JouRNAL OF BACTERIOLOGY, Feb. 1970, p. 517-524Copyright 0 1970 American Society for Microbiology

Vol. 101, No. 2Printed in U.S.A.

Defined Nongrowth Media for Stage II Developmentof Competence in Haemophilus influenzae

R. M. HERRIOTT, E. M. MEYER, AND M. VOGTDepartment ofBiochemistry, The Johns Hopkins University School ofHygiene and Public Health,

Baltimore, Maryland 2120S

Received for publication 7 November 1969

The composition of a defined nongrowth medium used in stage II development ofcompetence of Haemophilus influenzae affects the course of this development. Thedevelopment of competence in two nongrowth media, M-IV and M-V, is rapid,logarithmic, and independent of the cell concentration. This last property indicatesthat there is probably no transfer of a competence factor from competent to non-competent cells, in contrast to results reported for other organisms. Levels of com-petence reached in these completely defined media are such that 1 to 5% of the cellsare transformed in the presence of an excess of marked deoxyribonucleic acid. Themethod of evaluating competence, which depends on the frequency of multipleindependent transformations, has been reexamined. This and other methods are

compared on samples taken from a culture during development of competence.

Although the mechanism by which cells be-come competent and take up deoxyribonucleicacid (DNA) is the unique feature of genetictransformation in bacteria, this subject has re-ceived relatively little attention. Development ofcompetence in Haemophilus influenzae takesplace in two distinct stages. Stage I, the growthphase, has specific nutritional requirementswhich are necessary, not for growth, but for de-velopment of competence in stage II. At leasttwo of these specific nutrients, a purine nucleo-side (inosine being the best) and lactate or pyru-vate, have been' reported to be needed for theultimate development of competence (16) andhave been included in the defined growth medium(MI,) described in the preceding paper (8).Since a defined medium for stage II developmentwas described earlier (13, 18), conditions for theentire process have now been defined. A similarstudy of defined media for developing compe-tence in streptococci was described earlier (13a).

This paper describes two additional stage IImedia (M-IV and M-V) in which H. influenzaedevelop high levels of competence somewhatfaster than in M-II. The course of development ofcompetence in the various stage II media has beenexamined with particular attention to the earlyresponse and to the highest level of competencereached. In addition, the effect of cell concen-tration on the rate of competence developmenthas been examined. A direct dependence wouldindicate that competent cells release material

which aids noncompetent cells in becoming com-petent. Other organisms (1, 2, 14, 21) are knownto release substances during their development ofcompetence which enhance development ofcompetence in noncompetent cells.

Finally, the discrepancies (4, 7, 10, 15) foundin the values of f the fraction of a populationthat is competent, calculated from the multipletransformation frequency (6) have been reviewed,and some plausible explanations for the discrep-ancies are offered.

MATERIALS AND METHODSAll of the materials and equipment used in this

study have been described earlier (8). Two differentpreparations of transforming DNA were used in thiswork. One was isolated from H. influenzae cellsresistant to 2,000 ,ug of streptomycin per ml; the otherDNA was prepared from cells that were resistant to10 ,ug of erythromycin per ml. Competent cells wereprepared as follows.

Stage I: growth. H. influenzae cultures were dilutedin Difco Heart Infusion or in MI medium (8) to aconcentration of 107 to 2 X 107 cells/ml. A 35-mlamount of such a suspension in a 500-ml side-armErlenmeyer flask (Bellco Glass, Inc., Vineland, N.J.)was rotated at 150 rev/min at 37 C until the cellconcentration reached 5 X 108 to 7 X 108/ml, asjudged by turbidity measurements in a ColemanJunior spectrophotometer at 650 nm. The cells werecentrifuged at 2,000 to 3,000 X g for 5 min, resus-pended in the same volume of M-II, and then re-centrifuged and resuspended in the same or half thevolume of one of the stage II media.

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Stage II: competence development. Washed stage Icells were resuspended in 35 ml of M-IV or M-Vmedium; they were rotated in a 500-ml flask at 125rev/min for 70 to 100 min when the medium wasM-IV, and for 120 min when the medium was M-V.In each case, the cells had usually reached their peakof competence in the specified period.Competence was measured in the several different

ways described below. These differences were neces-sary because of the varied conditions and require-ments of the particular experiments.

Relative competence. The transformation frequencyunder any set conditions relative to the frequency atmaximal competence under the same conditions is thebasis of this ratio.

Per cent transformed. This measure of competenceis the percentage of the cell population that is trans-formed by an excess of transforming DNA. This ratiois a special case of relative competence ratio in whichthe cells are exposed to 1 ,ug of the high-level strepto-mycin-resistance marker per ml for 30 min in BrainHeart Infusion and then are diluted and plated. Theviable-cell count was sampled 5 min after mixing withDNA. This early sampling excludes cells which divideduring the 30-min uptake but are probably not com-petent.

Cell competence. The competence of individual cellsvaries (18). Early in the development of competence,most competent cells will take up one but not twodifferent pieces of transforming DNA. As develop-ment of competence proceeds, the capacity to takeup several pieces of DNA increases. This is shown bya 10-fold increase, during development of competence,in the ratio of cells transformed to two independentmarkers relative to the number transformed to oneof the markers.The change in the ratio of double to single trans-

formants when a culture is exposed to an excess ofmixed marked DNA preparations measures the cellu-lar competence.

Multiple transformations. This method (6) usesa mixture of DNA preparations carrying differentmarkers, and is based on the proposition that in fullycompetent cells the frequency of multiple transforma-tions is predictable from the frequencies of the in-dividual transformations. If competence in a cell isan "all or none" phenomenon, then once cells becomecompetent they should be fully transformable to allmarkers. In a mixed population of competent andnoncompetent cells where the two types cannot beidentified, the frequency of double transformationswill exceed the number calculated from the frequenciesof single transformants. This finding was the basisfor the suggestion that the multiple transformationprocedure provides a means of determining the frac-tion of competent cells in a culture. Data (6) onartificially mixed populations support this suggestion.

These assays of competence will be discussed laterin the text of this paper.

Plating. In general, dilutions of cells for platingwere made in Brain Heart Infusion-saline (1:10),and plating, in duplicate, was performed after firstmixing a sample of cells in the petri dish with 10 ml ofwarm (43 C) Brain Heart Agar containing 10 lAg of

hemin and 4 ,ug of nicotinamide adenine dinucleotide(NAD) per ml but no antibiotic. After hardening andincubation of these plates for 1.5 to 2 hr, they werecovered with a layer of 10 ml of similar Brain HeartAgar containing double the equilibrium concentrationof antibiotic. The plates were counted after 18 to 24hr of incubation at 37 C.

Preparation of various stage II media. The variousmedia, M-II through M-V, are quickly made up bydiluting sterile stock solutions of the components.The directions for making the final media follow thedescriptions of the stock solutions.

Stock solution 21 has the following composition:L-aspartic acid, 4.0 g; L-glutamic acid, 0.2 g; fumaricacid, 1.0 g; NaCl, 4.7 g; K2HPO4, 0.87 g; KH2PO4,0.67 g; and Tween 80, 0.2 ml. These compounds aredissolved in 850 ml of distilled water. The solution isadjusted to pH 7.0 with 48 ml of 1 N NaOH andthen is diluted to 1 liter with water. The resultingsolution is divided into 100-ml volumes and auto-claved.The components of stock solution 22 are as follows:

L-cystine, 0.04 g; L-tyrosine, 0.10 g; citrulline, 0.06g; L-phenylalanine, 0.20 g; L-serine, 0.30 g; and L-alanine, 0.20 g. The cystine and tyrosine are dis-solved in 10 ml of 1 N HCl at 37 C. The solution isdiluted with water to 100 ml and the other compo-nents are added. Sterilization is by filtration.

Stock solution 23 consists of 1.11 g of CaC12(anhydrous) dissolved in 100 ml of distilled water andautoclaved.

For stock solution 24, 0.60 g of MgSO4 (anhydrous)is dissolved in 100 ml of distilled water and auto-claved.

Stock solution 40 is 5.0 g of Difco Vitamin FreeCasamino Acids dissolved in 100 ml of water andsterilized by filtration.

Stock solution 51 has the following composition:L-glutamic acid, 0.20 g; fumaric acid, 1.0 g; NaCl,14 g; K2HPO4, 0.87 g; KH2PO4, 0.67 g; and Tween80, 0.2 ml. These components are mixed with 850 mlof distilled water, and 20.4 ml of 1 N NaOH is addedto adjust the pH to 7. Water is added to bring thevolume to 1,000 ml, and 100-ml volumes are auto-claved.The various nongrowth competence media are made

from the stock solutions as follows. M-II is made byadding 1 ml each of stocks 22, 23, and 24 to 100 ml ofstock 21. M-IV consists of 1 ml of stock 40 added to100 ml of M-II. M-V is made by adding 1 ml each ofstocks 22, 23, 24, and 40 to 100 ml of stock 51.

RESULTSEffect of composition of stage II nongrowth

medium on the course of competence development.Competence development in the defined mediumM-II involved a 30- to 60-min lag period beforeit was detectable and 2 hr more for full develop-ment (18). Addition of 0.05% Casamino Acids(17) reduced the time for full development toabout 70 to 100 min. M-II plus 0.05% CasaminoAcids is designated M-IV. Use of a mixture of

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individual amino acids simulating the CasaminoAcids also shortened the time period requiredfor development of competence, so that an un-known factor in the commercial preparation ofhydrolyzed casein did not seem likely. Of theamino acids in Casamino Acids, leucine wasfound to be most effective in shortening the lagperiod.

Recently, Donald Miller of this laboratoryfound that, in the presence of Casamino Acids,the high aspartate concentration of M-II is notneeded but the NaCl concentration must be in-creased. M-V is M-II minus aspartate but withthe addition of 0.05% Casamino Acids and 0.24M NaCl. The compositions of the various stageII media are shown in Table 1. The contributionof the added 0.05% Casamino Acids to thesefigures was calculated. Addition of the D form ofeither glutamic or aspartic acid failed to produceany detectable effects, although their presence inthe cell walls of some bacteria (9, 20) suggestedtheir possible involvement.The course of development of competence in

these media is shown in Fig. 1. The results indi-cate that the lag period is considerably reduced

when the additional amino acids are present andthat the number of competent cells increaseslogarithmically with time, reaching a maximumin a little over 1 hr. This finding was also reportedby Ranhand and Lichstein (17).

This logarithmic rise is probably significant, butwe are unable to interpret it. Perhaps the de-velopment of competence in a synchronized cul-ture would provide more meaningful information.

Kinetics of competence development. The previ-ous study with M-II (18) left the mechanism ofthe development of competence in doubt. Thedata appeared to indicate that development wasdependent on the cell concentration, yet a testfor transmissible factors was negative.With the finding that competence developed

rapidly in M-IV, the effect of cell concentrationon the development of competence was reex-amined (Fig. 2). Development of competence wasexamined at three different cell concentrations,but the levels of competence were measured atone cell concentration for this experiment. Itmay be seen that the rate of development of com-petence at 107, 108, and 109 cells/ml is essentially

TABLE 1. Stage II competence media"

Concn of componentsComponent

M-II M-IV M-V

pg/ml pg/ml pg/mi

L-Aspartic acid. 4,000 4,032 32L-Glutamic acid.................................. 200 314 314L-Arginine....................................... 0 21 21L-Citrulline. 12 12 12Glycine ....................................... 0 2.5 2.5L-Lysine ....................................... 0 35 35L-Methionine .................................... 0 18 18L-Serine ....................................... 30 65 65L-Leucine....................................... 0 61 61L-Tyrosine....................................... 10 42 42L-Histidine ...................................... 0 13 13L-Cystine....................................... 4 6 6L-Phenylalanine .................................. 20 46 46L-Threonine . 0 20 20L-Isoleucine ..................................... 0 33 33L-Valine.. . ....................................0 35 35L-Alanine....................................... 20 48 48L-Proline, hydroxyproline........................0 50 50Fumaric acid .................................... 1,000 1,000 1,000Tween 80....................................... 200 200 200

M M M

NaCl........................................ 0.08 0.08 0.24MgSO4....................................... 5X1 5 X 1(04 5 X 10-4CaC12............................................ 0.001 0.001 0.001KH2PO4-K2HPO4................................ 0.01 0.01 0.01

- Times for maximal competence development inM-IV, 80 to 100 min; M-V, 100 to 120 min.

the three media were as follows: M-II, 180 min;

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520 HERRIOTT, MEY

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30 60 90 120 150

TIME (MINUTES)FIG. 1. Development of competence in various

media. Wild-type H. influenzae strain Rd cells growingin ML, were used to inoculate 25 ml of Ml,. When thecells had grown to approximately 109/ml, they weresedimented and resuspended in 25 ml ofone ofthe stageII media. Samples (2 ml) were quickly pipetted intowarmed test tubes that were placed in a water bath at37 C. At sampling time, 0.1 mlof20;g/mlofDNAfromcells resistant to 2,000 ,g ofstreptomycin/ml was addedto a 2-ml sample and shaken gentlyfor 5 min, after which0.1 ml of 50 ,.g/ml of deoxyribonuclease in 0.1 MMgSO4 was added. After 2 min, samples were diluted ap-propriately andplated in Brain Heart Agar in duplicate.After 90 min of incubation, the plates were coveredwith a layer of 10 ml of Brain Heart Agar containing100 ug of streptomycin sulfate per ml. Incubation for18 to 24 hr was neededfor the colonies to be countable.All data represent transformants, and therefore com-petent cells, in the undiluted stage II medium. Symbols:A, cells in M-II; *, cells in M-IV; 0, cells in M-V.

the same-about a 1 log10 rise per 8 min or adoubling time of approximately 3 min.An analysis of the development of competence

in M-II was undertaken to determine why it wasthought earlier to be concentration-dependent.This analysis showed that, at a low cell concen-tration, the lag period for the development ofcompetence was shorter and competence was lostsooner than at higher cell concentrations. It seemslikely, therefore, that the lower levels of compe-tence reached by the lower concentrations of cellsin the earlier study (18) resulted from a loss in

ER, AND VOGT J. BACraERIOL.

competence after they had reached full compe-tence.The independence of rate of development of

competence from cell concentration is in accord

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TIME (MINUTES)FIG. 2. Effect of cell concentration on development

of competence. Agitation or rotation of a 500-mi flaskcontaining cells in 40 ml of ML, was stopped when thecell concentration reached approximately 10/ml, asjudged by turbidity. The cells were sedimented, washedonce by sedimentation in M-V, and finally resuspendedin 10 ml of M-V. The suspension was then seriallydiluted in M-V to contain 109, 108, and 107 cells per ml.All three suspensions were rotated at 125 rev/min at37 C. At appropriate times, samples were removed andtested for competence after dilution first to 10 cells/mlin M-V. To each 2 ml of cell suspension was added 0.1ml of20 ,g/ml ofDNA from cells resistant to 2,000 ,gofstreptomycin/ml, making thefinaIDNA concentration1 pg/ml. This mixture was agitated for S min and then0.1 ml of5 psg/ml deoxyribonuclease in 0.1 M MgSO4was added. After 2 min, samples were diluted andplated for streptomycin-resistant transformants. Con-trols in which the DNA was predigested with deoxy-ribonuclease for 2 min yielded no colonies on the plates.

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with the previous failure to detect a transmissiblefactor conferring competence (6, 18). Both resultssuggest that development of competence inHaemophilus cells is an internal phenomenon and,therefore, stands in clear contrast to the systemsin pneumococci (12, 21, 22), streptococci (14),and Bacillus subtilis (1).The earlier report (18) that cells from early and

late log phase of growth developed the same levelof competence in stage II was confirmed duringthe present study.

Competence of cells prepared in different media.The competence of cells prepared in differentstage I and stage II media is shown in Table 2.

In general, cells grown in MI, became compe-tent in all stage II media. The competence ofM10-grown cells was consistently lower than thatof cells grown in Heart Infusion. This result sug-gests that Heart Infusion contains a competencefactor. The growth rates in MI0 and Heart Infu-sion were essentially the same, and the concentra-tion of cells used at the initial stage of develop-ment of competence was the same; thus, it appearsthat the factor in Heart Infusion may be specificfor competence. Since doubling the constituentsof MI0, one at a time, did not correct the differ-ence, it is presumed that the factor(s) in HeartInfusion is not present in MI at a suboptimallevel.

Frequency of multiple transformations as ameasure of competence. When cells are fully com-petent and the uptake of one piece ofDNA doesnot interfere with the uptake of other pieces, the

frequency of multiple transformations should bepredictable from the frequencies of the individualtransformations. An earlier study suggested thatthis prediction was valid for Haemophilus (6). Itwas suggested that, since this relationship de-pended on the culture's being fully competent, itprovided a means for determining the fraction ofthe cells in a culture that was competent. Datafrom artificial mixtures of competent and non-competent cultures supported the suggestion.

Since this earlier report, many investigators, in-cluding us (4, 7, 10, 15), have noticed that somevalues of f0, the fraction of the cells which werecompetent, were well above one, so it was clearthat one or more facets of the problem were notunderstood.A contributing factor under some circumstances

was uncovered (18) when it was found that thecompetence of individual cells could vary as muchas 10-fold. This observation suggested that acell in early development might be able to takeup no more than one piece of DNA. Such cellscould not be multiply transformed, and this factwould produce a misleadingly high value off0 .

More significantly, perhaps, Cahn and Fox (4),pointed out that, whereas single marker trans-formations result from either donor strand goinginto the recipient DNA, so that there are twochances of success, double transformations bymarkers on different pieces ofDNA require thatboth marked strands be inserted into the samerecipient strand. This limitation reduces by twothe expected number of double transformants.

TABLE 2. Competence of H. influenzae cells prepared in different mediaa

MediumViable count (cells/ml) Transformants/ml transformants

Stage I Stage II

HPb M-II 9.6 X 108 3.5 X 107 3.6cMI0 M-II 1.3 X 109 5.9 X 10' 0.44

HI M-IV 9.3 X 108 4.6 X 107 5.0MIl M IV 1.3 X 109 3.7 X 107 2.8

HI M-V 8.8 X 108 4.2 X 107 4.8MI, M-V 1.3 X 109 3.6 X 107 2.8

a In these experiments, 35 ml of stage I medium in a 500-ml Erlenmeyer flask was inoculated with wild-type Rd H. influenzae to produce an initial cell concentration of approximately 107/ml. These wererotated at 150 rev/min at 37 C until the cell concentration rose to approximately 8 X 108/ml, after whichthey were sedimented, resuspended in an equal volume of stage II medium, resedimented, and resus-pended in the appropriate stage II medium so that the initial concentration was 109/ml. The flasks werethen rotated at 125 rev/min for 100 min in M-IV, for 120 min in M-V, and for 180 min in M-II, afterwhich 2-ml samples of the cells were mixed with 0.1 ml of 20 ,g/ml of DNA purified from cells resist-ant to 2,000 j,g of streptomycin/ml. After 30 min at 37 C, samples were diluted and plated for transform-ants in the usual manner.

b HI = Difco Heart Infusion plus hemin and NAD.¢ This figure is a mean of 11 separate values from reference 18.

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Porter and Guild (15) have extended this theory,adding other ideas, and have provided experi-mental support for the hypothesis includingtriple transformants. They introduced a factor, q,into the calculation to correct for this effect. Fordouble transformations, q = 2; for triple trans-formations, q = 4. The equation now becomes

fad = Ni,2

where fed is the fraction of the cells capable ofbeing transformed to two markers, N1, N2, andN1,2 are the numbers of transformants to any setof markers arbitrarily designated 1,2, and 1 and2, respectively, and C is the viable-cell concen-tration.

Since some values of fgd reached 5 to 10 (4,10), it is obvious that factors other than q mustbe responsible for this anomaly. As noted earlier,incomplete development of cellular competence,as distinct from cultural competence (fd), Willreduce the number of double transformants,thereby increasing the value off, as will also anystable clumps or chains of the competent cells.It has been reported (10) that competent pneu-mococci seemed to be "stickier" than noncompe-tent ones, and the competent cells showed astrong tendency to aggregate. Haemophilus, un-der the usual conditions of handling, does notexhibit this tendency.Table 3 contains a comparison of the different

means of evaluating competence in cultures, asit develops. Values of fLd are compared with therelative competence obtained as the ratio oftransformants of single or double markers, at anypoint in their development, relative to the trans-formants obtained at full development.

Several conclusions are evident from the resultsin Table 3. The fcd gives a misleading picture ofcompett.ice in the developing culture. The rela-tive frequency of single transformants is probablya more accurate figure. It is also clear that thecapacity of cells to take up DNA and undergotransformation to two markers develops afterdevelopment of that capacity for single markers,because the relative competence as measured bydouble transformations always trails similarmeasurements of single-marker transformants.This result confirms earlier findings (18).

Effect of the medium on transformation. Inprevious studies (3, 6), it was recognized that thecomposition of the medium played a role inDNAuptake by competent H. influenzae cells. The pH,salt concentration, and divalent cations seemedto be critical factors for interaction. Low levels ofTween 80, a commercially available sorbitanoleate, prevented loss of viability of the cells.

Recently, an occasional and elusive variabilityin response of competent cells to the surroundingmedium has been noticed. A 5- to 10-fold drop intransformants has occurred when the cells weremixed with DNA in M-II, or in a 1:10 dilution ofBrain Heart Infusion in 0.1 M saline solution in-stead of in undiluted Brain Heart Infusion. Thisevent has happened several times, yet freshlyprepared competent cells did not show such vari-ations. In general, transformations performed inundiluted Brain Heart Infusion or in MI. re-sulted in the highest frequency of transformation.

Preservation and wash solutions. M-II and MI,have been found to be superior to all other solu-tions for washing and preserving competent cells.Between 25 and 50% of the initial level of com-petence is retained by cells stored for 1 month at-65 C in M-II plus 17% glycerol. At 37 C,competence is retained in M-II for several hours.At 4 C, the competence of cells in M-II is notappreciably altered after 18 hr.Washing or dilution of competent cells in

buffered salts-Tween 80 (BSTw) made up of 0.1M NaCl, 0.02 M pQ4-2 (pH 7), 0.001 M Ca, and0.02% Tween 80 does not lead to a loss in com-petence within 30 min.

DISCUSSIONDefined media for the growth of H. influenzae

and for the development of competence by thecells have been prerequisite to any concertedattack on the metabolic processes contributing tocompetence or to the uptake of DNA. Compe-tence develops best under conditions which in-hibit cell duplication. Thus, high cell concentra-tion and reduced aeration (6) or the absence ofsuch essential growth factors as NAD, panto-thenate, or nucleosides, as in stage II media,promote competence development. Protein syn-thesis is the one macromolecular change that allworkers agree is necessary for the development ofcompetence. Removal of any one of severalamino acids [citrulline (arginine), cystine, orglutamate] from M-II during development bringsthe process to an abrupt halt (M. W. Herriott,unpublished results). In this same medium, valineat 1 ,ug/ml blocked development of competence(18). It is assumed that valine inhibits the leucineor isoleucine synthesis, since addition of eitheramino acid reversed the.inhibition. Chlorampheni-col likewise blocks development of competencewithout killing the cells (13, 18, 19). Similar ef-fects of chloramphenicol on the development ofcompetence have been noted in other bacterialsystems (1, 5).Ranhand and Lichstein (17) reported a peri-

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TABLE 3. Development of competence by cultures in different media(assayed at single and multiple marker level)a

Transformants/ml on medium containing Relative competence,TF(t)/TF(max)'

Stage II Time Cells/mlb fedmedium (min) Strpt y- Both

Streptomycin Erythromycin Both antibiotics mStrepo- mhry- Bothi

M-II 60 0.023 X 106 0.029 X 106 0.023 X 108 5.9 X 108 0.0240.0013 0.0014 0.000190 1.5 X 101 2.3 X 106 6.9 X 103 7.4 X 108 0.35 0.067 0.089 0.023120 9.5 X 106 13 X 106 91 X 108 7.5 X 108 0.9 0.43 0.49 0.30180 24 X 106 28 X 106 320 X 103 8.1 X 108 1.3 1.0 1.0 1.0210 21 X 106 27 X 106 340 X 103 9.6 X 108 0.85 0.74 0.80 0.9

M-IV 60 0.15 X 106 0.09 X 106 0.12 X 103 1.0 X 108 0.55 0.13 0.16 0.04380 0.45 X 106 0.26 X 106 0.47 X 10' 1.0 X 108 1.3 0.38 0.43 0.17100 0.88 X 106 0.48 X 106 1.1 X 10' 1.0 X 108 1.9 0.73 0.83 0.39120 1.1 X 106 0.52 X 106 1.5 X 10' 1.3 X t08 1.5 0.71 0.69 0.43140 1.2 X 106 0.68 X 106 2.7 X 103 1.2 X 108 1.3 0.85 1.0 0.82160 1.4 X 106 0.75 X 106 2.6 X 103 1.5 X 108 1.4 0.7 0.87 0.61180 1.5 X 106 0.75 X 106 3.0 X 108 1.5 X 108 1.2 0.85 0.87 0.72200 1.6 X 106 0.81 X 106 3.6 X 108 1.3 X 108 1.4 1.0 1.0 1.0

M-V 60 0.15 X 106 0.089 X 106 0.16 X 103 0.87 X 108 0.5 0.17 0.20 0.07290 0.53 X 106 0.26 X 106 0.71 X 10' 0.98 X 108 1.0 0.54 0.54 0.29120 0.72 X 106 0.47 X 106 1.5 X 10' 1.3 X 108 0.85 0.55 0.72 0.48140 0.84 X 106 0.48 X 106 1.6 X 103 0.97 X 108 1.3 0.88 1.0 0.69170 1.1 X 106 0.51 X 106 2.7 X 10' 1.1 X 108 0.95 1.0 0.92 1.0200 1.2 X 106 0.54 X 106 2.2 X 103 1.3 X 108 1.15 0.93 0.84 0.69

aThe details of the experiment with medium M-1I are similar to those reported in reference 18. Thedata were taken from H. T. Spencer (Sc.D. Thesis, Johns Hopkins School of Hygiene and Public Health, Baltl-more, Md., 1964). The initial cell concentration was 6 X 10s/ml. Cells were exposed to 1 jsg of DNA per ml for40 min, after which deoxyribonuclease and Mg++ were added to destroy unabsorbed DNA. For the other twoexperiments, cells were grown in Heart Infusion to 109/ml, centrifuged, and resuspended in M-IV or M-V.Another centrifugation and resuspension in M-IV or M-V at a concentration of 109/ml completed thepreparation. Samples (0.5 ml) were removed from the suspension, agitated at 37 C for predeterminedperiods, and diluted into 4.5 ml of Brain Heart Infusion (BHI) containing 0.3 ,ug of a mixture of DNAfrom streptomycin-resistant cells and DNA from erythromycin-resistant cells per ml. Incubation of thetransformation mixture was stopped after 5 min, and deoxyribonuclease and Mg+ were added to bringthe levels to 1 ,g/ml and 5 mm, respectively. Samples were then diluted appropriately in BHI-saline(1: 10), and known volumes were placed in six petri dishes and mixed with warm BHI Agar. After 1.5 to2 hr, two plates each were covered with layers of BHI Agar containing 100,g of streptomycin per ml,20,ug of erythromycin per ml, or both antibiotics at the above concentrations. Colony counts were madeafter 24 hr of incubation of the plates at 37 C.

b Other experiments have not shown the large increase in viable count in M-II suggested by the lastfigure, 9.6.

c TF(t) is the number of transformants at time t. TF(max) is the number of transformants at thehighest level.

odate-sensitive structure or function in H. in-fluenzae similar to that detected in B. subtilis(14a). This structure is not essential for viabilitybut is critical for interaction with DNA. A cor-relation of inosine uptake during growth withincreased ribonucleic content of the cells wasalso observed (J. M. Ranhand, Ph.D. Thesis,Univ. of Cincinnati, Cincinnati, Ohio, 1968).The present results support the earlier evidence

(6, 18) against the involvement of a macromolecu-lar transmissible comoetence factor in the Hae-

mophilus system. Barnhart's (2) factor appears tobe small and may be nutritional in nature.The need for 0.24 M NaCl in M-V to replace the

4 mg of aspartate per ml in M-II or M-IV wasunexpected. It is quite interesting that this isnearly the same level of chloride which Kohoutova(11) found necessary to raise the second cycle ofdevelopment of competence in pneumococci to alevel approaching that observed in the first cycle.At low cell concentrations, competence di-

minishes rapidly after reaching its peak (see Fig.

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2), and as the cell concentration increases the rateof deterioration decreases. Two explanations willaccount for this finding. The medium may containa low level of a destabilizing substance, such as agrowth stimulant, which is taken up by low con-centrations of cells but is not present in sufficientquantity to affect high concentrations of cells.The other explanation is that cells secrete a smallamount of stabilizing substance which increasesas the cell concentration increases.In the light of what we now know about the

calculation of competence from the frequencies ofmultiple transformations, it seems likely that thecultures analyzed previously (6) were not morethan 50% competent. The close correspondencebetween observed and calculated multiple trans-formants was due, in part, to corrections intro-duced to compensate for the loss of recipient cellmarkers through the displacement by the corre-sponding sensitive markers introduced on thedonor DNA.

ACKNOWLEDGMENTS

We are grateful to Sue Ellen Wolcott for her competent assistance in some of the experiments.

This investigation was supported by Public Health Servicetraining grant 5-TI-GM-73 from the National Institute GeneralMedical Sciences, by Public Health Service research grant Al-01218 from the National Institute of Allergy and InfectiousDiseases, and by Atomic Energy Commission contract AT(30-1)-1371 (NYO-1371-59).

LITERATURE CITED

1. Akrigg, A., S. R. Ayad, and G. R. Barker. 1967. The nature ofa competence-inducing factor in Bacillus subtilis. Biochem.Biophys. Res. Commun. 28:1062-1067.

2. Barnhart, B. J. 1967. Competence stimulating activity in sterilefiltrates from Hemophilus in,fluenzae. Biochim. Biophys.Acta 142:465.

3. Barnhart, B. J., and R. M. Herriott. 1963. Penetration ofdeoxyribonucleic acid into Hemophilus intfluenzae. Biochim.Biophys. Acta 76:25-39.

4. Cahn, F. H., and M. S. Fox. 1968. Fractionation of trans-formable bacteria from competent cultures of Bacillussubtilis on Renografin gradients. J. Bacteriol. 95:867-875.

5. Fox, M. S., and R. D. Hotchkiss. 1957. Initiation of bacterialtransformation. Nature (London) 179:1322-1325.

6. Goodgal, S. H, and R. M. Herriott. 1961. Studies on trans-formations of Hemophilus influenzae. I. Competence. J.Gen. Physiol. 44:1201-1227.

7. Hadden, C., and E. W. Nester. 1968. Purification ofcompetentcells in the Bacillus subtilis transformation system. J.Bacteriol. 95:876-885.

8. Herriott, R. M., E. Y. Meyer, M. Vogt, and M. Modan. 1970.Defined medium for growth of Haemophilus influenzae.J. Bacteriol. 101:513-516.

9. Ikawa, M., and E. E. Snell, 1960. Cell wall composition ofbacteria. J. Biol. Chem. 235:1376-1382.

10. Javor, G. T., and A. Tomasz. 1968. An autoradiographicstudy of genetic transformation. Proc. Nat. Acad. Sci.U.S.A. 60:1216-1222.

11. Kohoutova, M. 1965. Infection of the recipient cell by trans-forming DNA. The stimulation and inhibition of infection.Symp. Biol. Hung. 6:65-72.

12. Kohoutova, M., H. Brana, and I. Holubova. 1968. Isolationand purification of a substance inducing competence andinactivating transforming DNA in pneumococcus. Biochem.Biophys. Res. Commun. 30:124-129.

13. Leidy, G., J. Jaffee, and H. E. Alexander. 1962. Emergence ofcompetence (for transformation) of three Hemophilus spe-cies in chemically defined environment. Proc. Soc. Expt.Biol. Med. 111:725-731.

13a. Leonard, C. G., D. C. Corley, and R. M. Cole. 1967. Trans-formation of streptococci in chemically defined media.Biochem. Biophys. Res. Commun. 26:181-186.

14. Pakula, R., and W. Walczak. 1963. On the nature of compe-tence of transformable streptococci. J. Gen. Microbiol.31:125-133.

14a. Polsinelli, M., and S. Bariati. 1967. Effect of periodate oncompetence in Bacillus subtilis. J. Gen. Microbiol. 49:267-275.

15. Porter, R. D., and W. R. Guild. 1969. Number of transforma-ble units per cell in Diplococcus pneumoniae. J. Bacteriol.97:1033-1035.

16. Ranhand, J. M., and R. M. Herriott. 1966. Inosine and lactate:factors critical during growth for development of compe-tence in Haemophilus influenzae. Biochem. Biophys. Res.Commun. 22:591-596.

17. Ranhand, J. M., and H. C. Lichstein. 1966. Periodate inhibi-tion of transformation and competence development inHaemophilus influenzae. J. Bacteriol. 92:956-959.

18. Spencer, H. T., and R. M. Herriott. 1965. Development ofcompetence of Haemophilus influenzae. J. Bacteriol. 90:911-920.

19. Stuy, J. 1962. Transformability of Haemophilus influenzae.J. Gen. Microbiol. 29:537-549.

20. Toennies, G., B. Bakay, and G. D. Shockman. 1959. Bacterialcomposition and growth phase. J. Biol. Chem. 234:3269-3275.

21. Tomasz, A., and R. D. Hotchkiss. 1964. Regulation of thetransformability of pneumococcal cultures by macromolecu-lar cell products. Proc. Nat. Acad. Sci. U.S.A. 51:480-487.

22. Tomasz, A., and J. L. Mosser. 1966. On the nature of thepneumococcal activator substance. Proc. Nat. Acad. Sci.U.S.A. 55:58.

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