outer membrane complex was seen as a looselyattached layer. In addition, the chromatin-like material hadbecome fragmented and positioned at the periphery of thelarge body. The large protrusion may represent a bacillaryarm that had been cut tangentially. In what is interpreted asthe fragmentation process, the large body lost its roundedappearance and became irregular in shape (Fig. 2D). Inaddition to the irregular appearance, the cell envelope hadbecome thicker, and in places, invagination of the cellmembrane had occurred.To determine the fate of large-body forms, we used a slide

culture technique to follow any further development ofindividual large bodies. A broth culture was allowed todevelop to the large body stage, at which time a loopful ofculture was used to inoculate a small PYG agar block (seeabove). The development of three individual large bodieswas followed by phase microscopy over a 72-h period (Fig.3). The organism in the lower left corner of Fig. 3A throughFig. 31 became vacuolated (Fig. 3F) and did not developfurther. However, 2 h after transfer the two remaining largebodies developed protuberances which were pinched off andunderwent fission. In addition, large bodies themselvesdeveloped cleavages (Fig. 3B and C) and appeared tofragment (Fig. 3D through I). The fragmented large bodyseen in the lower right portion of Fig. 31 gave rise to at leastfive visible bacillary forms.Large body viability. In an effort to determine what

percentage of the large bodies retained their viability aftertransfer to fresh PYG medium, plate count dilutions wereperformed. A fresh culture was grown to an optical densityof 0.65 (ca. 9 h), and the number of large bodies and rods wascounted with a Petroff-Hauser counting chamber. Organismspossessing swollen centers were counted as large bodiessince previous observations indicated that once large bodydevelopment began it was not reversed by transfer ofswollen-centered organisms to fresh medium. Counts indi-cated that the culture contained 1.2 x 108 large bodies per mland 6.8 x 106 rods per ml, or 95% large bodies and 5% rods.The culture was serially diluted to 10-8, and 0.1 ml of eachdilution was plated in duplicate on PYG agar plates. Plateswere incubated for 48 h and counted. The 10-4 dilutionplates contained 546 and 650 colonies, whereas the 10-5dilution plates contained 60 and 61 colonies. If these data areexpressed in terms of number of colonies produced from 1ml of a 10-6 dilution, they represent 55, 65, 60, 61 coloniesproduced. One milliliter of a 10-6 dilution of the culturecontained 120 large bodies and seven rods, as determined bythe counting chamber. Assuming all seven rods were viable,the production of 60 colonies would mean that ca. 44% of thelarge bodies in the culture were viable. This indicated that

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PLEOMORPHIC FUSOBACTERIA 477

FIG. 1. Scanning electron micrographs of the rod to large body pleomorphic development of F. *varim,n. (A) Normal rod-shaped organisms;(B) initiation of central swelling in the rods; (C) early large body formation, (D) late large body formation before fragmentation. Bar = 1 I±m(for all panels).

large body formation was not entirely a dead-end phenome-non, i.e., some large bodies were capable of regeneratingviable cells.

Nutrition and large body formation. F. v'ariumi was grownon a variety of media in an attempt to determine the effect ofnutrients on large body formation. Organisms were grown ona number of conventional anaerobic media (15), includingrumen fluid-glucose-cellobiose agar, brain heart infusionagar, medium 10, veal heart infusion agar, and chopped meatbroth. In all cases the organisms developed large bodies inthe same manner as during growth on PYG medium. Inaddition, growth on PYG medium supplemented with 0.1, 1,or 10% cockroach gut extract had no effect on large bodyformation. Gut extract was made by suspending severalroach hindguts in 2 ml of phosphate-buffered saline, homog-enizing the guts with a tissue grinder, pelleting the debris by

centrifugation, and filter sterilizing the supernatant fluid.Large bodies also developed in PYG medium supplementedwith 0.1% Tween 80 or PYG medium incubated underatmospheres of various concentrations of CO,, H2, N,, orargon.

Since a variety of nutrient media seemed to have no effecton large body development, the possibility that a toxic endproduct was initiating the development was examined. Or-ganisms were grown to the rod stage in PYG medium, and asmall sample of the culture was used to inoculate a steriledialysis bag containing 10 ml of sterile PYG medium. Theculture was allowed to grow in the dialysis bag which wassuspended in PYG medium. Four changes of fresh, sterilePYG medium were made during the experiment. The devel-opment of the culture was followed by periodically removingsamples from the dialysis bag and examining them by phase

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478 FOGLESONG, CRUDEN, AND MARKOVETZ

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FIG. 2. Transmission electron micrographs of the rod to large body pleomorphic development of F. v,arium. (A) Rod stage of growth; (B)swollen center stage of growth; (C) large body stage with nuclear material localized in several areas at the periphery; (D) fragmentation stageof the large body. Bar = 1 .m in (B) and (D), 0.5 ,um in (A) and (C).

microscopy. In comparison with growth obtained by routineculturing in PYG medium, F. varium in the dialysis tubinggrew to a higher optical density before large body formationwas initiated. This observation indicated that the organismsin the dialysis bag were able to grow longer in the rod stage,owing either to the removal of a toxic end product or to thecontinued replenishment of a dialyzable nutrient by therepeated addition of fresh medium to the system.

Yeast extract has been shown to contain a number of oftentimes ill-defined growth factors, and for that reason, itseemed possible that normal development of F. variummight also depend on a factor present in yeast extract inlimiting amounts. PYG media containing 1, 3, 5, 7, and 10%

yeast extract were each inoculated with a standardizedinoculum of F. varium. The development of the organisms ineach culture was monitored by phase microscopy, andgrowth was monitored by following absorbance at 660 nm. Inthe case of 1, 3, 5, and 7% yeast extract, large bodyformation began in the late-log phase of growth with theamount of growth increasing with increasing concentrationsof yeast extract. However, with 10% yeast extract in PYGlarge bodies never developed.To further demonstrate the ability of large quantities of

yeast extract to prevent large body development, 50 ml of10% yeast extract was dialyzed for 60 h against six changes(1 liter each) of distilled water. The dialysate and nondialyz-

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PLEOMORPHIC FUSOBACTERIA 479

IdEL

FIG. 3. Phase-contrast micrographs of the development of large bodies of F. l!'arilul, over a 72-h period in slide culture. Bar = 10 p.m (for

all panels). Development times (hours) shown are as follows: A, 2; B, 6; C, 8; D, 12; B, 18; F, 24; G, 36; H, 48; l, 72.

able material were each concentrated to 50 ml in a flashevaporator and spray concentrator. Peptone (1%), glucose(1%), mineral salts, cysteine, and resazurin were added toeach 50-ml fraction and autoclaved. These represented twomedia in which yeast extract of PYG medium was replacedwith nondialyzable material in one case and by dialysate inthe other. Five milliliters of each medium was inoculatedwith F. variumn, and growth was followed for 40 h by phasemicroscopy. Cells grown in nondialyzable material-PG medi-um grew to an absorbance of only 0.04 and demonstratedabundant large body formation. On the other hand, cellsgrown in dialysate-PG medium reached an absorbance of 1.5and showed no evidence of large body formation. Theseresults indicate that yeast extract contains a dialyzablecomponent that allows for normal cell development. Deple-tion of this factor leads to pleomorphism.

DISCUSSIONPrevious reports from this laboratory document that the

hindgut of the cockroach possesses a complex microbialflora (3, 7, 8) predominantly composed of anaerobes (2).Numerous morphologically distinct forms have been consist-ently recognized and enumerated by electron microscopeanalyses (9). Most of the unique morphological forms havenot been isolated. Eleven of the numerous fusobacteriaisolated were identified as to species, and of this smallsampling, F. necrophorum was the most common species.Of these 11 species, 10 produced rods with swollen centersand large body forms when cultivated. However, examina-tion of several hundred micrographs accumulated over aperiod of years showed an absence of any recognizablepleomorphic forms of fusobacteria in the hindgut. This

indicates that the cause(s) for the pleomorphic development(swollen rods and large bodies) being examined is notmanifested in the complex microbial ecosysteni in the gut. Itappears that when the fusobacteria are deprived of interac-tions with other microbes or with the host by growth in pureculture a pleomorphic development occurs.The development of large bodies from normal rods of F.

i'arium followed by the regeneration of bacillary forms byfragmentation of the large body is somewhat similar toobservations made with F. necroplhoruln ("Bacteroides filn-duliformiss") (11, 22). Large bodies were reported to have atwofold potential, i.e., they produced ordinary bacteria or apeculiar pleomorphic L-type growth. Four to five heavilystained chromatin areas were noted in these large bodies,and segmentation to produce rods appeared to occur aroundthe chromatin granules. In some cases, small L-type cellsseemed to be produced from the chromatin granules. In thepresent study with F. "ariwn, chromatin-like material wasalso noted by electron microscopy around the periphery ofthe large body before fragmentation, but the production ofL-type cells was never observed.The induction of this pleomorphic series of events appears

to be caused by a lack of some nutritional factor present inyeast extract. An increase in yeast extract up to a concentra-tion of 10% progressively delayed pleomorphism, and at 10%yeast extract pleomorphism was not induced. Our resultsindicate that the factor(s) is dialyzable from yeast extract.The work of Dowell and Hill (Bacteriol. Proc. 1964, G84, p.29) with F. mortiferum ("F. necrophorium") demonstratedthat with the proper selection of dilution rates in a chemostatorganisms could be maintained in the classical rod, largebody, or intermediate stages of morphology. They conclud-

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480 FOGLESONG, CRUDEN, AND MARKOVETZ

ed that the development of large bodies was mediated byenvironmental changes. Our results would indicate that theenvironmental change is a depletion of a nutritional factor.As stated previously, we have never noted any of these

pleomorphic forms in electron micrographs of organisms ingut lumen contents or in organismns associated with the gutwall. This appears to be enigmatic since the hindgut cannotbe viewed as a continuous culture but as an unbalancedgrowth system in which nutrients are not continuouslysupplied, and under such conditions, growth factors couldconceivably be in short supply at various times. The pres-ence of storage granules in procaryotes is indicative ofunbalanced growth due to the presence of some nutrientsand the absence of others (10). The finding of such inclusionsin the cockroach hindgut (Cruden and Markovetz, Arch.Microbiol., in press) and the presence of significant numbersof sporulating bacteria in vivo (3, 9) seem to confirm thatunbalanced growth is characteristic of this microbial ecosys-tem. The lack of pleomorphism in F. varium when growingin vivo may indicate that it grows in close association withother microbes that continually provide the nutritional fac-tor. Alternatively, the host may be the source of continuoussupply. Our results indicate that pleomorphic developmentin fusobacteria may be only encountered when these orga-nisms are grown under laboratory conditions. This would besimilar to the production of L-phase variants of Streptobacil-lus moniliformis which occur most frequently under labora-tory conditions (25).

Glutamate-fermenting anaerobes produce butyrate fromglutamate by one of two pathways, i.e., the methylaspartatepathway first characterized in Clostridium tetanomorphum(1) and the hydroxyglutarate pathway found in P.asaccharolyticus ("P. aerogenes") (16, 24). Buckel andBarker (6) examined the occurrence of the two pathwaysamong glutamate-fermenting anaerobes and found that thehydroxyglutarate pathway was used by two strains of F.nucleatum and two strains of "F. fusiforme." The latterstrains were obtained from the VPI Anaerobe Laboratory,and these strains are now considered as F. nucleatum (18).Our results with F. varium indicate that the hydroxyglutar-ate pathway is used, and it appears that this route forglutamate fermentation is probably characteristic for thegenus Fusobacterium.

ACKNOWLEDGMENTThis work was supported in part by Public Health Service grant

AI-13990 from the National Institute of Allergy and InfectiousDiseases.

LITERATURE CITED1. Barker, H. A. 1961. Fermentation of nitrogenous compounds, p.

151-207. In I. C. Gunsalus and R. Y. Stanier (ed.), The bacte-ria, vol. II. Academic Press, Inc., New York.

2. Bracke, J. W., D. L. Cruden, and A. J. Markovetz. 1978. Effectof metronidazole on the intestinal microflora of the Americancockroach, Periplaneta americana. Antimicrob. Agents Che-mother. 13:115-120.

3. Bracke, J. W., D. L. Cruden, and A. J. Markovetz. 1979.Intestinal microbial flora of the American cockroach Peripla-neta americana L. Appl. Environ. Microbiol. 38:945-955.

4. Bradley, D. E. 1972. Ultrastructural studies of Sphaerophorus

varius and the infective process of an associated bacteriophage.Can. J. Microbiol. 18:1103-1111.

5. Bryant, M. P. 1972. Commentary on the Hungate technique forculture of anaerobic bacteria. Am. J. Clin. Nutr. 25:1324-1328.

6. Buckel, W., and H. A. Barker. 1974. Two pathways of glutamatefermentation by anaerobic bacteria. J. Bacteriol. 117:1248-1260.

7. Cruden, D. L., T. E. Gorrell, and A. J. Markovetz. 1979. Novelmicrobial and chemical components of a specific black-bandregion in the cockroach hindgut. J. B3acteriol. 140:687-698.

8. Cruden, D. L., and A. J. Markovetz. 1980. A thick-walledorganism isolated from the cockroach gut by using a spentmedium technique. Appl. Environ. Microbiol. 39:261-264.

9. Cruden, D. L., and A. J. Markovetz. 1981. Relative numbers ofselected bacterial forms in different regions of the cockroachhindgut. Arch. Microbiol. 129:129-134.

10. Dawes, E. A., and P. J. Senior. 1973. The role and regulation ofenergy reserve polymers in microorganisms. Adv. Microb.Physiol. 10:135-226.

11. Dienes, L., and W. E. Smith. 1944. The significance of pleomor-phism in Bacteroides strains. J. Bacteriol. 48:125-154.

12. Dowell, V. R., Jr., J. C. Loper, and E. 0. Hill. 1964. Constancyof deoxyribonucleic acid base composition in the transition ofSphaerophorus necrophorus from bacilli to large bodies. J.Bacteriol. 88:1805-1807.

13. Eggerth, A. H., and B. H. Gagnon. 1933. The bacteroides ofhuman feces. J. Bacteriol. 25:389-413.

14. Foglesong, M. A., and A. J. Markovetz. 1974. Morphology ofbacteriophage-like particles from Fusobacterium symbiosum. J.Bacteriol. 119:325-329.

15. Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.). 1977.Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Insti-tute Anaerobe Laboratory, Blacksburg, Va.

16. Johnson, W. M., and D. W. S. Westlake. 1972. Purification andcharacterization of glutamic acid dehydrogenase and a-ketoglu-taric acid reductase from Peptococcus aerogenes. Can. J.Microbiol. 18:881-892.

17. Kellenberger, E., A. Ryter, and J. Sechaud. 1958. Electronmicroscopic study of DNA-containing plasms. Il. Vegetativeand nmature phage DNA as compared with normnal bacterialnucleoids in different physiological states. J. Biophys. Biochem.Cytol. 4:671-678.

18. Moore, W. E. C., and L. V. Holdeman. 1974. Genus lI.Fusobacterium, p. 404-416. In R. E. Buchanan and N. E.Gibbons (ed.), Bergey's manual of determinative bacteriology,8th ed. The Williams & Wilkins Co., Baltimore.

19. Pearson, T. A., and E. Balish. 1970. Unusual Sphaerophorusspecies from the large intestine of man. Appl. Microbiol.19:458-462.

20. Phares, E. F. 1951. Degradation of labeled propionic and aceticacids. Arch. Biochem. Biophys. 33:173-185.

21. Reynolds, E. S. 1963. Lead citrate stain at high pH for electronmicroscopic work. J. Cell. Biol. 17:208-216.

22. Smith, W. E., S. Mudd, and J. Hiller. 1948. L-type variation andbacterial reproduction by large bodies as seen in electronmicrographic studies of Bacteroides funduliformis. J. Bacteriol.56:603-618.

23. Wahren, A., and T. Holme. 1973. Amino acid and peptiderequirement of Fusiformis necrophorus. J. Bacteriol. 116:279-284.

24. Whitely, H. R. 1957. Fermentation of amino acids by Micrococ-cus aerogenes. J. Bacteriol. 74:324-330.

25. Wittler, R. G., and S. G. Cary. 1974. Genus Streptobacillus, p.378-381. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey'smanual of determinative bacteriology, 8th ed. The Williams &Wilkins Co., Baltimore.

J. BACTERIOL.

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