MnSO4.H2O, 0.04 g; and FeSO4*7H20, g.

Transcription

1 LARGE CELL STAGE IN THE GENUS BACILLUS ROY M. JOHNSON Life Science Division, Arizona State University, Received for publication March 6, 1961 Tempe, Arizona ABSTRACT JOHNSON, Roy M. (Arizona State University, Tempe). Large cell stage in the genus Bacillus. J. Bacteriol. 82: Synchronous cultures of Bacillus polymyxa and Bacillus cereus produced a transitory large cell stage with increased nuclear material concomitant with a drop in viable count during growth in a defined medium. A variant of B. cereus that required no organic nitrogen for growth and sporulation was inhibited by serine and this inhibition reversed by equimolar amounts of threonine. Serine inhibition occurred before the large cell stage but not after. The significance of this large cell stage as regards transformation studies, nucleic acid analysis, and sporulation was considered. MATERIALS AND METHODS Cultures. The species used were obtained from Ruth Gordon, Rutgers University (B. polymyxa strain NRS 812 and B. cereus strain NRS 942). A variant of B. cereus capable of growth and sporulation without organic nitrogen was isolated after ultraviolet irradiation and designated as B. cereus strain M-1. Media. The inorganic base salt medium of Proom and Knight (1955) was used without ammonium molybdate and had the following composition 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, g. Ethylenediaminetetraacetic acid was sterilized separately and added aseptically to give a final concentration of 1 j,g per ml. Glucose was auto- In the course of studying nuclear changes in claved as a 50% stock solution and added aseptically to give a final concentration of 2.5 mg per synchronous cultures of Bacillus polymyxa, it was noted that large cells occurred at a regular period ml. Amino acids were prepared as 5 mg per ml in the growth cycle and showed increased solutions and added aseptically to give 0.05 mg amounts of nuclear material. per ml, final concentration. Medium A contained Large cells of Bacillus megaterium were reported by DeLamater (1953) to be associated L-cysteine, glycine, and L-lysine. All glassware the five amino acids, DL-alanine, DL-aspartic acid, with fusion. Large cells of Escherichia coli are was acid-cleaned and all water triple-distilled. associated with a diploid phase in that species Nuclear stain. The nuclear stain technique used (Lederberg, 1956). Large bodies were reported in was that of DeLamater (1951) with thionine. cultures of Proteus vulgaris by Stempen and Loopfuls of the culture to be stained were placed Hutchinson (1951) and shown to arise both from on coverslips, fixed with osmic acid vapors for swelling of individual bacilli and fusion of cells. 5 min, hydrolized in 1 N HCL at 60 C for 6 min, Similar large bodies were reported in luminous and subsequently stained for 8 hr. bacteria (Johnson and Gray, 1949; Warbasse and Synchronous cultures. Synchronous cultures Johnson, 1950) and associated with physiological were obtained by growing a culture to light turbidity. This culture was then alternated at 30- conditions of stress. Hardwick and Foster (1952) demonstrated that vegetative cells were "commited to sporulation" once the growth process min intervals between temperatures of 24 C and had passed a critical period. It has also been 35 C for 3.5 hr and used as inoculum for the shown for Bacillus cereus that there is an increase several experiments. in deoxyribonucleic acid per unit (dry weight) at Amino acid inhibition. The amino acids, DLserine, D-phenylalanine, L-cysteine, L-aspartic the early stationary phase as compared to the log phase of growth (Stuy, 1958). This paper deals acid, glycine, L-lysine, and DL-threonine, were with cytological and physiological characteristics prepared as stock solutions in base salt and added of the large cell stage in two species of the genus to give an equimolar concentration of 4.17 X 104 Bacillus. in the final medium. Optical density readings 418

2 ' ~~~~~~~~~~~~~~.. R....~~~~~~~~~~... ~~~~~~~~~~~~~~~~~~~~~~~... w *:~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. : LARGE CELL STAGE IN BACILLI 419. :..'....: _ A B D......eo:... ' ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..,l *2&:xtnR * *. R R l R R R~~~~~~~~~~~~~~~~~~~~... WiSMUr0 l E U *....;...;.o' ''..: :: e - :,,,_~J:, >,. e g....;..i.....^,^.. t...:b * ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~....,. li' 2,..'; FIG. 1. Nuclear stains of normal and large cells of Bacillus polymyxa. A to D-normal diplobacilli and streptobacilli. E and F- large cells. El and F,, phase photos8 of E and F, respectively, to show cell shape more clearly.

3 420 JOHNSON [VOL. 82..'1e s^- 0 ^xv ỵ,f t0 FIG2.Nucearstansof n.pesece.f.srin..l.ad...phse.ho ary cels f Bciluscerus ormd pciey t hwcelsaean iemoecery.n ra clscnbese n,b n D for comparison. were made on a spectronic 20 spectrophotometer at a wavelength of 550 m,. Photomicrography. A phase microscope was used with a 35-mm camera attachment. Photographs were taken on high contrast copy film and enlarged four times to give a final print magnification of 7760 X:. RESULTS Figure 1 shiows nuclear stains made of a synchronous culture of B. polymyxa. Large cells were found at 6 and 8 hr, and the increase in nuclear material was noted. At 10 hr, the culture showed no large cells and subsequently sporulated normally 30 hr from the onset of synchrony. The percentage of large cells on a given smear is quite small, perhaps 1 per 1,000 cells or less. A synchronous culture of B. cereus under similar conditions showed large cells at 12 hr and prior to any significant chain formation. In testing a variant strain of B. cereus (M-1) for the effect of amino acids on growth and sporulation, it was noted that serine retarded growth, with large cells eventually (48 hr) occurring. Figure 2 shows the nuclear stains made of this form when grown in the presence of serine. A series of amino acids was added in equimolar concentration to the base salt-serine medium to ascertain its effect on serine inhibition and large cell formation (Fig. 3). Threonine reversed the serine growth inhibition. However,

4 9611 LARGE CELL STAGE IN BACILLI ,'Z 18/ 2H ~~~~j 0~~~~~~~~ I..~ ~ ~ ~ ~ ~ ~ I 6.5'~~~~~~~~~~~~q A. t~~a Effect of amino acids on growth of Bacillus cereus FIG C0TRO SER I NE.05~~~~~~~~~~~~J LArE SCRINE A 0. O SEI., NE s FIG I HOVRS Correlation of growth, large cell formation, and serine inhibition for Bacillus cereus

5 422 A1.1",fE ~~~~~~~~~~~~~~~~~~~~~~~~~. : : y..-k... *:~~~~~~~~~~~~~~~~~~ JOHNSONT B B ' *.... :: - e s *X=1 1E; *iild «C...i r..:... S PU.X X l' ^ W.. *-.,..X.:.!.. ^' R; :. *.Rx _ '',$g: B1.... woxg EE+.s B' 3..:,E,..,... ::.. *::,.ft,e..f5.,5<.,-o'c,..,.....i...b;!49;:g g y: e 9 gy,5.#.q M9.. 3 'fl c..>...,sj, *.,js 6-,.8i.fp]-:-:.... 6,.> g9'< 4 Yp,;.j. ;.-, +-5e355 ;. '53 #S3isF: *.: w 1.'. ;5.;..*. :,.: [VOL. 82 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 cells for comparison. phenylalanine and lysine caused even slower growth, 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, turbidity measurements, and cytological examinations were made on several synchronous cultures of B. cereus strain MI-1, with serine added to the

6 19611 LARGE CELL STAGE IN BACILLI 423 cells per ml concentration. However, when serine was added after large cell formation, no such inhibition was noted. The mechanism of serine inhibition is unknown and deserves further investigation. Serine has been shown to produce elongated cells in E. coli (Grula, 1960). However, that phenomenon was not observed here B. CEREUS B. POLYMYX_A_ LARQE CELLS * O) 15 HOURS FIG. 6. Typical relationship of concentration of initial inoculum to time of large cell formation for Bacillus polymyxa and Bacillus cereus. base medium at the start and then after large cell formation (Fig. 4). It was first apparent that the size of the synchronous inoculum played a role in time of appearance of large cells. Large cells (Fig. 5) appeared concomitant with a viable count drop shortly after a concentration of 106 cells per ml was reached. Chain formation (as observed on simple smears) was not adequate to account for the drop in count. The drop in viable count also correlated with a continued increase in optical density. Figure 6 shows the effect of synchronous inoculum concentration on large cell formation for both B. cereus strain M-1 and B. polymyxa. lvhen the initial inoculum was above 3 to 4 million cells per ml, no large cells were observed, and this stage had presumably been passed in the culture used for synchrony. Serine appeared, on the basis of viable counts, to inhibit the culture when it approached the 106 DISCUSSION As cited earlier, large cells have been reported in a number of species, including members of the genus Bacillus. With the exception of E. coli forms, their significance has been controversial. DeLamater and Hunter (1953) associated their formation in B. megaterium with nutritional factors in blood. As with the genus Proteus (Stempen and Hutchinson, 1951), our experience would suggest these cells arise primarily by swelling of normal cells. However, the tendency of bacilli to pair leaves some suggestion of fusion, particularly when associated with the viable count drop. All attempts to observe nuclear material connected between two adjacent cells have, however, been negative. Three independent reports would suggest a modification of metabolism at a point coincident with our findings on large cell formation. Spizizen and Anagnostopoulos (1960) reported an increased transformation rate for Bacillus subtilis at the end of log phase, Stuy's (1958) increase in deoxyribonucleic acid in B. cereus at the early stationary phase, and the inhibition of sporulation for B. megaterium and B. polymyxa by periodate (Johnson and Cords, 1961). The effect of serine on growth, reported here, before and after the appearance of large cells further indicates this period to be a distinct phase of growth. This stage may also explain the apparently contradictory reports that alanine inhibited sporulation for B. cereus (Foster and Heiligman, 1949) whereas it improved growth and enhanced sporulation 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 a cell density of 1 to 3 million cells per ml. A fragility of large cells has been suggested in our work since large cells seen on simple strains near the end of this stage are not found on simultaneous nuclear stain preparations, presumably because of the HCL treatment in the procedure. Their relationship to sporulation is unknown. However, in view of the ability to transform

7 424 JOHNSON [VOL. 82 asporogenic cultures with wild type deoxyribonucleic acid (Schaeffer, Ionesco, and Jacob, 1959), the possibility of autolysis of large cells and "autotransformation" prior to sporulation as the point at which cells are "committed to sporulation" may be suggested. It is also of interest that, although 13 amino acids have been reported as necessary for various species of Bacillus, serine, threonine, and lysine have, to the best of the author's knowledge, never been shown to be required by any species of this genus. This would suggest a basic essential mechanism by which these were synthesized, or their complete exclusion from Bacillus metabolism. ACKNOWLEDGMENT This research was supported in part under a contract (Nonr 2794(01)) with the Office of Naval Research. LITERATURE CITED DELAMATER, E. D A staining and dehydrating procedure for the handling of microorganisms. Stain Technol. 26: DELAMATER, E. D Cytologic evidence for the occurrence of cellular and nuclear fusion in Bacillus megaterium. Bull. Torrey Botan. Club 80: DELAMATER, E. D., AND M. E. HUNTER Preliminary studies into the conditions controlling cellular fusion and secondary colony formation in Bacillus megaterium. J. Bacteriol. 65: FOSTER, J. W., AND F. F. HEILIGMAN Biochemical factors influencing sporulation in a strain of Bacillus cereus. J. Bacteriol. 57: GRELET, N Growth limitation and sporulation. J. Appl. Bacteriol. 20: GRULA, E. A Cell division in a species of Er2irnia. II. Inhibition of division by D-amino acids. J. Bacteriol. 80: HARDWICK, W. A., AND J. W. FOSTER On the nature of sporogenesis in some aerobic bacteria. J. Gen. Physiol. 35: JOHNSON, F. H., AND D. H. GRAY Nuclei and large bodies of luminous bacteria in relation to salt concentration, osmotic pressure, temperature and urethan. J. Bacteriol. 58: JOHNSON, R., AND C. CORDS The effect of periodate on sporulation in a synthetic medium for the Bacillus genus. J. Ariz. Acad. Sci. 1: LEDERBERG, J Conjugal pairing in Escherichia coli. J. Bacteriol. 71: PROOM, H., AND B. C. J. G. KNIGHT The minimal nutritional requirements of some species in the genus Bacillus. J. Gen. Microbiol. 13: SCHAEFFER, P., H. IONESCO, AND F. JACOB Sur le determinisme g6netique de la sporulation bacterienne. Compt. rend. 249:577. SPIZIZEN, J., AND C. ANAGNOSTOPOULOS Transformation of indol and sucrose characters in Bacillus subtilis, strain 168. Bacteriol. Proc. 1960:185. STEMPEN, H., AND W. G. HUTCHINSON The formation and development of large bodies in Proteus vulgaris OX-19. IL. Comparative cytology of bacilli and large bodies. J. Bacteriol. 61: STUY, J. H The nucleic acids of Bacillus cereus. J. Bacteriol. 76: WARBASSE, W. W., AND F. H. JOHNSON The influence of penicillin on large body production by luminous bacteria. J. Bacteriol. 60: