- Email: [email protected]
Growth cycles of filamentous Escherichia coli
Zbl. Bakt. Abt. II, Bd.128, S. 261-267 (1973)
[Dairy Industry, Strakonice, CSSR]
Growth cycles of filamentous Escherichia coli II. Induced filaments
Eva Jiclnskli With 2 figures
Summary The effect of inhibition of cell division by gentian violet and thymine deficiency on the course of division of two E.coli strains, growing in batch cultures, was studied. These factors inhibited division and induced filament formation only in the pre-log phase and the exponential phase of growth. In the late logarithmic phase, the division was spontaneously derepressed even in the presence of inhibitor or thymine deficiency, and the filaments broke up into rods. Division of the filaments in the early log phase can be induced in the presence of the dye or thymine deficiency by transfering the filaments to media without any added carbon- or nitrogen source. In the thymutant, filament formation was suppressed during growth in a synthetic minimal medium with a low thymine concentration. Derepression of cell division under growth-limiting conditions is not caused by adaptation, selection of mutants or impermeability for the dye. The hypothesis is expressed that inhibition of the cell division, which can be reversed by growth-limiting conditions, acts indirectly on the division mechanism by way of unbalanced macromolecular synthesis. The preceding report described filamentous mutants of Escherichia coli which, during growth in a batch culture, displayed morphological changes similar to the growth cycles of coryneform bacteria and Nocardias. The population in the exponential phase consists mainly of elongated rods and filaments; as it enters the stationary phase of growth, the filaments break up into short rods and coccobacilli. Transfer of the bacteria to a growthlimiting medium significantly stimulates fragmentation of the filaments. Derepression of cell division under starvation conditions is not limited to filamentous mutants. It also occurs in filamentous cultures, induced by external factors, presumably inhibiting DNA or cell envelope synthesis. The following experiments show that cultures of Escherichia coli in which cell division was partly blocked by gentian violet or by thymine deficiency exhibit similar morphological changes in batch cultures as conditional filamentous mutants.
Methods Bacteria: Escherichia coli 15T- (thy- agr- ura-); Escherichia coli strain 32 which was used in the previous study for isolating mutants. The nutrient media and the methods employed for cultivation, measuring growth, and determining protein and DNA were described in the previous paper (JICINSKA 1972). Chemicals; gentian violet (MERCK, Darmstadt), thymine (LACHEMA Brno).
262
Eva JiiSinsk:i
Results
Growth cycle of E. coli 32 in the presence of gentian violet. Gentian violet was supplied at 2.5 fig/ml. final concentration to the medium lVI 9 C before starting cultivation. This concentration of the dye still allowed good growth, but it inhibited the division and induced filament formation. The logarithmic phase population consisted of elongated rods and filaments of varying length. In the early exponential phase, most of the filaments displayed only initial stages of septum formation. Septal rudiments were often localized at regular intervals, corresponding to the length of the rods of the untreated control. In the late log phase, many of the filaments had complete, thickened cross walls, and were commited to division. This was manifested after transferring the filaments to fresh nutrient media, containing gentian violet. Within 2-3 h. of incubation the late log phase filaments broke up into rods, which only then, after the filaments had disintegrated, grew again into new filaments, whereas cells in the early exponential phase continued with filamentous growth. 10
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Fig. 1. Growth cycle of E.coli 32 in M9C with added gentian violet and in :M9C. Curve 1. M9C, average cell length. Curve 2. M9C, specific growth rate. Curve 3. M9C plus gentian violet, average cell length. Curve 4. :M9C plus gentian violet, specific growth rate. The outset of the stationary phase is characterized by filament fragmentation. The stationary phase cultures show a predominance of rods and pairs of coccoid cells with a few autolysing filaments (Fig. 1). To determine whether adaptation or selection of resistant mutants (which might lead to a preponderance of rods and to a decrease of filament formation in the late phase of growth) occurred during the experiments, stationary phase cultures were filtered through Synspore 1 membrane filters, and fresh gentian violet media were inoculated with the filtrates (0.1 ml. per 20 mI.). Microscopic examination revealed only short rods in the filtrate. Growth in the second and third subculture followed the same course as in the primary culture (Table 1): rods in the inoculum grew in the logarithmic phase into filaments which broke up again in the stationary phase.
263
Growth cycles of filamentous Escherichia coli Table l
Serial transfers of strain 32 to M9-medium, containing gentian violet. Cells were grown in M9C plus 2.5 [lg gentian violet/m!. at 25°C to stationary phase, filtered, and the filtrate employed as inoculum for the next subculture. Subculture No.
Average cell length, [lm Mid-log phase
1 2 3
11.6
Stationary phase 3.1 2.8 3.0
9.8 8.7
Inoculation of gentian violet-lactose agar plates with a stationary phase culture failed to show the presence of resistant non-filamenting mutants. Effect of starvation conditions. 5 ml. of an exponentially growing filamentous culture in M9C, supplemented with gentian violet, or 15 ml. of a rod culture in M9C, was filtered through a Synpore 1 membrane filter. The amount of rod culture, required to obtain about the same cell density, is larger, since few rods are captured on the loose filter. The filter was washed with 10 ml. of sterile starvation medium, and a further 10 ml. portion was used to wash the bacteria off the filter. The suspension was diluted with the starvation medium to a density of about 10' cells/ml., and 10 ml. aliquots in 100 ml. ERLE~MEYER-flasks were incubated in the same way as the batch cultures. The starvation media used were nitrogen-free .M9 (.M9-N), supplemented with 2.5 pg of gentian violet per ml., and .M9-N without added gentian violet. The transfer to starvation media resulted in an extensive disintegration of the filaments into rods and coccoid pairs within 3 h. of incubation, with a concomitant increase in viable count, DNA content per ml. of culture, and a decrease in DNA to the protein ratio. However, the increase in viable count was always higher in the absence of the dye. This indicates that gentian violet impairs cell metabolism even under starvation conditions; thus the derepression of cell division is not due to impermeability for the dye under these conditions. The effects of starvation conditions On filamentous and rod cultures are summarized in Table 2 and Fig. 2. Table 2 Induction of cell division in cultures starved of nitrogen. Cells were grown to early exponential phase in M9C plus 2.5 [lg gentian violet/m!. or in M9C, transferred by filtration to M9-N with or without added gentian violet and shaken for 3 h . at 25°C. Numbers in columns are per cent of the initial value (initial value = 100). Culture
Induction medium
Filaments early log phase, M9-N plus M9C plus gentian violet gentian violet Filaments early, log phase, M9-N M9C plus gentian violet Rods early log phase, M9-N M9C
Viable count per unit bacterial protein
DNA/m!. culture
Protein/m!. Average cell culture length
409
184
104
26
482
189
107
22
217
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III
61
264
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Fig. 2. Increase in viable count in cultures starved of nitrogen. Curve"I. Control: rods from M9C, transferred to M9-N. Curve 2. Filaments from M9C, containing 2.5 fLg gentian violet/ml., transferred to M9-N with equal concentration of gentian violet.
Effect of thymine deficiency. In M9 C medium, supplemented with uracil (20 /kg/mI.), the growth rate as well as the cell morphology of the strain 15T- are dependent on thymine concentration. In the presence of a low thymine concentration, 5 flg/mI., the exponential phase was characterized by the growth of elongated rods and filaments with partly formed or complete thickened septa. During the cycle the filaments broke up into short fragments, and at the outset of the stationary phase the population was composed of short rods and some autolysing filaments. The transfer of early log phase filaments to nitrogen-free M9, containing 5 /kg thymine/mI., induced fragmentation of the filaments. The presence of the other two growth factors was also required for division; if thymine alone was supplied, the division of filaments was incomplete. In the absence of thymine, the filaments underwent autolysis, and there was a rapid reduction in the viable count. Filaments were also formed in the presence of 20 /kg thymine/mI., but only in the early exponential phase. In the second half of the log phase, the population consisted of rods, though occasional filaments appeared throughout the whole of the cycle. In M9 medium, supplemented with 20/kg uracil, 50 /kg arginine, and 5 /kg thymine/mI., bacteria grew throughout the cycle in the form of rods 2-3 /km long; no filaments were present in the exponential phase. The growth ofthe mutant 15T- is saturated with thymine already at 5/kg thymine/ml. in this medium; however, the growth rate is substantially decreased, as compared to the growth rate in M9C, supplemented with 5 /kg thymine/ml. (Table 3). All stationary phase cultures were currently checked for the presence of thymine prototrophs. The results described above were obtained with cultures where no such thy+ revertants have been detected.
265
Growth cycles of filamentous Escherichia coli
Table 3 Growth rates and morphology of the strain 15T- under different growth conditions. II. Cells were grown to early log phase in :M:9C plus 5 fLg thymine/ml., transferred to :M:9-N pillS 5 fLg thymine/m!. (supplemented with arginine and uracil), and incubated for 3 h . at 25°C with shaking.
1. Batch cultures-logarithmic phase Average cell length pm
:M:ldium
9.2 4.5 2.9 3.1
M9C, 5 f.lg thymine/ml, M9C,20 f.lg thymine/m!. M9, 5fJg thymine/m!. M9, 20f.lg thymine/m!.
Specific growth rate, h- 1 0.46 0.89 0.33 0,34
II. Nitrogen-starved cultures. Viable count per unit bacterial protein
DNA/m!.
325
198
Protein/m!. 122
Average cen length 34,6:
Initial values = 100 per cent.
Discussion The formation offilamentous cells by Escherichia coli and other bacteria is a response to many different unfavourable environmental factors (HUGHES 1956) and is probably the most common morphological reaction of bacteria to such conditions. Inhibition of cell division with subsequent filament formation is also caused by numerous mutations. Recent evidence indicates that many of these factors block the cell division by mechanisms which impair DNA or cell envelope synthesis. The regulatory significance of the arrest of cell division in the absence of DNA synthesis is borne out by experiments on E. coli and B. subtilis mutants, which are unable to stop dividing when DNA replication is inhibited, and consequently produce DNA-less cells (ADLER et al. 1967; INOUYE and GUTHRIE 1969; HUANG and GOODMAN 1970; DONAOHIE et al. 1971; INOUYE 1971; SPRATT and RowBURY 1971). Filament formation may thus have an adaptive character, protecting the cells from some deleterious effect of a metabolic unbalance. Crystal violet-induced filamentous cells of the E. coli lon- mutants are capable of dividing in a minimal medium, yet not in enriched broth in the presence of the dye (WALKER et al. 1971). The results of experiments on chemically induced filaments, and observations on lon- conditional mutants (WALKER and PARDEE 1967; MJASNIK 1971) and filamentous mutants of E. coli (J IOINSKA 1972) show that inhibition of cytokinesis is, in some cases, closely correlated to growth conditions. It can be derepressed in the stationary phase of growth, under conditions preventing growth, and in poor or nutrientdeficient media. These are also conditions which preferentially promote DNA and/or cell envelope synthesis (KJELDGAARD et al. 1958; HERBERT 1961; WINDER and O'HARA 1962; SUD and SCHAECHTER 1964; SHOOKMAN 1965; KOUKALOVA et al. 1970), whereas protein and RNA syntheses are inhibited or become growth rate-limiting factors (MANDELSTAM 1958, 1960; HARRISON and LAWRENOE 1963; GONZALEZ et al. 1968; GOLDBERG 1971; NATH and KOCH 1971). Several authors described or suggested mechanisms of division inhibition in E. coli which are dependent upon, or activated by inhibition of DNA synthesis (WITKIN 1967; HIROTA et al. 1968; GREEN et al. 1969). It is thus possible
266
Eva Jicinska
that various types of mutations and inhibitors, whose effect on bacterial division is influenced by growth conditions, act on the cytokinesis indirectly by means of some common mechanism, which is associated with unbalanced growth. The author is indebted to Dr. VONlJHEJS for strain 15T-. The teehnieal assistanee of Mrs. H. SLAVIKOVA is aeknowledged.
Zusammenfassung Die Einwirkung von Gentianaviolett und Thyminmangel auf die Hemmung der Zellteilung und den Veriauf derselben von Escherichia coli in Bateh-Kulturen wurde studiert. Diese Faktoren hemmen die Zellteilung und induzieren Fadenbildung nur in der pre-logarithmischen und logarithmischen Phase des Wachstums; in der spaten logarithmischen Phase wurde die Zeliteilung spontan reaktiviert auch unter den Bedingungen eines Thyminmangels oder bei Anwesenheit eines Inhibitors. Die Filamente fragmentieren unter Bildung von Kurzstabchen. Durch Ubertragung der fadenf6rmigen Zellen aus der fruhen logarithmisehen Phase in ein Stickstoff- oder Kohlenstoff-freies Minimalmedium konnte Filamentteilung bei Anwesenheit eines Inhibitors oder bei Thyminmangel induziert werden. Die Neuerwerbung der Teilungsfahigkeit in der spaten logarithmischen Phase wurde nicht durch Adaptation, Selektion von Mutanten oder Impermeabilitat fur Gentianaviolett verursacht.
Literature AD LEE, H.1., FISHEH, ,"V. D., COHEN, A., and HARDIGREE, A.A.: :Miniature Escherichia coli cells deficient in DNA. Proe. Natl. Acad. Sei. U. S. 57 (1967), 321. - DONACHIE, W.D., MARTIN, D. T. M., and BEGG, K. J.: Independence of cell division and DNA replication in Bacillus subtilis. Nature (New BioI.) Lond. 231 (1971), 274. - GOLDBERG, A.L.: A role of aminoacyl-tRNA in the regulation of protein breakdown in Escherichia coli. Proe. Natn. Acad. Sei. U. S. 68 (1971),362. GONZALEZ, N. S., GOLDENBERG, S. H., and ALOEA:'iATI, 1. D.: Protein synthesis and ribosomal distribution at different growth stages in Bacillus stearothermophilu8. Biochim. Biophys. Acta 166 (1968), 760. - GREEN, M.H.L., DO:'iCH, J., and GEEENBEEG, J.: An immediate effect of inhibition of DNA synthesis on cell division in a Ion strain of Escherichia coli. Mut. Res. 8 (1969), 409.HAHRISON, P.A., and LAWRENCE, F.R.: Phenotypic, genotypic and chemical changes in starving populations of Aerobacter aerogenes. J. Bacteriol. 85 (1963), 742., - HEHBERT, D.: The chemical composition of micro-organisms as a function of their environment. In: Microbial Reaction to Environment. 11th Symp. Soc. gen. Microbiol. (ed. MEYNELL, G. G., and GOODER, H.): Cambridge Univ. Press, 1961,391. - HIRO'rA, Y., JACOB, F., RYTii:R, A., BUTTIN, G., and NAKAI, T.: On the process of cellular division in Escherichia coli. 1. Asymmetrical cell division and production of deoxyribonucleic acid-less bacteria. J. Mol. BioI. 35 (1968), 175. - H1:ANG, P., and GOODMAN, R.N.: Morphology and ultrastructure of normal rod-shaped and filamentous forms of Erwinia amylovora. J. Bacteriol. 102 (1970), 862. - HUGHES W.H.: The structure and development of induced long forms of baeteria. In: Bacterial Anatomy. 6th Symp. Soc. gen. Microbiol. (ed. SPOONER, E. T. C., and KAPLAN, N. 0.), Cambridge Un,iv. Press 1956, 341. - INOUYE, M.: Pleiotropic effect of the recA gene in uncoupling of cell division from deoxyribonucleic aeid replication. J. Bacteriol. 106 (1971), 539. - INOUYE, M., and GUTHRIE, J.P.: A mutation which changes a membrane protein of E.coli. Proc. Natn. Acad. Sci. U. S. 64 (1969), 957. - JICINSKA, E.: Growth cycles of filamentous Escherichia coli. 1. Filamentous mutants. Zbl. Bakt. II 128 (1973), 252. - KJELDGAARD, N.O., MAALOE, 0., and SGHAECHTER, M.: The transition between different physiologieal states during balanced growth of Salmonella typhimurium. J. gen. Microbiol. 19 (1958),607. - KOURALOVA, B., SOSRA, J., and REICH, J.: Excessive DNA synthesis in Lactobacillus acidophilus during amino acid starvation. Folia Microbiol. 14 (1970), 536. - MANDELSTAM, J.: Turnover of protein in growing and non-growing populations of Escherichia coli. Bioehem. J. 69 (1958), 110. -1\L~NDELSTAM, J.: The intracellular turnover of protein and nucleic aeids and its
Growth cycles of filamentous Escherichia coli
267
rolc in biochemical differentiation. Bact. Rev. 24 (1960), 289. - M,JASNIK, 1"LN.: 0 prirode vlijanija genov fil+, uvr- i exr- na radiocuvstvitel'nost' Escherichia coli. Genetika 7 (1971), 59. NATH, K., and KOCH, A. L.: Protein degradation in Escherichia coli. II. Strain differences in the degradation of protein and nucleic acid resulting from starvation. J. BioI. Chern. 246 (1971), 6956.SHOCK!\IAN, G.D.: Unbalanced cell wall synthesis: autolysis and cell-wall thickening. Bact. Rev. 29 (1965), 345. - SPRA'l'T, B. G., and ROWBlJRY, R. J.: Cell division in a mutant of Salmonella typhimurium which is temperature-sensitive for DNA synthesis. J. gen. Microbiol. 65 (1971), 305. - SUD, J. J., and SCHAECHTER,.M.: Dependence of the content of cell envelopes on the growth rate of Bacillus megatherium. J. Bacteriol. 88 (1964), 1612. - WALKEI(, J.R., and PARDEE, A. B.: Conditional mutations involving septum formation in Escherichia coli. J. Bacteriol. 93 (1967), 107. - VVALKER, J.R., SHAFIQ, N.A., and ALLE:", R. G.: Bacterial cell division regulation. Physiological effects of crystal violet on Escherichia col'i lon+ and lon- strains. J. Bacteriol. 108 (1971), 1296. - WINDEH, F. G., and O'HARA, C.: Effects of iron deficiency and zinc deficiency on the composition of XI ycobacterium smegrnatis. Biochem. J. 82 (1962), 98. - WITKIN, E..M.: The radiation sensitivity of Escherichia coli B. A hypothesis relating filament formation and prophage production. Proc. Natn. Acad. Sci. U. S. 57 (1967), 1275.
Authors' address: Dr. Eva Jicinska, Dairy Industry, Strakonice (eSSR).
[Dairy Industry, Strakonice, CSSR]
Growth cycles of filamentous Escherichia coli II. Induced filaments
Eva Jiclnskli With 2 figures
Summary The effect of inhibition of cell division by gentian violet and thymine deficiency on the course of division of two E.coli strains, growing in batch cultures, was studied. These factors inhibited division and induced filament formation only in the pre-log phase and the exponential phase of growth. In the late logarithmic phase, the division was spontaneously derepressed even in the presence of inhibitor or thymine deficiency, and the filaments broke up into rods. Division of the filaments in the early log phase can be induced in the presence of the dye or thymine deficiency by transfering the filaments to media without any added carbon- or nitrogen source. In the thymutant, filament formation was suppressed during growth in a synthetic minimal medium with a low thymine concentration. Derepression of cell division under growth-limiting conditions is not caused by adaptation, selection of mutants or impermeability for the dye. The hypothesis is expressed that inhibition of the cell division, which can be reversed by growth-limiting conditions, acts indirectly on the division mechanism by way of unbalanced macromolecular synthesis. The preceding report described filamentous mutants of Escherichia coli which, during growth in a batch culture, displayed morphological changes similar to the growth cycles of coryneform bacteria and Nocardias. The population in the exponential phase consists mainly of elongated rods and filaments; as it enters the stationary phase of growth, the filaments break up into short rods and coccobacilli. Transfer of the bacteria to a growthlimiting medium significantly stimulates fragmentation of the filaments. Derepression of cell division under starvation conditions is not limited to filamentous mutants. It also occurs in filamentous cultures, induced by external factors, presumably inhibiting DNA or cell envelope synthesis. The following experiments show that cultures of Escherichia coli in which cell division was partly blocked by gentian violet or by thymine deficiency exhibit similar morphological changes in batch cultures as conditional filamentous mutants.
Methods Bacteria: Escherichia coli 15T- (thy- agr- ura-); Escherichia coli strain 32 which was used in the previous study for isolating mutants. The nutrient media and the methods employed for cultivation, measuring growth, and determining protein and DNA were described in the previous paper (JICINSKA 1972). Chemicals; gentian violet (MERCK, Darmstadt), thymine (LACHEMA Brno).
262
Eva JiiSinsk:i
Results
Growth cycle of E. coli 32 in the presence of gentian violet. Gentian violet was supplied at 2.5 fig/ml. final concentration to the medium lVI 9 C before starting cultivation. This concentration of the dye still allowed good growth, but it inhibited the division and induced filament formation. The logarithmic phase population consisted of elongated rods and filaments of varying length. In the early exponential phase, most of the filaments displayed only initial stages of septum formation. Septal rudiments were often localized at regular intervals, corresponding to the length of the rods of the untreated control. In the late log phase, many of the filaments had complete, thickened cross walls, and were commited to division. This was manifested after transferring the filaments to fresh nutrient media, containing gentian violet. Within 2-3 h. of incubation the late log phase filaments broke up into rods, which only then, after the filaments had disintegrated, grew again into new filaments, whereas cells in the early exponential phase continued with filamentous growth. 10
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Fig. 1. Growth cycle of E.coli 32 in M9C with added gentian violet and in :M9C. Curve 1. M9C, average cell length. Curve 2. M9C, specific growth rate. Curve 3. M9C plus gentian violet, average cell length. Curve 4. :M9C plus gentian violet, specific growth rate. The outset of the stationary phase is characterized by filament fragmentation. The stationary phase cultures show a predominance of rods and pairs of coccoid cells with a few autolysing filaments (Fig. 1). To determine whether adaptation or selection of resistant mutants (which might lead to a preponderance of rods and to a decrease of filament formation in the late phase of growth) occurred during the experiments, stationary phase cultures were filtered through Synspore 1 membrane filters, and fresh gentian violet media were inoculated with the filtrates (0.1 ml. per 20 mI.). Microscopic examination revealed only short rods in the filtrate. Growth in the second and third subculture followed the same course as in the primary culture (Table 1): rods in the inoculum grew in the logarithmic phase into filaments which broke up again in the stationary phase.
263
Growth cycles of filamentous Escherichia coli Table l
Serial transfers of strain 32 to M9-medium, containing gentian violet. Cells were grown in M9C plus 2.5 [lg gentian violet/m!. at 25°C to stationary phase, filtered, and the filtrate employed as inoculum for the next subculture. Subculture No.
Average cell length, [lm Mid-log phase
1 2 3
11.6
Stationary phase 3.1 2.8 3.0
9.8 8.7
Inoculation of gentian violet-lactose agar plates with a stationary phase culture failed to show the presence of resistant non-filamenting mutants. Effect of starvation conditions. 5 ml. of an exponentially growing filamentous culture in M9C, supplemented with gentian violet, or 15 ml. of a rod culture in M9C, was filtered through a Synpore 1 membrane filter. The amount of rod culture, required to obtain about the same cell density, is larger, since few rods are captured on the loose filter. The filter was washed with 10 ml. of sterile starvation medium, and a further 10 ml. portion was used to wash the bacteria off the filter. The suspension was diluted with the starvation medium to a density of about 10' cells/ml., and 10 ml. aliquots in 100 ml. ERLE~MEYER-flasks were incubated in the same way as the batch cultures. The starvation media used were nitrogen-free .M9 (.M9-N), supplemented with 2.5 pg of gentian violet per ml., and .M9-N without added gentian violet. The transfer to starvation media resulted in an extensive disintegration of the filaments into rods and coccoid pairs within 3 h. of incubation, with a concomitant increase in viable count, DNA content per ml. of culture, and a decrease in DNA to the protein ratio. However, the increase in viable count was always higher in the absence of the dye. This indicates that gentian violet impairs cell metabolism even under starvation conditions; thus the derepression of cell division is not due to impermeability for the dye under these conditions. The effects of starvation conditions On filamentous and rod cultures are summarized in Table 2 and Fig. 2. Table 2 Induction of cell division in cultures starved of nitrogen. Cells were grown to early exponential phase in M9C plus 2.5 [lg gentian violet/m!. or in M9C, transferred by filtration to M9-N with or without added gentian violet and shaken for 3 h . at 25°C. Numbers in columns are per cent of the initial value (initial value = 100). Culture
Induction medium
Filaments early log phase, M9-N plus M9C plus gentian violet gentian violet Filaments early, log phase, M9-N M9C plus gentian violet Rods early log phase, M9-N M9C
Viable count per unit bacterial protein
DNA/m!. culture
Protein/m!. Average cell culture length
409
184
104
26
482
189
107
22
217
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Fig. 2. Increase in viable count in cultures starved of nitrogen. Curve"I. Control: rods from M9C, transferred to M9-N. Curve 2. Filaments from M9C, containing 2.5 fLg gentian violet/ml., transferred to M9-N with equal concentration of gentian violet.
Effect of thymine deficiency. In M9 C medium, supplemented with uracil (20 /kg/mI.), the growth rate as well as the cell morphology of the strain 15T- are dependent on thymine concentration. In the presence of a low thymine concentration, 5 flg/mI., the exponential phase was characterized by the growth of elongated rods and filaments with partly formed or complete thickened septa. During the cycle the filaments broke up into short fragments, and at the outset of the stationary phase the population was composed of short rods and some autolysing filaments. The transfer of early log phase filaments to nitrogen-free M9, containing 5 /kg thymine/mI., induced fragmentation of the filaments. The presence of the other two growth factors was also required for division; if thymine alone was supplied, the division of filaments was incomplete. In the absence of thymine, the filaments underwent autolysis, and there was a rapid reduction in the viable count. Filaments were also formed in the presence of 20 /kg thymine/mI., but only in the early exponential phase. In the second half of the log phase, the population consisted of rods, though occasional filaments appeared throughout the whole of the cycle. In M9 medium, supplemented with 20/kg uracil, 50 /kg arginine, and 5 /kg thymine/mI., bacteria grew throughout the cycle in the form of rods 2-3 /km long; no filaments were present in the exponential phase. The growth ofthe mutant 15T- is saturated with thymine already at 5/kg thymine/ml. in this medium; however, the growth rate is substantially decreased, as compared to the growth rate in M9C, supplemented with 5 /kg thymine/ml. (Table 3). All stationary phase cultures were currently checked for the presence of thymine prototrophs. The results described above were obtained with cultures where no such thy+ revertants have been detected.
265
Growth cycles of filamentous Escherichia coli
Table 3 Growth rates and morphology of the strain 15T- under different growth conditions. II. Cells were grown to early log phase in :M:9C plus 5 fLg thymine/ml., transferred to :M:9-N pillS 5 fLg thymine/m!. (supplemented with arginine and uracil), and incubated for 3 h . at 25°C with shaking.
1. Batch cultures-logarithmic phase Average cell length pm
:M:ldium
9.2 4.5 2.9 3.1
M9C, 5 f.lg thymine/ml, M9C,20 f.lg thymine/m!. M9, 5fJg thymine/m!. M9, 20f.lg thymine/m!.
Specific growth rate, h- 1 0.46 0.89 0.33 0,34
II. Nitrogen-starved cultures. Viable count per unit bacterial protein
DNA/m!.
325
198
Protein/m!. 122
Average cen length 34,6:
Initial values = 100 per cent.
Discussion The formation offilamentous cells by Escherichia coli and other bacteria is a response to many different unfavourable environmental factors (HUGHES 1956) and is probably the most common morphological reaction of bacteria to such conditions. Inhibition of cell division with subsequent filament formation is also caused by numerous mutations. Recent evidence indicates that many of these factors block the cell division by mechanisms which impair DNA or cell envelope synthesis. The regulatory significance of the arrest of cell division in the absence of DNA synthesis is borne out by experiments on E. coli and B. subtilis mutants, which are unable to stop dividing when DNA replication is inhibited, and consequently produce DNA-less cells (ADLER et al. 1967; INOUYE and GUTHRIE 1969; HUANG and GOODMAN 1970; DONAOHIE et al. 1971; INOUYE 1971; SPRATT and RowBURY 1971). Filament formation may thus have an adaptive character, protecting the cells from some deleterious effect of a metabolic unbalance. Crystal violet-induced filamentous cells of the E. coli lon- mutants are capable of dividing in a minimal medium, yet not in enriched broth in the presence of the dye (WALKER et al. 1971). The results of experiments on chemically induced filaments, and observations on lon- conditional mutants (WALKER and PARDEE 1967; MJASNIK 1971) and filamentous mutants of E. coli (J IOINSKA 1972) show that inhibition of cytokinesis is, in some cases, closely correlated to growth conditions. It can be derepressed in the stationary phase of growth, under conditions preventing growth, and in poor or nutrientdeficient media. These are also conditions which preferentially promote DNA and/or cell envelope synthesis (KJELDGAARD et al. 1958; HERBERT 1961; WINDER and O'HARA 1962; SUD and SCHAECHTER 1964; SHOOKMAN 1965; KOUKALOVA et al. 1970), whereas protein and RNA syntheses are inhibited or become growth rate-limiting factors (MANDELSTAM 1958, 1960; HARRISON and LAWRENOE 1963; GONZALEZ et al. 1968; GOLDBERG 1971; NATH and KOCH 1971). Several authors described or suggested mechanisms of division inhibition in E. coli which are dependent upon, or activated by inhibition of DNA synthesis (WITKIN 1967; HIROTA et al. 1968; GREEN et al. 1969). It is thus possible
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that various types of mutations and inhibitors, whose effect on bacterial division is influenced by growth conditions, act on the cytokinesis indirectly by means of some common mechanism, which is associated with unbalanced growth. The author is indebted to Dr. VONlJHEJS for strain 15T-. The teehnieal assistanee of Mrs. H. SLAVIKOVA is aeknowledged.
Zusammenfassung Die Einwirkung von Gentianaviolett und Thyminmangel auf die Hemmung der Zellteilung und den Veriauf derselben von Escherichia coli in Bateh-Kulturen wurde studiert. Diese Faktoren hemmen die Zellteilung und induzieren Fadenbildung nur in der pre-logarithmischen und logarithmischen Phase des Wachstums; in der spaten logarithmischen Phase wurde die Zeliteilung spontan reaktiviert auch unter den Bedingungen eines Thyminmangels oder bei Anwesenheit eines Inhibitors. Die Filamente fragmentieren unter Bildung von Kurzstabchen. Durch Ubertragung der fadenf6rmigen Zellen aus der fruhen logarithmisehen Phase in ein Stickstoff- oder Kohlenstoff-freies Minimalmedium konnte Filamentteilung bei Anwesenheit eines Inhibitors oder bei Thyminmangel induziert werden. Die Neuerwerbung der Teilungsfahigkeit in der spaten logarithmischen Phase wurde nicht durch Adaptation, Selektion von Mutanten oder Impermeabilitat fur Gentianaviolett verursacht.
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Authors' address: Dr. Eva Jicinska, Dairy Industry, Strakonice (eSSR).