Morphological changes in Bacillus subtilis with genetic and phenotypic origins

9 ELSEVIER Paris 1985

Ann. Inst. Pasleur/Mierobiol. 1985, 136 A, 77-84

MORPHOLOGICAL WITH

CHANGES

GENETIC

AND

IN

BACILLUS

PHENOTYPIC

SUBTILIS ORIGINS

by H. J. Rogers (*) Nalional Institute/or Medical Research, Mill Hill, London N W 7 1AA

Cylindrically shaped bacteria growing at a constant exponential rate divide at a cell length which is nearly constant from generation to generation and maintain a constant diameter. Variations in growth rate lead to changes in length but not usually in diameter. These morphological parameters are maintained by growth in two cell regions, one at the developing septum which forms the pole, the other within the cylinder which extends the length. Genetic lesions upsetting this growth pattern, with consequent changes in cell morphology, have been described in a number of species. In Bacillus subtilis, conditional rod mutations have been studied in some detail [17]. These cause the cells to be either spheroid or rod-shaped according to the growth conditions used. A possible approach to investigating the molecular mechanisms underlying these changes is to find inhibitors or other substances of known action that cause similar changes. Phenotypic morphological changes of the wild type similar to those in Escherichia coli caused by ~-lactams, in particular mecillinam [23, 24], which mimic these genetically inspired changes, have not been described in B. subtilis or in other Gram-positive bacteria apart from Arlhrobacter. At the present time, no clear picture can be drawn of the changes in molecular architecture resulting from genetic lesions and giving shape alterations, although a number of ideas have been put forward. The burden of the present article will be to attempt to bring together the molecular basis of two phenotypic changes and some molecular and physiological facts associated with the genetic lesions. The role o[ negatively charged wall polymers An early example of a phenotypic morphological change, not of a wild type microorganism but of phosphogluco-mutase negative (pgm-) mutants, was demonstrated when B. licheni[ormis pgm- was grown under phosphate limitation [8]; corresponding mutants (gt-C) B. sublilis behaved in the same way. Spherical cells resulted from organisms which formed neither teichoic acid, because of the nutritional phosphate limitation, nor teichuronic acid, which phosphate-limited wild type organisms would otherwise have done, because of the phosphogluco-mutase lesion. In both their shape and wall composition, the phosphate-limited pgmcoccal-shaped bacteria closely resembled one type of rod mutant called tag-1 by Boyland and Mendelson [2] and Boylan el al. [3], or rodA by Karamata el al. [10] and Rogers el al. [22] of B. sublilis. Moreover, the morphological similarities extended even to the ultrastructure, in which septa appeared to run at right angles within the coccal-shaped cells [6, 8], thus differentiating them from rodB mutants [4]. The curious, folding, comma-like shapes observed when the pgmManuscrit re~u le 12 septembre 1984. (*) Present address: University of Kent at Canterbury, Biological Laboratory, The Universily, Canterbury, Kent CT2 7NJ (UK).

78

H.J.

ROGERS

mutants were in the process of change from phosphate to Mg2§ growth, providing further support of Mendelson's [12] suggestion for the origin of spherical shapes by the folding of two rod-shaped cells in a hairpin bend at the division septum. Some caution should perhaps be applied, however, before this hypothesis can be accepted as the primary mechanism for the derivation of coccal shapes from rods, since, when double rodA lyl-2 mutants were grown at 45 ~ C to give coccal shapes, the presence of the lyl gene prevented separation between the initially rod-shaped cells which changed at 45 ~ C to a perfectly regular chain of cocci. These only later appeared as a tangled mass of larger cocci with septa at right angles [18]. At its face value, the above evidence strongly suggests t h a t the presence of negatively charged polymers, such as teichoic and teichuronic acid attached to the peptidoglycan in the walls, is necessary to maintain the growth of regular rod-shaped bacilli. However, we do not know enough about the membranes of either rodA mutants or of pgm- mutants under various growth conditions to be dogmatic. Nor do we know whether lipoteichoic acids in the mutants are modified in amount or type. The reasons of these cautions will be more apparent from the next section. J.0-i

2.S

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84

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The effect o] eerulenin on the aacial ralio o[ t3. s u b l i l i s 16~'.

a c i d - h y d r o l y z e d c a s e i n ; 7.5 t z g / m l c e r u l e n i n w a s a d d e d a t z e r o t i m e ( e ) 22-h incubation (o). The results represent the means from measurements c h o s e n a t r a n d o m , e a c h i n a d i f f e r e n t m i c r o s c o p i c field. T h e m a g n i f i c a t i o n measurements were made with a Walson's split image eyepiece.

MORPHOLOGICAL CHANGES IN B A C I L L U S S U B T I L I S

79

Membrane lipids and shape modification Other evidence has suggested that disturbances of lipid synthesis can be correlated with altered bacterial cell division [13, 25, 27]. Cerulenin is an antibiotic that stops fatty acid synthesis [14, 26] and hence phospholipid formation. It also inhibits export of bacterial proteins [1, 5, 7, 9, 11, 15, 16], and has now been found to cause gross changes in the shape of B. subtilis cells as well as inhibiting autolysin formation. The change in axial ratios of cells of B. subtilis trp 168 growing in a minimal salts glucose medium containing 7.5 ~g/ml cerulenin is shown in figure 1. This change in shape was completely reversed when the cerulenin was removed. It could be prevented by the addition of Tween 20 or Tween 80 (5%) at the same time as the cerulenin. The rate of growth of the organism was reduced by about 60% by the antibiotic. After about 2-3 generations, at this rate, the phospholipid content had been reduced by 50% without an effect on protein or peptidoglycan content, and the appearance of the cells at 1,000 times magnification was much as those of a rod mutant growing in its coccal form. Measurements showed, however, that the diameter of the spheroid cells was less than t h a t of those from rod mutants. When the change in cell diameter was compared with the change in cell length (fig. 2) it was clear that eerulenin caused a major reduction in average cell length

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(HRS)

Fro. 2 . - - The effect o[ cerulenin on the d i m e n s i o n s o/ B. subtilis 168. The growth was in either a minimal-salts-glucose ( c o ) or a casein hydrolysate medium (AA) 7.5 ~g/ml eerulenin was added at time zero. The lengths are shown by the solid symbols and the diameters by the e m p t y ones. The results are expressed as eyepiece units ( E P U ) read from a split image eyepiece. The results are expressed as in figure 1.

80

H.J.

ROGERS

rather than the cell diameter. The change from rod to coccus in a rodB m u t a n t is, on the other hand, largely due to an increase in cell diameter [4]. The correlation between inhibition of lipid synthesis and change of shape made it of interest to investigate any changes t h a t may occur in lipid content during the change from rod to coccal morphology in a rod mutant. Rod B104 was investigated [10, 19]. Lipid was measured by the radioactivity incorporated from 2J4C-glycerol or 2-3H-glycerol into the cell fraction extracted with chloroform-methanol. The results were compared in a number of instances with those of direct total phosphorus measurements on the same lipid extracts. The results agreed reasonably well (table I) both showing that, per unit dry weight of bacteria, the cocci had 3-4 times as much lipid as the rod-shaped organism. The shape change was effected in these experiments by adding M/15 K B r to the growth medium during incubation at 35 ~ C [19]. The rate of increase in content of phospholipid related rather well with t h a t of change in shape, as measured by the axial ratios of the cells. This was, of course, the opposite of the result which might have been expected from the cerulenin studies and would not be predicted from the surface/volume relationship for the rods and cocci, the latter having values 30-40% lower than the former. TABLE I. - - The concentrations of phospholipids and membrane proteins in rod and coecal forms of ~ B. subtilis met rodB104 >>.

Glycerol -- 2-14C-glycerol (nmoles/A4~0) Total lipid p h o s p h o r u s (nmoles/A4ao) Axial ratios of cells % p h o s p h a t i d y l glycerol (of total PL) % p h o s p h a t i d y l e t h a n o l a m i n e (of total PL) Membrane protein (*) (mg/A4so)

Rods

Cocci

5.28 2.98 1.92 71 7 46.5

12.1 8.68 1.04 63 10 31.2

The salts glucose m e d i u m [21] was supplemented either with M/15 K B r to give rod forms or w i t h o u t to give cocci and contained 0.5 txmole/ml glycerol of specific activity 0.4 ~Ci/~mole as 2-1aC-glycerol. The cultures were incubated at 35 o C and shaken to aerate. Flasks contained 0.2 v o l u m e of medium. (*) Isolated from 1-1 batches of culture harvested at A43o 1.30.

The proportions of types of phospholipids, measured from thin-layer chromatograms, in the coccal and rod forms of the m u t a n t were slightly different but, in both, 60-70% appeared as phosphatidylglycerol. Variations in the total amount of lipid in the coccal and rod-shaped rod mutants could correspond either to more membrane per unit weight of cell or more lipid per unit weight of membrane. Measurements of the amount of membrane in the two types of cell (table I) showed t h a t there was more membrane in the rod-shaped cells than in the cocci, as would be expected from their surface-to-volume ratios (J. M. Fox and H. J. Rogers, unpublished work). The ratio of membrane protein isolated from rods to t h a t from cocci was 1.49 compared with a surface-to-volume ratio of 1.54 [4]. Examinations of the rates of lipid synthesis and turnover provided what seemed to be a further paradox. The rate of lipid synthesis was measured from ~H/14C ratios after the incorporation during 2-min pulses of 2-3H-glycerol into the lipid extractable from microorganisms already completely labelled with 2-14C-glycerol. During transition from a coccus to a rod, the rate of lipid synthesis increases markedly, even though there is eventually a net loss in the total amount of lipid (fig. 3). How then can this loss of lipid be explained. One factor appears to be t h a t turnover of lipid, as measured by the loss of radioactively labelled glycerol from completely labelled bacteria

MORPHOLOGICAL CHANGES IN BACILLUS SUBTILIS

81

when growing in media containing unlabelled glycerol, is faster for the rod-shaped cells than for the cocci. In a preliminary experiment, rods lost rather more than 50% of their label per generation, whereas the coccal-shaped cells lost less than 25%. It may then be that the increased rate of synthesis shown during transition from coccus to rod does not lead to accumulation of lipid because of increased hydrolysis, due perhaps to the induction of a phospholipase. In any case, it is tempting to draw further parallels between the action of cerulenin, which inhibits lipid synthesis and alters the shape of wild type cells to cocci and the decreased rate of synthesis of lipid in the rod mutant during change in the same morphological direction.

251

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T h e rate of l i p i d s y n t h e s i s in rod B 1 0 4 changing f r o m a coccus to a rod.

The m u t a n t was grown at 35 ~ C for 10 generations in a salts-glucose medium [21] containing 0.5 ~moles/ml 2-t4C-glycerol of specific activity 0.4 ~Ci/#mole. At time zero, M/15 K B r was added to the m e d i u m and at the intervals indicated, 4-ml samples were w i t h d r a w n and 8 FCi of 2-all-labelled glycerol of specific activity 500 ~Ci/~mole were added. After 2 rain, four 0.8-ml samples were filtered t h r o u g h glass fibre filters. The organisms were washed with Tris buffer p H 7.5 containing 10 mM MgSOa and 10 I n g / m l unlabelled glycerol. The discs were t h e n dried over P~Os and the lipids extracted with CHCIs-MeOH by the s t a n d a r d method. These extracts were dried down, mixed with toluene-based liquid scintillation fluid and counted with a differential setting to distinguish between I4C and all. The ratio 3H/14C measures the rate of synthesis. All bacterial cultures were incubated at 350 C and contained in conical flasks 5 times their volmne. They were shaken to aerate. The shape of the cells was measured as in figure 1. A n n . ~Iicrobiol. (Inst. Pasteur), 136 A,

n ~ 1, 1 9 8 5 .

6

82

H.J.

ROGERS

It would seem t h a t the relationship between the total phospholipid content of membranes and morphology in the cerulenin effect and the rod mutation are the opposite of each other. It is known t h a t after cerulenin treatment the membranes of bacilli are phospholipid-depleted [15]. Further support for the lack of correlation between total phospholipid content of membranes and cell shape is provided by an experiment in which the coccal form of rodB104 was grown with 7.5 ~g/ml of cerulenin (see table II). The antibiotic reduced the lipid content by 41%, as would be expected, and failed to alter the axial ratios of the cells. Both the lengths and diameters of the cells, however, increased b y about 20%. Addition of cerulenin to rodB104 growing as a rod with Br' reduced its lipid content by 58% in 1.5 generations of growth and its axial ratio from 1.91 to 1.10 by decrease in length (--23%), b u t also b y an increase in diameter ( + 2 9 % ) (see fig. 1 for the wild type). Removal of both cerulenin and Br' led to an increase in lipid content by 42% in less than 1 generation of growth, and this value, which is somewhat lower than even the rod form usually contains, remained constant for a further generation of growth. TABLE II.

Medium

No Br" + cerulenin +Br" +cerulenin No Br' No cerulenin

~ t ~ I ~ ~

--

The

action of cerulenin on rod and coeeal forms o f (( r o d B 1 0 4 >~.

Time (h)

Lipid dpm/A4a o • 10 -4

Cell d i m e n s i o n s Length Width

0 4 0 3 0 4

4.212 2.505 2.270 0.911 1.000 1.490

E P U (*) 86.6 81.9 101.4 96.0 93.6 49.2 72.0 65.2 72.0 65.2 78.6 74.8

Acial ratios

1.06 1.06 1.91 1.10 1.10 1.05

(*) Eyepiece u n i t s as m e a s u r e d b y a s p l i t - i m a g e eyepiece. Cerulenin (7.5 9 g / m l ) was a d d e d to t h e m i n i m a l salts glucose m e d i u m [21] either with M/15 K B r to p r o d u c e rods or w i t h o u t to p r o d u c e cocci. T h e m e d i a c o n t a i n e d 0.5 ~tmoles/ml glycerol with specific a c t i v i t y of 0.4 ~Ci/~mole as 2-3H-glycerol. I n c u b a t i o n was at 35 ~ C with s h a k i n g to aerate flasks c o n t a i n i n g 0.2 • v o l u m e of m e d i u m .

Thus, the cells remained coccal-shaped (axial ratio 1.10) without regaining their characteristic high lipid content. Both the axial dimensions of the cells increased (length 14%, diameter 23%) slightly. The ~ excess ), lipid was only slowly regained on longer cultivation. Thus, it would seem t h a t the rod mutation has two separate effects which are the conditional regulations of (1) a step in lipid biosynthesis and (2) a phospholipid hydrolase or an other enzyme removing phospholipid. It would be fascinating to discover whether the membrane protein characteristic of rod mutants which appeared to be processed [20] during changing from cocci to rods is indeed concerned with lipid metabolism. A further point of interest in this work is the apparently wide variation in membrane lipid content that the bacterial cell can tolerate. MOTS-CLI~S : Morphogen~se bact6rienne, Lipide; B. sublilis, C6rul6nine.

REFERENCES

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MORPHOLOGICAL CHANGES 1N B A C I L L U S

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SUBTILIS

83

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[21] ROOEI~S, H. J., THURMAN, P. F. & BUXTON, R. S., Magnesium and anion requirements of rod B mutants of Bacillus sublilis. J. Bacl., 1976, 125, 536-564. [22] RO6ERS, H. J., THURMAN, P. F. ~r REEVE, J. i . , Mucopeptide synthesis by rod mutants of Bacillus sublilis. J. gen. Microbiol., 1974, 85, 335-350. [23] SPRATT, B. G., Distinct penicillin binding proteins involved in the division, elongation and shape of E. colA. Proc. nat. Acad. Sci. (Wash.), 1975, 72, 2999-3003. [24] SPRATT, B. S., Penicillin binding proteins and the future of ~-lactam antibiotics. J. geu. Microbiol., 1983, 129, 1247-1260. [25] STARKA,J. • MORAVOVA,J., Phospholipids and cellular division of Escherichia colA. J. gen. Microbiol., 1970, 60, 251-257. [26] VANCE, D., GOLDBERG, I., MITSUHASHI, O., BLOCH, K., 0MURA, S. 3r NOMUt~A, S., Inhibition of fatty acid synthetases by the antibiotic cerulenin. Biochern. biophgs. Res. Commun., 1972, 48, 649-656. [27] VANDER~VINKEL, E., DE VLIEGHERE, M., FONTAINE, D. C., DENAMUR, G., VANDEVOORDE, D. • DE KEGEL, D., Septation deficiency and phospholipid perturbation in E. colA genetically constitutive for the D-oxidation pathway. J. Bacl., 1976, 127, 1389-1399.