LARGE CELL STAGE IN THE GENUS BACILLUS

ROY M. JOHNSONLife Science Division, Arizona State University, Tempe, Arizona

Received for publication March 6, 1961

ABSTRACT

JOHNSON, Roy M. (Arizona State University,Tempe). Large cell stage in the genus Bacillus.J. Bacteriol. 82:418-424. 1961.-Synchronouscultures of Bacillus polymyxa and Bacillus cereusproduced a transitory large cell stage with in-creased nuclear material concomitant with a dropin viable count during growth in a defined me-dium. A variant of B. cereus that required noorganic nitrogen for growth and sporulation wasinhibited by serine and this inhibition reversedby equimolar amounts of threonine. Serine inhibi-tion occurred before the large cell stage but notafter. The significance of this large cell stage asregards transformation studies, nucleic acidanalysis, and sporulation was considered.

In the course of studying nuclear changes insynchronous cultures of Bacillus polymyxa, it wasnoted that large cells occurred at a regular periodin the growth cycle and showed increasedamounts of nuclear material.

Large cells of Bacillus megaterium were re-ported by DeLamater (1953) to be associatedwith fusion. Large cells of Escherichia coli areassociated with a diploid phase in that species(Lederberg, 1956). Large bodies were reported incultures of Proteus vulgaris by Stempen andHutchinson (1951) and shown to arise both fromswelling of individual bacilli and fusion of cells.Similar large bodies were reported in luminousbacteria (Johnson and Gray, 1949; Warbasse andJohnson, 1950) and associated with physiologicalconditions of stress. Hardwick and Foster (1952)demonstrated that vegetative cells were "com-mited to sporulation" once the growth processhad passed a critical period. It has also beenshown for Bacillus cereus that there is an increasein deoxyribonucleic acid per unit (dry weight) atthe early stationary phase as compared to the logphase of growth (Stuy, 1958). This paper dealswith cytological and physiological characteristicsof the large cell stage in two species of the genusBacillus.

MATERIALS AND METHODS

Cultures. The species used were obtained fromRuth Gordon, Rutgers University (B. polymyxastrain NRS 812 and B. cereus strain NRS 942).A variant of B. cereus capable of growth andsporulation without organic nitrogen was isolatedafter ultraviolet irradiation and designated as B.cereus strain M-1.

Media. The inorganic base salt medium ofProom and Knight (1955) was used without am-monium molybdate and had the following com-position per liter: KH2PO4, 1.5 g; (NH4)2HP04,7.0 g; MgSO4.7H20, 0.5 g; CaCl2-2H20, 0.3 g;MnSO4.H2O, 0.04 g; and FeSO4*7H20, 0.025 g.Ethylenediaminetetraacetic acid was sterilizedseparately and added aseptically to give a finalconcentration of 1 j,g per ml. Glucose was auto-claved as a 50% stock solution and added asep-tically to give a final concentration of 2.5 mg perml. Amino acids were prepared as 5 mg per mlsolutions and added aseptically to give 0.05 mgper ml, final concentration. Medium A containedthe five amino acids, DL-alanine, DL-aspartic acid,L-cysteine, glycine, and L-lysine. All glasswarewas acid-cleaned and all water triple-distilled.

Nuclear stain. The nuclear stain technique usedwas that of DeLamater (1951) with thionine.Loopfuls of the culture to be stained were placedon coverslips, fixed with osmic acid vapors for5 min, hydrolized in 1 N HCL at 60 C for 6 min,and subsequently stained for 8 hr.

Synchronous cultures. Synchronous cultureswere obtained by growing a culture to light tur-bidity. This culture was then alternated at 30-min intervals between temperatures of 24 C and35 C for 3.5 hr and used as inoculum for theseveral experiments.Amino acid inhibition. The amino acids, DL-

serine, D-phenylalanine, L-cysteine, L-asparticacid, glycine, L-lysine, and DL-threonine, wereprepared as stock solutions in base salt and addedto give an equimolar concentration of 4.17 X 104in the final medium. Optical density readings

418

on Decem

ber 30, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

19611 LARGE CELL STAGE IN BACILLI 419

...

. ::...'....~~~~~~~~~~~~~~~~~~~~~~~.... ... ......eo:...

'w ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..... .,.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..,l..........A B.: D_ §

*2&:xtnR....... ..

..0........... .** . .. R R | l R RR~~~~~~~~~~~~~~~~~~~~...................

*. .....;...;

.o'°''.*:~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.|WiSMUr0 l E U

.:::..1 ... e - : , > ,. e g,,,_~J:........;..i ..... . R. . ..~~~~~~~~~~..... .....^,^. . t...:B

*: ' ~~~~~~~~~~~~~~. ..

li' 2, ..';

FIG. 1. Nuclear stains of normal and large cells of Bacillus polymyxa. A to D-normal diplobacilli andstreptobacilli. E and F- large cells. El and F,, phase photos8 of E and F, respectively, to show cell shapemore clearly.

on Decem

ber 30, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

JOHNSON

..'1e s^- 0 ^xv y.,f t0FIG2.Nucearstansof ary cels f Bciluscerus ormdn.pesece.f.srin..l.ad...phse.ho

pciey t hwcelsaean iemoecery.N ra clscnbese n ,B n

D for comparison.

were made on a spectronic 20 spectrophotometerat a wavelength of 550 m,.

Photomicrography. A phase microscope wasused with a 35-mm camera attachment. Photo-graphs were taken on high contrast copy film andenlarged four times to give a final print magnifi-cation of 7760 X:.

RESULTS

Figure 1 shIows nuclear stains made of a syn-chronous culture of B. polymyxa. Large cells werefound at 6 and 8 hr, and the increase in nuclearmaterial was noted. At 10 hr, the culture showedno large cells and subsequently sporulated nor-mally 30 hr from the onset of synchrony. The

percentage of large cells on a given smear is quitesmall, perhaps 1 per 1,000 cells or less.A synchronous culture of B. cereus under simi-

lar conditions showed large cells at 12 hr andprior to any significant chain formation. In test-ing a variant strain of B. cereus (M-1) for theeffect of amino acids on growth and sporulation,it was noted that serine retarded growth, withlarge cells eventually (48 hr) occurring. Figure 2shows the nuclear stains made of this form whengrown in the presence of serine.A series of amino acids was added in equi-

molar concentration to the base salt-serine me-dium to ascertain its effect on serine inhibitionand large cell formation (Fig. 3). Threonine re-versed the serine growth inhibition. However,

420 [VOL. 82

on Decem

ber 30, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

9611 LARGE CELL STAGE IN BACILLI 421

.30

,'Z 18/2H

~~~~j0~~~~~~~~I..~~ ~ ~ ~ ~ ~ ~ I

A. t~~A

FIG. 3. Effect of amino acids on growth of Bacillus cereus

7.55 C0TROSER INELArE SCRINE A

7.0 SEI.,NE°s

6.5'~~~~~~~~~~~~q

6.0

.05~~~~~~~~~~~~J

0. O 20 0400 60.0

0 2 4 6 8 I0 1 2 1 6 20 24 30HOVRS

FIG. 4. Correlation of growth, large cell formation, and serine inhibition for Bacillus cereus

on Decem

ber 30, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

JOHNSONT

B ... .......... .....*:~~~~~~~~~~~~~~~~~~.........1",fE4 ..

A1

[VOL. 82

B '

* . .......:: - e s

*X=1 1E;

*iilD «C...i | r............

.:.... S

| | PU

.X X l'^ | W..*-. ,.

.X.:.! .. : :^' R; :.

*.Rx

... .....

_ '',$g:

B1

B' 3..:,E,.., .... :: ..*:: ,.ft,E ..f5.,5<.,-o'c, ..,........ . woxg EE+.s

..i...B;!49;:g g

y: 9 gy,5.# M9..e .q §3

'fl c..>...,Sj,

*. ,js6-,.8i.fp]-:-:.... 6,.>..5 ......

g9'< 4

Yp,;° .j.

;.-, +-5e355 ;.'53 #S3isF:*.: w

.'.

.:

;5.;..*.:,

1~~~~~~~~~~~~~~~~~~~~~~~~~. y.

FIG. 5. Bacillus cereuts. A, B, and B'-crystal violet stain of diplobacilli and stireptobacilli large cells.A1 and B1-nuclear stain of diplobacilli and streptobacilli large cells. C-nuclear stain of normal cellsfor comparison.

phenylalanine and lysine caused even slowergrowth, with large cells occurring in all cultures.All cultures eventually sporulated normally.Toward the end of relating serine inhibition

with large cell formation, viable counts, turbiditymeasurements, and cytological examinationswere made on several synchronous cultures ofB. cereus strain MI-1, with serine added to the

422

.-K

on Decem

ber 30, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

LARGE CELL STAGE IN BACILLI

-6.0

5.0B. CEREUSB.POLYMYX_A_LARQE CELLS *

O) 15HOURS

FIG. 6. Typical relationship of concentration ofinitial inoculum to time of large cell formation forBacillus polymyxa and Bacillus cereus.

base medium at the start and then after large cellformation (Fig. 4). It was first apparent that thesize of the synchronous inoculum played a role intime of appearance of large cells. Large cells(Fig. 5) appeared concomitant with a viablecount drop shortly after a concentration of 106cells per ml was reached. Chain formation (as ob-served on simple smears) was not adequate toaccount for the drop in count. The drop in viablecount also correlated with a continued increase inoptical density. Figure 6 shows the effect of syn-chronous inoculum concentration on large cellformation for both B. cereus strain M-1 and B.polymyxa. lVhen the initial inoculum was above3 to 4 million cells per ml, no large cells were ob-served, and this stage had presumably beenpassed in the culture used for synchrony.

Serine appeared, on the basis of viable counts,to inhibit the culture when it approached the 106

cells per ml concentration. However, when serinewas added after large cell formation, no such in-hibition was noted. The mechanism of serine in-hibition is unknown and deserves further investi-gation. Serine has been shown to produceelongated cells in E. coli (Grula, 1960). However,that phenomenon was not observed here.

DISCUSSION

As cited earlier, large cells have been reportedin a number of species, including members of thegenus Bacillus. With the exception of E. coliforms, their significance has been controversial.DeLamater and Hunter (1953) associated theirformation in B. megaterium with nutritional fac-tors in blood. As with the genus Proteus (Stempenand Hutchinson, 1951), our experience wouldsuggest these cells arise primarily by swelling ofnormal cells. However, the tendency of bacilli topair leaves some suggestion of fusion, particu-larly when associated with the viable count drop.All attempts to observe nuclear material con-nected between two adjacent cells have, however,been negative.

Three independent reports would suggest amodification of metabolism at a point coincidentwith our findings on large cell formation. Spizizenand Anagnostopoulos (1960) reported an in-creased transformation rate for Bacillus subtilisat the end of log phase, Stuy's (1958) increase indeoxyribonucleic acid in B. cereus at the earlystationary phase, and the inhibition of sporulationfor B. megaterium and B. polymyxa by periodate(Johnson and Cords, 1961). The effect of serineon growth, reported here, before and after the ap-pearance of large cells further indicates thisperiod to be a distinct phase of growth. Thisstage may also explain the apparently contra-dictory reports that alanine inhibited sporulationfor B. cereus (Foster and Heiligman, 1949)whereas it improved growth and enhanced sporu-lation for B. cereus var. mycoides (Grelet, 1957).Under the growth conditions reported here,

this stage of growth is 3 to 4 hr in duration with acell density of 1 to 3 million cells per ml. A fra-gility of large cells has been suggested in ourwork since large cells seen on simple strains nearthe end of this stage are not found on simultane-ous nuclear stain preparations, presumably be-cause of the HCL treatment in the procedure.Their relationship to sporulation is unknown.However, in view of the ability to transform

19611 423

on Decem

ber 30, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

JOHNSON

asporogenic cultures with wild type deoxyribo-nucleic acid (Schaeffer, Ionesco, and Jacob, 1959),the possibility of autolysis of large cells and"autotransformation" prior to sporulation as thepoint at which cells are "committed to sporula-tion" may be suggested.

It is also of interest that, although 13 aminoacids have been reported as necessary for variousspecies of Bacillus, serine, threonine, and lysinehave, to the best of the author's knowledge, never

been shown to be required by any species of thisgenus. This would suggest a basic essential mech-anism by which these were synthesized, or theircomplete exclusion from Bacillus metabolism.

ACKNOWLEDGMENT

This research was supported in part under a

contract (Nonr 2794(01)) with the Office of NavalResearch.

LITERATURE CITED

DELAMATER, E. D. 1951. A staining and dehy-drating procedure for the handling of micro-organisms. Stain Technol. 26:199-204.

DELAMATER, E. D. 1953. Cytologic evidencefor the occurrence of cellular and nuclearfusion in Bacillus megaterium. Bull. TorreyBotan. Club 80:289-298.

DELAMATER, E. D., AND M. E. HUNTER. 1953.Preliminary studies into the conditions con-

trolling cellular fusion and secondary colonyformation in Bacillus megaterium. J. Bacteriol.65:739-743.

FOSTER, J. W., AND F. F. HEILIGMAN. 1949. Bio-chemical factors influencing sporulation in a

strain of Bacillus cereus. J. Bacteriol. 57:639-646.

GRELET, N. 1957. Growth limitation and sporu-lation. J. Appl. Bacteriol. 20:315-324.

GRULA, E. A. 1960. Cell division in a speciesof Er2irnia. II. Inhibition of division byD-amino acids. J. Bacteriol. 80:375-385.

HARDWICK, W. A., AND J. W. FOSTER. 1952.On the nature of sporogenesis in some aerobicbacteria. J. Gen. Physiol. 35:907-927.

JOHNSON, F. H., AND D. H. GRAY. 1949. Nucleiand large bodies of luminous bacteria inrelation to salt concentration, osmotic pres-sure, temperature and urethan. J. Bacteriol.58:675-688.

JOHNSON, R., AND C. CORDS. 1961. The effectof periodate on sporulation in a syntheticmedium for the Bacillus genus. J. Ariz. Acad.Sci. 1:116-118.

LEDERBERG, J. 1956. Conjugal pairing inEscherichia coli. J. Bacteriol. 71:497-498.

PROOM, H., AND B. C. J. G. KNIGHT. 1955. Theminimal nutritional requirements of somespecies in the genus Bacillus. J. Gen. Micro-biol. 13:474-480.

SCHAEFFER, P., H. IONESCO, AND F. JACOB. 1959.Sur le determinisme g6netique de la sporula-tion bacterienne. Compt. rend. 249:577.

SPIZIZEN, J., AND C. ANAGNOSTOPOULOS. 1960.Transformation of indol and sucrose char-acters in Bacillus subtilis, strain 168. Bac-teriol. Proc. 1960:185.

STEMPEN, H., AND W. G. HUTCHINSON. 1951.The formation and development of largebodies in Proteus vulgaris OX-19. IL. Com-parative cytology of bacilli and large bodies.J. Bacteriol. 61:337-344.

STUY, J. H. 1958. The nucleic acids of Bacilluscereus. J. Bacteriol. 76:179-184.

WARBASSE, W. W., AND F. H. JOHNSON. 1950.The influence of penicillin on large body pro-duction by luminous bacteria. J. Bacteriol.60:279-282.

424 [VOL. 82

on Decem

ber 30, 2019 by guesthttp://jb.asm

.org/D

ownloaded from