Growth cycles of filamentous Escherichia coli

ZbI. Bakt. Abt. II, Bd.128, S.252-260 (197:1)

[Dairy Industry, Strakonice, CSSR]

Growth cycles of filamentous Escherichia coli I. Filamentous mutants Eva

Jicinska

With 9 figures

Summary Growth cycles of four filamentous mutants of Escherichia coli are described. Strains 10-5, 32-17, and 43-7 are conditional division mutants: cell division of these strains is derepressed under starvation or growth-limiting conditions. Batch cultures exhibit growth cycles, characterized by a filamentous phase and a proliferation phase in which the bacterial filaments undergo gradual fragmentation into short rods and coccoid cells. Fragmentation of filamentous cells occurs in cultures which are entering the stationary phase of growth. Comparable morphological changes also take place in ageing cultures on agar media. Fragmentation can be induced in young, rapidly growing cultures by transferring the filaments to media without a carbon or a nitrogen source. Two of the strains reverted to the rod-like morphology, when grown at a slow rate in a glucosesalt medium. Filaments of the strain 21-28 do not divide at the end of active growth, and fragmentation is not induced in carbon- or nitrogen-free media. The growth cycles of filamentous mutants are compared with life cycles of some naturally occurring filamentous prokaryotes, and adaptations pre-requisite for the evolution of filamentous forms are discussed. Morphological changes which occur in bacterial cultures upon ageing during transition from the exponential phase of growth to the stationary phase are usually designated as growth cycles. In some more highly evolved filamentous prokaryotes, the division in the logarithmic phase is more or less repressed and postponed to the phase of decreasing growth rate. Thus, their growth cycles are characterized by the alteration of a multi-cellular(vegetative) phase and a unicellular (proliferation) phase. Assuming that these higher forms originated from unicellular bacteria, the pre-requisite properties for the evolution of filamentous multi-cellular organisms would be the ability of the organism to grow in the absence of cell division (or cell separation), and to divide in the absence of growth. Both these pre-adaptations are already present in rod-shaped bacteria. The ability to form filaments is very widespread among unicellular bacteria (HUGHES 1956). Filamentous strains also develop as a result of mutations; many of these filamentous mutants appear to possess

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different genotypes as well as phenotypes (KOHIYAMA et al. 1966). On the other hand, cell division does occur in the absence of net growth under conditions of nitrogen starvation or shift down (KJELDGAARD et al. 1958). Both these capabilities are probably the result of a certain degree of mutual independence of the synthesis of macromolecular compounds - DNA, RNA, protein, and cell envelope - in bacteria. To combine these pre-adaptations into a growth cycle, a further condition must be met: the derepression of cell division when the growth rate declines. Evidence obtained in experiments with UV-induced filaments of E. coli B and chemically induced filaments of E. coli 32 and 15T- indicates that division is derepressed in poor media or at super-optimal temperatures in the lon- strains (WALKER and PARDEE 1967; .!\lJASNIK 1971), or under starvation conditions as well as during transition to the stationary phase of growth in the two other strains (JICINSKA 1972). It appears that in these filamentous forms cell division is repressed indirectly, the repression of division being mediated by partial inhibition of DNA and/or membrane synthesis. Thus, it can be assumed that some filamentous mutants of E. coli, in which division is only partly and indirectly blocked, would exhibit growth cycles, morphologically comparable to those of some higher filamentous bacteria. In this paper, isolation and some properties of such mutants are described.

Methods Bacterial strains. E. coli, strains 10, 32, and 43, originally isolated from milk. The strains were labelled with biochemical mutations. Morphological mutants wera isolated from these labelled strains and checked for the presence of the labe!. Labelled strains: lO-l(thi-), 32-3(met- or B 12-), 43-5(his-). E. coli strain 21, a natural variety "flava", forming a yellow pigment when grown at lower temperatures. Media. Meat extract-peptone-Iactose agar (Immuna), mineral salt-glucose medium M9 (ANDERSON 1946). M9C: M9 supplemented with casein hydrolysate (vitamin-free; KOCH and LIGHT). Casein and glucose were sterilized separately and added at a final concentration of 0.5 per cent (w/v) to the medium. M9-G: basal salt medium without added glucose. JYl9-N: M9 without added NH 4 Cl. Methods of culture. Stock cultures were maintained on meat extract-peptone agar slants, stored at 5°C, and subcultured monthly. Overnight growth on lactose agar slants, incubated at 30 DC, was employed as inoculum for batch cultures. Bacteria were suspended in saline, filtered through Synpore 1 membrane filter, and the filtrate was appropriately diluted to give a viable count of about 107 cells/m!. 0.5 m!. aliquots of filtrates were inoculated into 50 m!. M9C in 250 m!. ERLE":l'lE YER-flasks. The cultures were incubated at 25 DC with aeration provided by shaking the flasks on a reciprocating shaker-water bath at 90 oscillations/min. Plate counts. Samples of the cultures were diluted in steps in M9-G and plated on lactose agar. The colonies were counted after 48 h. of incubation at 30 DC. For each value of viable count, plating was done from at least four individual dilutions, adjusted to give between 50-300 colonies per plate. Staining and measurement of the cell length. Dried smears were fixed with formaldehyde vapours, which preserve well the cross walls, and stained with LOFFLER'S methylene blue. WEBB'S method was used for demonstrating cell walls and septa (\VEBB 1954). Estimations of the average filament lengths were made by means of a projection MP-microscope, equipped with a calibrated scale. Values in tables and graphs are means of 100 individual measurements. Estimation of protein and DNA. Samples of the cultures were chilled in an ice bath, and tr;chloroacetic acid added to a final concentration of 5 Fer cent. After 10 min., the samples were

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Eva Jicinskli

centrifuged at 5000 rpm. in the cold, washed twice with ice-cold distilled water, frozen at -20°C, and kept for further analysis. For protein determination, the pellets were treated with 1 m!. of 1 per cent sodium deoxycholate in 0.2 N NaOH for 5 min. at 100°C. Protein was assayed colorimetrically according to the procedure of LOWRY et a!. (1951). DNA was extracted with 1N perchloric acid at 70°C for 30 min. with regular shaking and determined colorimetrically with'modified DISCHE'S diphenylamine method (GILES and MYERS 1965). Specific growth rates are expressed as the rate of synthesis of bacterial protein. They were calculated from semilogarithmic plots of protein per 1 m!. of culture against time. Isolation of filamentous mutants. The cells were grown to log phase in M9C at 30°C, centrifuged, and re-suspended in 0.2M sodium acetate buffer (pH 5.0). 1 m!. amount of N-methyl-N'nitro-N-nitrosoguanidine solution (4 mg./m!.) was added to 10 m!. of bacterial suspension, and the mixture was incubated at 30°C for 3 h. The bacteria were then diluted 1: 100 into M9C and grown at 30°C for 8 h. Selection of filamentous mutants was performed as described by VAN DE PUTTE et a!. (1964), using membrane filters Synpore 1 (average pore diameter 4 [.Lm; Synthesia Semtin).

Results

Strain 32-17 was derived from the rod strain 32-3. The log phase population of batch cultures, growing in M9 C medium, is composed of filaments of various lengths (up to 50-60,um) and long rods. In the early log phase, septa are incomplete in most filaments; septal rudiments are usually spaced at "unit cell" distance along the length of the filaments. During transition to the stationary phase, the cells in the filaments become separated by thick cross walls, and the filaments break up into rods which divide further to produce short rods and coccoid cells (Fig. 1, 2, 3). The growth cycle in batch cultures is represented in Fig. 4.

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Fig. 1. Strain 32-17, late exponential phase in M9C. Fig. 2. Strain 32-17, early stationary phase in M9C.

Colonies on lactose agar are semi-transparent with irregular spreading margins, composed of long filaments. Central parts of colonies contain mostly short rods and fragments of filaments.

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Fig. 3. Strain 32.17, fragmentation in M9C.

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Colonies of the mutant 10-5 resemble those of 32 -17 : they are semi-transparent and rapidly spreading at the periphery. The population in the spreading area is polymorphous, composed of thick filaments, large club-shaped cells, ovoid cells, and a smaller number of rods. The centres of colonies include short rods and cocci. In M9C, the mutant has the following growth cycle: in the first half of the exponential phase the population is polymorphous, in the second half the number of short rods increases, and the filaments and large cells gradually divide into rods and coccoid cells (Fig. 5 and 6). Strain 43-7 has been derived from an amorphous (sphaerical) mucoid mutant of the strain 43-5 by selection of the semi-transparent, filament-forming type of colonies. In the early log phase, the cells are polymorphous. The population consists of large rods (8-12

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Eva Jicinska

x 1-3,um), often forked at the poles (Y-shaped cells), ovoid cells, and thick filaments. Transverse septa are either completely absent or only incipient. In the course of exponential growth, septated rods and filaments and shorter rods gradually appear in the culture. These break up into shorter fragments and rods which continue dividing to form large coccoid cells (Fig. 6, 7) in the stationary phase.

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Fig. 5. Strain 10-5, mid-log phase in M9C. Fig. 6. Strain 10-5, stationary phase in M9C.

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Fig. 7. Strain 43-7, stationary phase in M9C. Fig. 8. Strain 43-7, early log phase in M9C. Magnification X 1000. Stationary state cultures of filamentous mutants were filtered through Synpore 1 filters, and the filtrates, which contained only rods and cocci, were plated on lactose agar. No rod-shaped or coccoid revertants were found. Strain 21-18 produces filamep.ts and long rods during the exponential phase of growth. Most of the filaments are septated after prolonged incubation. Septa are distributed at equal intervals, corresponding to the length of the rods of the parent strain. The ratio rods/filaments increases somewhat in the stationary phase; however, no extensive

257

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fragmentation occurs at this stage, and growth terminates in autolysis of the filaments. The growth cycle of this mutant is represented in Fig. 9.

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Fig. 9. Growth cycle of E. coli 21-18 in M9C. Curve 1. Specific growth rate. Curve 2. Average cell length. Effect of carbon and nitrogen starvation. When bacteria of strains 10-5, 32-17 and 43-7, growing in exponential phase, are transferred to media whithout a carbon or a nitrogen source, cell division is stimulated, and the filaments fragment into rods and coccoid elements. Concomitantly, there is an increase in viable count and DNA content per unit volume of culture. The increase in viable count under these conditions is caused by at least three different effects: 1. the "shift-down effect", i. e., DNA synthesis and celldivision in the absence of net proteosynthesis (KJELDGAARD et al., 1958), 2. disintegration of bacterial clusters, formed as a result of the tendency of the bacteria to agglomerate and of the manipulation during transfer to starvation media, and 3. fragmentation of the filaments. The first two effects were also manifested in rod-shaped strains, but the third effect was evident in filamentous cultures, in which the increase in viable count was usually significantly greater than in the relevant parental strain, while the increase in DNA content was about the same in both strains (Table 1). The reason for this could be that fragmentation of the filaments is due merely to completion of cytokinesis, without division of the nuclei, and the rods, thus formed, further divide by the shift-down effect to smaller cells (according to microscopic observations, sometimes already inside the dis" integrating filaments). 17

Zhl. Bakt. Aht. II Bd.12H

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Table 1 Induction of cell division in cultures starved of nitrogen. Strain

Viable count per unit DNA/m!. bacterial protein culture

Protein/m!. culture

Mean cell length

10-5 10-1 32-17 32-3 43-7 43-5 21-18 21

285 172 367 212 172 180 188 210

103 110 109 116 102 112 115 118

31 47 29 59 44 62 78 56

206 187 196 192 139 203 205 209

Cells were grown to early log phase in M9C, filtered through a Synpore 1 filter at a low negative pressure, washed on the filter with equal volume of M9-N, and re-suspended in M9-N at a concentration of about 107 cells/m!. The suspensions were incubated for 3 h . at 25°C with shaking. Numbers in columns are per cent of initial values (initial values = 100). Average of 3 sets of experiments. Cells of the strain 43-7 tend to autolyse if transferred to liquid starvation media, and the increase of viable count is usually less than that of the parent strain. However, when cultures grown on membrane filters on the surface of M9C-agar are transferred to M9-N or M9-C agar, intensive fragmentation to coccoid cells is observed within 2-3 hours. Growth in M9 medium. The growth rate of the large-celled strain 43-7 is diminished in M9, supplemented with 50 flg histidine/ml., and at the same time the morphology of the mutant is completely normalized. Exponentially growing bacteria are short rods, indistinguishable from the parent strain cells growing in the same medium. Strain 10-5 grows in M9 with added thiamine (2 flg/ml.) as small coccoid cells; no club-shaped cells or filaments are formed. Contrary to this, mutant 32-17 remains filamentous in the logarithmic phase in M9 at all methionine concentrations which support growth (2-100 flg/ml.), though the growth rate is also decreased in this medium, as compared to the growth rate in M9C. Discussion

Filamentous mutants 10-1, 32-17, and 43-7 are characterized by partial repression of cell division in the exponential phase of growth and a greater correlation of the ability t~ divide the environmental conditions which limit growth. In this respect they resemble certain conditional lon- mutants, studied by WALKER and PARDEE (1967), in which division also is derepressed under specific environmental conditions. To growth-limiting conditions which occur in exhausted media, and which are produced by the bacteria themselves, the mutants react by activating the division; their growth cycles thus exhibit elementary "vegetative" and "reproductive" phases. The PQssible cause of this entrainment of cell division to growth-limiting conditons might be the inhibition of cell division by unbalanced synthesis of different macromolecular components (unbalanced growth). Indeed, several authors have expressed the view that- in some mutants at least - it is this unbalance per se rather than any specific primary effect which leads to division inhibition and filament formation (KANTOR and DEERING 1968;

Growth cycles of filam( ntcus Escherichia coli

259

MJASNIK 1971). Many of the filament-forming mutants, so far studied, appear to have an impaired DNA or envelope synthesis (HIROTA et al. 1968, 1970; HUANG and GOODMAN 1970; KARAMATA and GROSS 1970; MACH and ENGELBRECHT 1970). If these syntheses are partly inhibited by mutations, a metabolic unbalance may develop during rapid growth in a rich medium. This type of inhibition will be abolished under conditions favouring preferentially synthesis of the deficient macromolecular component, i. e., DNA and cell envelope. The rate of synthesis of these cellular components appears to be relatively accelerated under starvation conditions. This point is discussed in greater detail in connection with growth cycles of induced filaments. It is probable that mutants, carrying a defect in the division mechanism itself, cannot be activated by growth-lirniting conditions. Strain 21-18 may represent such a mutation.

Zusammenfassung Wachsturnszyklell vier filament6ser Mlitallten von Escherichic! coli in Batch-Kulturen wurden studiert. Wachstumszyklen dreier dieser Mlitanten (Stamme 10-5, 32-17 und 43-7) bestehen aus einer filament6sell und einer Vermehrungsphase, in der Kurzstabchen und Cocci durch eine allmahliche Fragmentation der filamentosen Zellen gebildet werden. Filamentteilung und l!'ragmentation findet am Anfang der stationaren Phase statt. Dieselben morphologischen Verande. rungen kOlmten auch in alteren Agarkulturen beobachtet werden. Man kann die Filamentfrag. mentation in logarithmisch wachsenden Klilturen induzieren, indem man die filament6sen Zellen aus diesen Kulturen in ein Stickstoff- odeI' Kohlenstoff-Mangelmedium iibertragt. 1m Glucose. Salzmedium, in dem das Wachstum del' Mutanten verlangsamt wird, bilden zwei Stamme (10.5 und 43-7) nur Kurzstabchen auch in del' logarithmischen Phase. ZellteiJung und Fragmentation konnte in den Filamenten der Mutante 21-18 weder durch Eintritt del' stationaren Wachstumsphase noch durch Stickstoff oder KohJenstoffmangel induziert werden. Die Wachstumszyklell der filamentiisen Mutanten werden mit den :Lebenszyklen hoherer filamentoscr Prokaryonten verglichen und die fi.ir filamen.tcse Lehndolm erfOlderlichen Preadaptationnl erwogen.

Literature ANDEHSON, E.H.: Growth requirements of virus-resistant mutants of E.coli. Proe. Natn. Acad. Sei. WaEh 32 (19'16),120. - GILES, W.K., and MYEHS, A.: An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature, Lond. 206 (1965), 93. - HIROTA, Y, RYTER, A., and .TACOB, F.: Thermosensitive mutants of E. coli affected in the process of DNA synthesis and cell division. Cold Spring Harbor Symp. Quant. BioI. 33 (1968), 677. - HIROTA, Y, MORDOR, J., and JACOB, F.: On the proccss of cellular division in Escherichia coli. III. Thcrmosensitive mutants of Escherichia coli, altered in the process of DNA initiation. J. Mol. BioI. 53 (1970), 369. - HUANG, P., and GOODMAN, R.N.: :Morphology and ultrastructure of normal rodshaped and filamentous forms of Erwinia, amylovora. J. Bacteriol. 102 (1970), 862. - HUGHES, W. H.: The structure and development of induced long forms of bacteria. In: Bacterial Anatomy. 6th Symp. Soc. gen. :Microbiol. (ed. SPOONER, E. T. C., and KAPLAN, N. 0.): Cambridge Univ. Press 1956, 341. - J ICINSKA, E.: Growth cycles of filamentous Escherichia coli. II. Induced filaments. Zbl. Bakt. II 128 (1973), 261. KANTOR, J., and DEERING, R. A.: Effect of nali. dixic acid and hydroxyurea on division ability of Escherichia coli fiJ'- and Jon- strains. J. Bacteriol. 95 (1968),520. - KARAMATA, D., and GHOSS, J.D.: Isolation and genetic analysis oftemperature. sensitive mutants of B.subtil-is, defective in DNA synthesis. Mol. Gen. Genet. 108 (1970),277.KJELDC1AARlJ, N. 0., MAALOE, 0., and SCHAECHTEH, M.: The transition between different physiological states eluring balanced growth of Salmonella typhimurimn. J. gen. Microbiol. 19 (1958), 607. - KOHIYAMA, M., CoriSIN, D., RI'TEll, A., and JACOB, F.: Mutants thermosensibles d'Escherichia coli K12. 1. Isolement et caracterisation rapide. AnnIs. Inst. PASTEUR no (1966), 41i5. _ 17'

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LOWRY, D.H., ROSEBROUGH, N.J., FARR, A.L., and RA:'!DALL, R.J.: Protein measurement with the FOLIN phenol reagent. J. BioI. Chem.193 (1951),265. - MACH, F., and ENGELBRECHT, H.: Isolation und erste Charakterisierung einer temperatursensitiven filament6sen Mutante von Bacillus subtilis SB19. Z. Allg. Mikrobiol. 10 (1970), 383. - MJASNIK, M.N.: 0 prirode vlijanija genov fil+, uvr- i exr- na radiocuvstvitel'nost' Escherichia coli. Genetika 7 (1971), 59. - VAN DE PUTTE, P., VAN DILLEWIJN, J., and RonscH, A.: The selection of mutants of Escherichia coli with impaired division at elevated temperatures. Mut. Res. 1 (1964), 121. - WALKER, J. R., and PARDEE, A.B. Conditional mutations involving septum formation in Escherichia coli. J. Bacteriol 93 (1967),107. - WEBB, R.B.: A useful bacterial cell wall stain. J. Bacteriol. 67 (1954), 252.

Author's address: Dr. Eva Jicinska, Dairy Industry Strakonice (CSSR)