JOURNAL OF BACTERIOLOGY, Jan. 1976, p. 282-289Copyright © 1976 American Society for Microbiolog-Y

Vol. 125, No. 1Printed in U.S.A.

Isolation and Description of a Menaquinone Mutant fromBacillus licheniformis

STEVEN R. GOODMAN, BARRY L. MARRS, ROMAN J. NARCONIS, AND ROBERT E. OLSON*Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104

Received for publication 6 October 1975

A menaquinone mutant (SG1) of Bacillus licheniformis has been isolated byselecting for colonies that are resistant to low levels of kanamycin (1.5 ,g/ml) butsensitive to the same concentration of kanamycin in the presence of shikimate(25 jg/ml). The wild type (IU1) contained 0.38 + 0.02 nmol of menaquinone-7(MK-7) per mg (dry weight) of cells when grown ± shikimate, whereas SG1 had<0.01 nmol of MK-7 per mg (dry weight) of cells when grown without shikimateand 0.30 ± 0.02 nmol of MK-7 per mg (dry weight) of cells when grown in thepresence of shikimate. SG1 had a generation time of 85 min, as compared to 24min for IUl grown ± shikimate. SG1 doubled with a generation time of 28 minwhen grown in the presence of shikimate. IUl consumed 02 at various ratesdepending on the stage of growth. A triphasic 02 consumption curve withmaxima at mid-exponential phase, the transition from exponential to stationaryphase, and early stationary phase was found for IUl ± shikimate and SG1 +shikimate. SG1 grown without shikimate consumed 02 at a low level (10 to 20% ofIUI). Normal respiration could be restored to SG1 8.5 min after shikimateaddition, whereas normal growth was not restored until 40 min after shikimateaddition. Electron microscopic studies of SG1 and IUl have indicated amorphological alteration in the mutant. SG1 is a dwarf cell as compared toIUI, when grown without shikimate. However, SG1 grown with shikimatebecame morphologically indistinguishable from IUl.

It has been established that vitamin K2 is acomponent of the electron transport chain inmany bacterial species. Other functions havebeen reported, such as involvement in pyrimi-dine synthesis in Escherichia coli (15), sphingo-lipid synthesis in Bacteroides melaninogenicus(13), and the ability to regulate sporulation inBacillus subtilis (19). Bacillus licheniformis isan organism which has well-characterizedmembranes, contains menaquinone as the solequinone, and possesses the ability to grow aiiaer-obically. We undertook studies on this organismto determine whether menaquinone might playa role in determining membrane composition inaddition to serving as an electron carrier. Tothis end we have isolated mutants of B.licheniformis deficient in menaquinone biosyn-thesis and report on the isolation, growth, 02consumption, and cell morphology of one suchauxotroph.

(This work was taken in part from a disserta-tion submitted by S. R. G. to St. Louis Uni-versity, St. Louis, Mo., 1976, in partial fulfill-ment of the requirements for the Ph.D. degree.)

MATERIALS AND METHODSBacterial strains. A wild-type B. licheniformis

(IU1) was received from H. Gest. Our menaquinone-deficient mutant (SG1) was isolated as describedbelow.

Chemicals. All organic solvents used were reagentgrade, except for the spectral-grade hexane which wasused for ultraviolet (UV) spectra and was purchasedfrom EM Laboratories. The menaquinone homologues(MK-2 to MK-10) used as markers were the gift ofHoffmann-La Roche, Basel, Switzerland. Shikimateand kanamycin sulfate were purchased from SigmaChemical Co., and ethyl methane sulfonate waspurchased from Eastman Chemicals. The tryptose,beef extract, and tryptose blood agar base (TBAB)were purchased from Difco. Thin-layer chromatogra-phy (TLC) plates (0.25 mm) without fluorescentindicator were purchased from Brinkmann Instru-ments.Media. The minimal salts medium (SMM) was

that of Anagnostopoulos and Spizizen (1). The richmedium (TMG) contained, per liter: 10 g of tryptose,5 g of NaCl, and 3 g of beef extract. Enough sterileglucose was added to give a final concentration of0.25% glucose. The solid equivalent to TMG mediais TBAB, which was purchased as premixed packs.Rich medium (TBABG) plates were made by adding33 g of TBAB (Difco) to 1 liter of water. Glucose wasadded to a final concentration of 0.25% after auto-claving.

Isolation of mutants. JUl was grown to 80 Klettunits in 20 ml ofTMG at 37 C and harvested at 10,000

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MENAQUINONE MUTANT FROM B. LICHENIFORMIS 283

rpm for 8 min, and the cells were suspended in 2 ml of50 mM tris(hydroxymethyl)aminomethane, pH 7.2.The cell suspension was mixed with 8 ml of 3% ethylmethane sulfonate in 50 mM tris(hydroxymethyl)-aminomethane, pH 7.2. This mixture was incubatedwith shaking at 37 C for 12 min, at which time.99.9%of the cells were killed. The cells were spun downand washed 3x with 20 ml of SMM, suspended in 2ml of SMM, and used to inoculate 18 ml of TMG.These cells were then incubated at 37 C for 6 h. Thistreatment resulted in a 700-fold increase in thefrequency of mutants resistant to 1.5 ug of kanamycinsulfate per ml.

Kanamycin-resistant colonies were replica platedonto TBABG, TBABG + shikimate (25 #g/ml),TBABG + kanamycin (1.5 Ag/ml), and TBABG +kanamycin (1.5 ug/ml) + shikimate (25 Ag/ml). Thosecolonies that grew on TBABG, TBABG + kanamycin,and TBABG + shikimate, but would not grown onTBABG + kanamycin + shikimate, were selected forfurther study.

Growth. The rich medium supports growth bothof wild-type (IU1) and men- (SG1) mutants at 37 C.Since SG1 is blocked prior to shikimate in themenaquinone synthetic pathway, the rich mediummust supply the other end products derived from thecommon aromatic pathway (aromatic amino acidsand p-aminobenzoic acid). Cells were grown at 37 Cin 10 to 30 ml of rich medium contained in 250-mlnephelometer flasks. Cell growth was determinedturbidimetrically using a Klett-Summerson colorime-ter equipped with a red filter. Both SG1 and IUl werealways pregrown in 20 ml ofTMG media + shikimate(25 jg/ml) to 80 Klett units. The cells were harvestedat 10,000 rpm for 8 min in a Sorvall centrifuge (10 C)and washed 3 x with 10 ml of SMM. The washed cellswere resuspended in 20 ml of SMM, and 0.5 ml wasused to inoculate 30 ml of TMG without shikimateand 30 ml of TMG with shikimate (25 gg/ml). Thecells were allowed to grow with shaking at 37 C.Assays for menaquinone-7 (MK-7). IU1 and SG1

strains, which were pregrown in 20 ml of TMG +shikimate, were harvested, washed 3x with 10 ml ofSMM, and suspended in 10 ml of SMM. This cellsuspension (2 ml) was used to inoculate 500 ml offresh TMG media in a 4-liter Erlenmeyer flask. Atotal of 1 liter of cells grown to 1 h into stationaryphase was used for each menaquinone determination.The cells were harvested, washed with 20 ml of SMM,and then freeze dried to a constant weight. Theresulting powder was homogenized with 20 ml ofacetone, and then an additional 80 ml of acetone wasadded and the extraction was allowed to proceed at4 C for 6 h. The mixture was centrifuged at 10,000rpm for 10 min, and the supernatant fluid was filteredto remove any remaining cell debris. The acetone wasremoved on a rotary evaporator, and the acetone-extractable lipids were dissolved in 10 ml of diethylether. The diethyl ether was removed under N2, andthe lipid was dissolved in 0.5 ml of methylene chlorideand spotted on a TLC plate along with marker MK-7.In a developing system of isooctane-diethyl ether(100:30, vol/vol), the vitamin K2 migrates to an R, of0.52. The spot was visualized with a UV lamp,scraped from the plate, and extracted in diethyl ether.

The silica gel was removed by low-speed centrifuga-tion, and the supernatant fluid was decanted andevaporated under N2. The vitamin K2 was dissolvedin 3 ml of spectral-grade hexane, and the UV spectrumfrom 200 to 320 nm was obtained in a Beckman DBspectrophotometer. An ether extract of scrapings ofthe TLC plate above the solvent front was used as ablank. The extinction coefficient at the 248-nm ab-sorption maximum was 1.9 x 104 liters/mol per cm,and this value was used to quantitate MK-7 concen-tration. In control experiments in which radioactivevitamin K was added to the acetone extract and thentaken through all the described steps, 95 to 97% of theradioactivity was recovered.Oxygen consumption. Measurement of 02 con-

sumption was carried out at 37 C using a Clark-typeoxygen electrode (Yellow Springs Instruments Co.).

Electron microscopy. IUl and SG1 cells, whichwere pregrown and washed as described above, wereused to inoculate fresh TMG media + shikimate (25Ag/ml). The cells were grown at 37 C to mid-exponen-tial growth stage (100 Klett units), harvested, andfixed at 4 C for 45 min in a solution of 30 mM glucose,0.15 M sodium phosphate buffer, pH 7.3, and 0.45mM CaCl2, containing 5% glutaraldehyde. The fixedcells were washed four times with the glucose-phos-phate-CaCl2 solution, postfixed at 4 C for 45 min inthe same solution containing 2% OSO4, and washedonce with the glucose-phosphate-CaCl2 solution. Thefixed cells were ethyl alcohol dehydrated, embeddedin epon-araldite embedding resin, cut on an LKB-Huxley ultramicrotome, and double stained withuranylacetate and lead citrate. Micrographs weretaken in a Philips 300 electron microscope at 60 kV.

RESULTSAnalysis of menaquinone from B. licheni-

formis. Salton and Schmitt (16) have suggestedMK-7 to be the homologue present in B.licheniformis, but their criterion was comigra-tion with a standard on thin-layer plates devel-oped in benzene or isooctane-diethyl ether(100:30, vol/vol), where one observes very poorresolution between MK-6, -7, and -8. Therefore,we extracted the UV-absorbing band that comi-grates with MK-7 in isooctane-diethyl ether(100:30) at R, = 0.52 (Fig. 1) for analysis onparaffin-impregnated plates, which were devel-oped in acetone-water (92:8, vol/vol) (7). Thissystem gives clear resolution of the menaqui-nones, in particular MK-4 through -9. It can beclearly seen that vitamin K2 from B.licheniformis comigrates with MK-7 at Rf =0.36.Mass spectrometry. The mass spectra of

both the bacterial and authentic MK-7 areshown in Fig. 2. Each spectrum shows themolecular ion at m/e 648 and a base peak atm/e 69 characteristic of menaquinones (5).Peaks at m/e 579, 511, 443, 375, 307, and 239represent the loss of one terminal (69 massunits) and five internal (68 mass units) isoprene

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284 GOODMAN ET AL.

groups, respectively. The peak at m/e 225corresponds to the chromylium ion formed frommenaquinones by cyclization of the first iso-

Reverse Phase TLCAcetone / H2092/8 (Vol /Vol)

Straight Phase TLCIsoOctane/Diethyl Ether

100/30 (Vol /Vol)

Menaquinone Wild MK-7(2-10) Type

ExtractFIG. 1. TLC of menaquinones. The cold acetone

extract of 1 liter of cells grown into stationary phase(T1) was spotted on a TLC plate along with MK-7marker and developed in isooctane-diethyl ether(100:30, vol/vol). The UV-absorbing band that comi-grates with MK-7 was extracted from the plate withdiethyl ether and rerun on a paraffin-impregnatedplate in a developing system of acetone-water (92:8,vol/vol) with menaquinone markers (MK-2 to MK-7).

prene unit and loss of the remainder of theside chain (6). The mass spectral data indicatefurther that the MK-7 from B. licheniformishas a normal isoprenyl side chain.

Isolation of vitamin K2 auxotrophs. Amino-glycoside antibiotic resistance has been widelyused to select for mutants blocked in someaspect of energy metabolism (8, 9, 11, 12, 14,17-19). It has been suggested that these energymetabolism auxotrophs are unable to activelytransport the aminoglycoside across their mem-brane (8).We have sought menaquinone-deficient mu-

tants among colonies resistant to 1.5 ug ofkanamycin sulfate per ml. Only a small per-centage of kanamycin-resistant mutants aremenaquinone auxotrophs, and therefore furtherscreening was necessary. If a cell is blocked inmenaquinone synthesis and is kanamycin re-sistant because of this block, then one should beable to make that cell kanamycin sensitive byadding back an intermediate after the block,thereby allowing menaquinone synthesis andkanamycin transport. This is the rationale be-hind the replica-plating technique described inMaterials and Methods.Using this procedure, we have isolated several

shikimate auxotrophs, and the phenotype of onesuch mutant is described in Table 1. In this andall other experiments to be described, IU1 andSG1 are always pregrown in the presence ofshikimate (25 gg/ml) and then harvested andwashed 3x with 10 ml of SMM. Samples of cellsuspensions were used to inoculate two cultures,one containing TMG and the other containing

B4TERIAL MENAOUINONE-7Dwe Pube, W0C 70ev

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AUTHENTIC MENAQUNONE-7Dired Probe, KX0C,70v

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307 373 443 Sit 573 646

280 320 360 400 440 440 520 560 600 640m/e

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FIG. 2. Mass spectra of bacterial and authentic menaquinone. Mass spectra of menaquinone isolated by TLCof a cold acetone extract of B. licheniformis were run at 100 C, with an electron impact of 70 eV, using the directprobe.

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MENAQUINONE MUTANT FROM B. LICHENIFORMIS 285

TABLE 1. Growth of shikimate auxotroph on TBABG platea

Growth on TBABG plates (CFU/Klett unit)

Mutant No addition +Shikimate +Kanamycin +Kanamycin+shikimate

Wild type 510,000 (±+40,000) 530,000 (±+20,000) 7 (±1) 11 (±i 1)

Wild type + shiki- 500,000 (±40,000) 510,000 (-20,000) 14 (±1) 8 (±2)mate

Men- 920,000 (±30,000) 970,000 (±40,000) 900,000 (±30,000) 750 (+50)

Men- + shikimate 500,000 (±20,000) 490,000 (±40,000) 498 (±14) 456 (±15)

a Wild type (IU1) and Men- (SG1) were grown + shikimate (25 Ag/ml) to 100 Klett units in TMG media at37 C. The cells were then plated on TBABG plates, with shikimate (25 ug/ml) and kanamycin (1.5 yg/ml)added in conjunction and separately, and the plates were allowed to incubate for 15 h at 37 C before colonieswere counted. Values presented are the mean of triplicate plates. Numbers in parentheses represent the range ofthree determinations. CFU, Colony-forming units.

TMG + shikimate (25 jg/ml), to give an initialcell density of 1 Klett unit. In the experimentdescribed in Table 1, the cells were allowed togrow at 37 C to 100 Klett units and then were

plated. The data in Table 1 show that: (i) IU1 isunaffected by having shikimate present ineither the liquid or solid media; (ii) IU1 issensitive to kanamycin (1.5 ug/ml); (iii) SG1 isresistant to kanamycin (1.5 Ag/ml); (iv) SG1 issensitive to kanamycin (1.5 jg/ml) in the pres-ence of shikimate (25 jug/ml), but is not killedby shikimate (25 ,g/ml) alone; (v) SG1, whengrown with shikimate (25 ug/ml) in the liquidmedium, behaves in a manner similar to MUl;(vi) there is -1.8 times as many colony-formingunits per Klett unit for SG1 grown withoutshikimate as compared to IU1 i shikimate orSG1 + shikimate.

Results (i) through (v) suggest that SG1 is a

menaquinone auxotroph blocked prior to shiki-mate in the synthetic pathway, whereas (vi)suggests that a morphological alteration alsoexists in SG1.MK-7 content of IUI and SG1. The concen-

tration of MK-7 in IU1 and SG1 grown +shikimate is presented in Table 2. IU1 grownwith or without shikimate had -0.38 nmol ofvitamin K2 per mg (dry weight) of cells. How-ever, for SG1 grown without shikimate, therewas no detectable menaquinone in an acetoneextract of cells derived from a 1-liter cultureharvested 1 h into stationary phase. There was

no spot comigrating with MK-7 on TLC viewedwith a UV lamp, and, when the region whereMK-7 should run was scraped from the plateand eluted, no vitamin K2 spectra were ob-served on scanning the UV range (200 to 320nm) with a Beckman DB spectrometer. The

TABLE 2. MK-7 in IUI and SG1 grown i shikimatea

Mutant ~~MK-7 (nmol/mgMutant [dry wt] of cells)

Wild type ...................... 0.38 + 0.02 (5)Wild type + shikimate (25

'Ug/ml) ...................... 0.37 ± 0.02 (3)Men . ....................... <0.01 (4)Men- + shikimate (25 Ag/

ml) ....................... 0.30 ± 0.02 (3)

a One-liter batches of cells were grown at 37 C to 1 hinto stationary phase. The cells are harvested,washed, freeze dried, and then acetone extracted. Thevitamin K2 was obtained as described in Materialsand Methods. Wild type (IU1) ± shikimate and Men-(SG1) + shikimate yield 155 ± 10 mg (dry weight) ofcells per liter of culture grown to stationary phase.Men- (SG1) yield 120 ± 10 mg (dry weight) of cellsper liter of culture grown to stationary phase. Num-bers in parentheses represent number of determina-tions.

limit of accurate detection using this techniqueis 0.01 optical density units at 249 nm becauseof trace UV-absorbing materials endemic tocommercially available TLC plates. Therefore,we can assign a value of <0.01 nmol of MK-7per mg (dry weight) of cells for SG1. When SG1is grown with 25 jg of shikimate per ml presentin the liquid medium, the MK-7 concentrationis -0.30 nmol of MK/mg of cells, indicating ablock prior to shikimate in the menaquinonesynthetic pathway of SG1.Growth and 02 consumption. Cultures for

02 consumption and growth measurementswere pregrown in the presence of shikimate,suspended in TMG i shikimate as described inMaterials and Methods, and then allowed togrow with shaking at 37 C (Fig. 3a and b).

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GROWTH AT 370 C

TIME IN HOURS

FIG. 3. (a) Growth curves for wild type and Men-mutant of B. licheniformis. Men- (SG1) and wildtype (IUI) were pregrown in 20 ml of TMG +shikimate (25 tsg/ml) to 80 Klett units and then were

harvested, washed 3x with SMM (10 ml), and resus-

pended in 20 ml of SMM. Enough cells (0.5 ml) were

added to 30 ml of TMG shikimate (25 ug/ml) to givean initial Klett reading of 1. Cells were allowed togrow at 37 C with readings taken on a Klett-Summer-son colorimeter every 0.5 h. (b) 02 consumption ofwild type (IUI) and men- (SG1) mutants of B.licheniformis as a function of time.

The growth curve (Fig. 3a) for IU1 was

unaffected by the presence of 25 Ag of shikimateper ml. IU1 had a generation time of 24 min andgrew to a final cell density of "290 Klett units.SG1 grew just like IU1 for the first hour ofshikimate starvation, and it was not until 1.5 hthat we observed a deviation in growth (at thistime, the cell density had increased from 1 to 6Klett units). From this point to the onset ofstationary phase, the mutant doubled, with a

generation time of 85 min. SG1 reached a finalcell density of -120 Klett units; however, itshould be remembered that the number ofcolony-forming units per Klett unit was 1.8times greater in SG1 than in IUL. SG1 grown inthe presence of shikimate had a growth curveresembling IU1, with a generation time of 28min and a final cell density of -290 Klett units.The oxygen consumption curves for this ex-

periment are shown in Fig. 3b. IU1 was unaf-fected by the presence of shikimate, and atriphasic 02 consumption curve was observed,with maxima at the mid-exponential growthphase (1.7 h), the transition from exponential tostationary phase (3.7 h), and early stationaryphase (7.5 h). During the 11-h growth periodstudied, IU1 varied from a maximum 02 con-sumption of 0.52 ,ug-atom of 0/min per Klettunit to a minimum of 0.24 Ag-atom of 0/min perKlett unit. SG1 02 consumption was similar tothat of IU1 until 1.5 h of shikimate starvation ina manner similar to growth. At 5.5 h (60 Klettunits), the 02 consumption began to slowlydecrease towards a constant value of 0.05 gg-atom of 0/min per Klett unit, a value which is10 to 20% of the values observed for IU1 overthe same growth period. SG1 grown in the pres-ence of shikimate had an 02 consumptioncurve resembling the triphasic curve of IUL.To observe the kinetics of recovery from

menaquinone starvation, SG1 was pregrown inTMG + shikimate (25 ,g/ml) and then har-vested and washed; enough cells were added to30 ml of fresh TMG to give a Klett reading of 1Klett unit. These cells, starved for shikimate,were allowed to grow to 20 Klett units, at whichtime the culture was split and one-half receivedenough shikimate in 25 gl of water to give a finalconcentration of 25 ug/ml, whereas the otherhalf received only 25 ul of sterile water. Theresults shown in Fig. 4 indicate that the portionof the culture receiving only water continuedgrowing unaffected, with a generation time of 85min. The portion receiving shikimate was unaf-fected for the first 10 min, showed increasedgrowth at 20 min, and began its maximalgrowth rate (generation time, 28 min) at 40 min.In a parallel study, TUl was unaffected byshikimate addition (data not shown).When the same cells that had reacted 20

Klett units during shikimate starvation wereplaced in an 02 electrode chamber, they con-sumed 02 at a steady low level (0.12 ,g-atomof 0/min per Klett unit). This low rate of 02consumption was only slightly affected by addi-tion of 5 ,l of sterile water. But addition ofenough shikimate in 5 ;il of water to give a finalconcentration of 25 ug/ml gave a large and rapid

TIME IN HOURS

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MENAQUINONE MUTANT FROM B. LICHENIFORMIS 287

effect (Fig. 4). Increased 02 consumption wasseen 2 min after shikimate addition, and amaximal level of 0 2 consumption (0.34 ,ug-atom of 0/min per Klett unit) was reached in 8.5min. Therefore, maximal respiration was re-sumed 8.5 min after shikimate addition,whereas normal growth was not resumed until40 min. In a parallel study, 02 consumption ofIU1 was found to be unaffected by shikimate(data not shown).Electron microscopy. Electron microscopic

studies of SG1 and IU1 i shikimate revealedthat the SG1 mutant is a dwarf cell withdimensions of 1.4 to 1.6 ,um (length) and -0.5,lm (width) (Fig. 5). IUl 4 shikimate and SG1+ shikimate are large cells with dimensions of2.5 to 2.9 um (length) and 0.6 um (width). Theseresults, giving the relative sizes of cells har-vested at 100 Klett units, are consistent withour results shown in Table 1 which indicatedthat there were 1.8 times as many colony-form-ing units per Klett unit for SG1 as for IU1 ishikimate or SG1 + shikimate.

DISCUSSIONThe first menaquinone auxotrophs in B.

licheniformis have been isolated by selecting forcolonies resistant to low levels of kanamycin(1.5 ug/ml) but sensitive to kanamycin (1.5tg/ml) in the presence of shikimate (25,gg/ml).This is a technique which should have generalapplication for the isolation of auxotrophsblocked elsewhere in the menaquinone biosyn-thetic pathway, simply by varying the interme-diate used in replica plating.Mass spectral analysis has indicated that

MK-7 is the vitamin K2 homologue present inB. licheniformis. The concentration of MK-7 inIUl B. licheniformis was found to be 0.38 -fi 0.02nmol/mg (dry weight) of cells, and this MK-7concentration was unaffected by the presence of25 ,ug of shikimate per ml. SG1 was found tocontain <0.01 nmol of MK-7 per mg (dryweight) of cells when grown without shikimateand 0.30 + 0.02 nmol of MK-7 per mg (dryweight) of cells when grown in the presence of 25Ag of shikimate per ml.02 consumption for IU1 followed a triphasic

curve, with maxima at mid-exponential growthphase, the transition from exponential to sta-tionary phase, and early stationary phase. Simi-larly, Farrand and Taber (9) found two maximafor B. subtilis in mid-exponential and transitiongrowth stages, corresponding to our first twomaxima; they did not report on the consump-tion of stationary-phase cultures. SG1 whenstarved for shikimate has less than 3% of theJUl level of vitamin K,, but still consumes 02 at

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10 .10-10 0 10 20 30 40 50 60 70 80

TIME IN MINUTES

FIG. 4. Restoration of normal 02 consumption andgrowth after addition of shikimate to SG1. Men-(SG1) was pregrown in 20 ml of TMG + shikimate (25Ag/ml). The cells were harvested, washed, and reinoc-ulated into 30 ml of fresh TMG without shikimate asdescribed in Materials and Methods to an initialKlett reading of 1. The cells were allowed to grow to 20Klett units in the absence of shikimate, and then theculture was split, with one-half receiving 25 gl ofenough shikimate to give a final concentration of 25ug/ml and the other half receiving 25 ul of sterilewater. Klett readings were taken every 10 min. 02consumption of men- (SG1) grown to 20 Klett unitswithout shikimate was followed polarigraphically be-fore and after addition of 5 Ail of enough shikimate togive a final concentration of 25 Mg/ml. A controlreceived 5 MI of sterile water.

10 to 20% of the IU1 rate. There are twoexplanations for this residual 02 consumption:(i) B. licheniformis can carry on a low level ofrespiration independent of menaquinone and/or(ii) trace amounts of MK-7 can support this lowlevel of 02 consumption. In support of the firstexplanation, a vitamin K-independent oxidase,which has an artificial bypass from reducednicotinamide adenine dinucleotide dehydrogen-ase to the termihal oxidase, has already beensuggested (3) to explain residual reduced nico-tinamide adenine dinucleotide oxidase activity.This activity is not destroyed by irradiation orlipid extraction techniques, which reduce men-aquinone in Mycobacterium phlei (3) andMicrococcus lysodeikticus (10) to undetectablelevels. In addition, succinoxidase has beenfound not to have a direct quinone requirementin several bacterial species (2, 9). Therefore, it isreasonable that our SG1 mutant might carry ona low level of respiration in the absence ofmenaquinone, although a contribution fromminute levels of MK-7 cannot be ruled out atpresent.Of great interest was the fact that there is a

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288 GOODMAN ET AL.

..i. A

A Men + ShikimateB=Men-

B C= Wild TypeD= Wild Type +Shikimate

lii 2p 3p

C

D

FIG. 5. Electron micrographs of men- (SG1) and wild type (IUI) grown + shikimate (25 ug/ml). Techniquesare described in Materials and Methods.

lag period of about 32 min between the times forrestoration of normal respiration and normalgrowth after shikimate addition to SG1.Clearly, restoration of normal respiration is notsufficient to restore the IU1 phenotype to SG1.Some other changes either directly or indirectlyrelated to the menaquinone mutation mustoccur during this lag period before normalgrowth can be restored.

Finally, we have described a morphologicalalteration related to the menaquinone muta-tion. SG1 is a dwarf cell as compared to IU1 +shikimate and SG1 + shikimate. The questionof whether this morphological alteration is adirect effect of the menaquinone mutation or a

secondary effect due to altered respiration or

growth is currently under investigation by tak-ing advantage of the fact that B. licheniformis isa facultative anaerobe.

ACKNOWLEDGMENT

This work was supported by Public Health Service grantAM-09992 from the National Institute of Arthritis, Metabo-lism, and Digestive Diseases.

LITERATURE CITED

1. Anagnostopoulos, C., and J. Spizizen. 1961. Require-ments for transformation in Bacillus subtilis. J. Bacte-riol. 81:741-746.

2. Brodie, A. F. 1966. Oxidative phosphorylation in fraction-ated bacterial systems. XXII. The effect of nearultraviolet irradiation on the succinate oxidase path-way of Mycobacterium phlei. J. Biol. Chem.241:4016-4022.

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MENAQUINONE MUTANT FROM B. LICHENIFORMIS 289

3. Brodie, A. F., and J. Ballantine. 1960. Oxidative phos-phorylation in fractionated bacterial systems. II. Therole of vitamin K. J. Biol. Chem. 235:226-231.

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