Variability of Bacillus thuringiensis under various growth conditions

When a lysogenic culture of Bacillus thuringiensis subsp. galleriae 69-6 was grown under the batch conditions, 93-99% of cells in the population produced R-form colonies and ca. 1% yielded S-form colonies. The amount of spore-forming cells was 99% in R-variants and 8% in S-variants. The quantity of S-variants rose abruptly to 99% when the culture was grown under the chemostat conditions. The number of S-variants increased with the rate and the duration of growth. The process was influenced by growth-limiting factors. Temperate phage variants capable of host culture lysis on solid media (i.e. h-mutants) were not found under the conditions of batch cultivation. However, such phage particles (h-mutants) appeared under the conditions of chemostat. The titre of these phage particles reached 10(8), 10(7) and 10(4) particles per 1 ml at limitation with yeast extract, glucose and phosphorus, respectively. Under the conditions of chemostat, the particles behaved as temperate ones and their growth was not found. Irrespective of the limitation, the phage titre did not correlate with the ratio of R and S-forms in the population. When the growth was limited with phosphorus, the quantity of S-forms increased abruptly while the spontaneous induction of the phage was inhibited. The quantity of cells capable of spore formation decreased in the cultures isolated from the chemostat and grown on MPA: 69-80% of the cells in R-forms and merely 8% in S-forms.     

Dynamic interaction of virulent Pseudomonas aeruginosa bacteriophages with the host bacteria

The population interactions of Pseudomonas aeruginosa virulent bacteriophages phi kF77 and phi mnF82 with host bacterial cells were studied in dynamics under the conditions of continuous cultivation in the chemostat regime with glucose limitation. Two different types of maintaining the bacterium and its specific bacteriophages in the population were detected. When P. aeruginosa was cultivated with phage phi mnF82, such a maintenance was realized due to the successive appearance of bacterial mutants resistant to the phage and of phage mutants overcoming this resistance. When P. aeruginosa was cultivated with phage phi kF77, these were maintained owing to the ability of P. aeruginosa to form unstable phage-resistant variants with the segregation of phage-sensitive cells.     

Dynamics of the interaction between Pseudomonas aeruginosa bacteriophage phimF81 and host bacteria]

The population interactions of Pseudomonas aeruginosa virulent bacteriophage phi mF81 with host bacterial cells were studied in dynamics under the conditions of continuous cultivation in the chemostat regime with glucose limitation. It was detected that a maintenance of the bacterium and its specific bacteriophage in the population was realized due to the successive appearance of bacterial mutants resistant to the phage and of phage mutants overcoming this resistance.     

Production of active mutants of a new species of rifamycin producer resistant to specific actinophage]

Selection of a phage-stable strain of a new species of the rifamycin-producing organism was carried out. The phage-stable mutants were selected with respect to the virulent phage 2739 isolated from a lysogenic culture of the rifamycin-producing organism. Spontaneous phage-stable mutants formed at a rate of 0.8 per cent. Most of them belonged to the morphological colony type with a decreased activity level. No shifts in variation with respect to the property of the antibiotic production were noted under the action of phage 2739. 62 per cent of the phage-stable variants isolated from the secondary growth colonies after infection with the phage were lysogenic and liberated phage 2739 to the culture fluid. Induction of mutations with MNNG, UV and gamma(Co30) rays increased the frequency of the phage-stable mutanta by 1.5 times. Active phage-resistant mutants stable to the phage because of its adsorption and liberating no phage 2739 into liquid media during its cultivation were selected.     

Production in an Actinomyces levoris culture under the action of an actinophage specific to it of variants with stable preservation of its resistance to the phage]

Act. levoris 28, an organism producing levorin was treated with an actinophage virulent to it. Variants of the organism were isolated from the secondary growth of the culture. As a result of lysogenization with the above phage the variants acquired stability to it which was preserved during the further generations. In the previous experiments carried out by the authors the variants isolated from the secondary growth of the culture after its exposure to the same phage lost their stability to the phage as a result of loosing the prophage by it during the subsequent passages. The phage stable variants did not differ from the initial culture either in the activity of levorin or the levorin composition. The phages found in the initial culture 28, and the virulent mutant were identical with respect to the particles morphology and antigenic properties which confirmed their relation.     

The nature of the competitive ability of spontaneous staphylocoagulase-negative mutants of Staphylococcus aureus with respect to growth of the parent strains in continuous culture

During prolonged cultivation of S. aureus strains 104 and NCTC 8178 in continuous culture, staphylocoagulase-negative mutants arose and accumulated progressively in increasing proportions. The resulting loss of production of staphylocoagulase was accompanied by a simultaneous loss of production of -haemolysin and PV-leucocidin. Characterization of the strains revealed no further differences in biotype, exoenzymes, phage pattern and plasmid content.Cultivation in batch cultures showed that the maximal specific growth rates and specific oxygen-consumption rates of the mutant strains were slightly higher than those of the parent strains, whereas the production of total extracellular protein of the mutant strains had decreased significantly.From competition experiments between parent and mutant strains in chemostat cultures at different dilution rates and cultivation temperatures, it was concluded that the underlying mechanism of accumulation of staphylocoagulase-negative mutants in the chemostat is based on differences in affinity for the limiting substrate(s) rather than on differences in the production rates of total extracellular proteins. The complete repression of three exoenzymes, a partial repression of the total extracellular protein production, and an increased affinity for the limiting substrate(s) suggested that a mutation in a regulatory gene is involved. The possible role of a transposon in this mutation is discussed.     

Behaviour of Nif− mutants of Rhodobacter capsulatus in photosynthetic, ammonia-limited chemostat cultures

Abstract Nif⁻ mutants of Rhodobacter capsulatus carrying mutations either in the nifR4 regulatory gene or in the nifH structural gene both outgrew the wild-type strain B10 in mixed chemostat cultures under conditions favouring nitrogenase-mediated H₂ production by the wild-type (ammonia as limiting nutrient, inert argon atmosphere, light as energy source), whereas under aerobic conditions in the dark, or in batch culture, the growth of Nif⁻ mutants was not favoured. Nitrogenase-mediated H₂ production therefore appears to be detrimental to the growth of R. capsulatus in nitrogen-limited continuous culture, as may also be the case for other nitrogen fixers.     

Comparative characteristics of spore-forming and asporogenic strains of Bacillus thuringiensis

Comparative characteristics of sporogenous and asporogenous Bacillus thuringiensis strains is carried out. Asporogenous strains are found to differ from wild type strains in a number of criteria, including colony morphology, character of growth on rich and poor media and UV-sensitivity. Sporogenous strains form R colonies, they are more stable and more rare produce variants forming S colonies. S colonies are typical for asporogenous mutants, and under the cultivation in unfavourable conditions (elevated temperature, a shift of pH, a change of an incubation regime) asporogenous strains dissociate with a high frequency into R form. Initial strains, which are multiple auxotrophs, under certain conditions can form "prototrophic" revertants which are unstable when incubated on rich media. Suppressor mutation is supposed to be a possible mechanism of the origination of "prototrophs".     

Comparative study of the phage sensitivity of actinomycetes and their Nocardia-like variants

The work was aimed at studying the resistance of three streptomycetes (Streptomyces chrysomallus, S. azureus and S. roseoflavus var. roseofungini) and their spontaneous Nocardia-like variants lacking aerial mycelium and spores against nine polyphages isolated mainly from soil. Some Nocardia-like variants were found to differ from their parent cultures in the resistance against certain actinophages. S. chrysomallus VKM Ac-590 and Ac-628 variants lost resistance against the phages. S. azureus VKM Ac-719 and S. roseoflavus var. roseofungini VKM Ac-770 variants became resistant to the phages. The changed phage resistance of the streptomycetes and their Nocardia-like variants was attributed to the disorganised process of adsorption (8 and 7%, respectively, against 70 and 90% for the parent strains).     

pH oscillations and constant low pH delay the appearance of highly branched (colonial) mutants in chemostat cultures of the quorn(R) myco-protein fungus, Fusarium graminearum A3/5

At pH 5.8, highly branched (colonial) mutants appear in glucose-limited chemostat cultures of Fusarium graminearum A3/5 after ca. 400 h (ca. 107 generations) of growth. The appearance of these mutants was delayed by up to 144 h (45 generations) when the culture was switched at intervals of 120 h between pH 4.8 and 6.6. The concentration of cycloheximide-resistant macroconidia in the culture was used as an indicator of the periodic selection of advantageous mutants and it was found that, in chemostat populations subjected to pH oscillations, the interval (210 +/- 20 h) between peaks was nearly double that observed in chemostat populations cultured at constant pH (124 +/- 12 h at constant pH 5.8 and 120 h +/- 17 h at constant pH 4.5), indicating that the population evolved more slowly under oscillating pH than under constant pH. When grown in mixed culture with the parental strain (A3/5), the selective advantage of two colonial mutants isolated from chemostat cultures grown under conditions of oscillating pH was found to be pH dependent. Compared to cultures grown at constant pH 5.8, a delay of ca. 312 h (87 generations) in the appearance of colonial mutants was observed when F. graminearum A3/5 was grown in glucose-limited chemostat culture at constant pH 4.5. (c) 1996 John Wiley & Sons, Inc.     

Colonial heterogeneity of Thiobacillus versutus.

In acetate-limited chemostat cultures started with single-colony cultures of Thiobacillus versutus, a mutant appeared after approximately 85 volume changes. The inhomogeneity of the culture was detected by the development of two different types of colonies on agar plates. When a pure culture of the mutant was grown in a chemostat, parent colonies appeared after almost the same period of time. Electron micrographs of the mutant grown on butyrate showed the presence of fibrils surrounding the cells. The cells of the parent strain were bald when grown under the same conditions. The growth kinetics of the parent and the mutant were investigated in batch cultures with a variety of substrates and were found to be identical. Major differences between the two strains were observed during growth on mannitol; the mutant attained a lower yield and excreted large amounts of extracellular polysaccharides.     

Spontaneous deletion of a 209-kilobase-pair fragment from the Escherichia coli genome occurs with acquisition of resistance to an assortment of infectious phages

To breed resistance to an assortment of infectious phages, continuous cultures of Escherichia coli JM109 grown in a chemostat were exposed to phage mixtures prepared from sewage influent. Four sequential chemostat-grown cultures were each infected with a different phage mixture. At the end of a chemostat run, one phage-resistant colony was isolated and used to inoculate the subsequent culture. This process was repeated, and increased phage resistance of the input bacterial strain resulted from the successive challenges with different phage cocktails. Multiple mutations apparently accumulated progressively. A mutant isolated at the end of the four runs, designated D198, showed resistance to 38 of 40 phages that infect the parent strain, JM109. D198 produced less outer membrane protein C (OmpC) than JM109. However, restoration of the OmpC protein by plasmid-mediated complementation did not completely restore the susceptibility of D198 to the 38 phages. Therefore, alterations beyond the level of OmpC protein production contribute to the phage resistance of D198. PCR-based genetic analysis revealed that D198 has a genome that is 209 kbp (about 200 genes) smaller than JM109. The deletion includes the chromosomal section from ompC to wbbL that encodes the rhamnosyl transferase involved in lipopolysaccharide biosynthesis. Strains D198 and JM109 were comparable in their growth characteristics and their abilities to express a recombinant protein.     

Continuous cultivation in a chemostat of the phototrophic procaryote,anacystis nidulans,under nitrogen-limiting conditions

Anacystis nidulans was grown photoautotrophically in a chemostat in the presence of light, air and CO2 as the sole carbon source. Either the amount of the nitrogen source in the medium or light intensity were used as growth-limiting parameters. 1. Cells of high glycogen content obtained by pre-incubation under nitrogen starvation conditions maintained their glycogen content during continuous cultivation. Both growth rate and the amount of cell-mass and of glycogen depended on the nitrate content of the medium and the light intensity. The values for the growth rate, the maximal rates of glycogen synthesis and of cell mass formation were 0.1 h-1, 6 mg/l.h and 17 mg/l.h, respectively. 2. Cells without glycogen which had been transferred from an exponentially growing batch culture to chemostat conditions showed increasing rates of growth and of cell mass formation when the light intensity was increased. A determination of specific values resulted in 0.15 h-1 for growth rate and 23 mg/1.h for cell mass formation. 3. The chemostat apparatus is described in detail.     

Heterogeneity of <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> 1CP as a Response to Stress Induced by Chlorophenols

Dissociation of Rhodococcus opacus 1CP during cultivation in different media (containing phenol and its monochlorinated derivatives as the sole source of carbon and energy) was studied. Three variants of strain 1CP (S1, S2, and R) differing in the morphology of cells and colonies, lipid composition, and manner of growth on phenol and monochlorophenols were isolated. It was shown that 2- and 4-chlorophenols were most actively degraded by the smooth (S) forms of the culture, and that the rough (R) form predominated when the culture was grown in a rich medium. The S forms differed from the R forms of the strain by an increased content of cardiolipin, fatty acids, and phosphatidylethanolamine.     

Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 +/- 8 mg (mean +/- S.E.) glucoamylase (GAM) L-1 in batch culture and 373 +/- 9 mg GAM L-1 in maltodextrin-limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (qp) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (qp, 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 +/- 12 to 496 +/- 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 +/- 0.4 to 16.4 +/- 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production.     

Adaptation of Salmonella typhimurium mutants containing uncoupled enzyme IIGlc to glucose-limited conditions.

Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP):glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. As a result of the ptsG mutation, the apparent Km of the system for glucose transport is increased about 1,000-fold (approximately 18 mM) compared with wild-type PTS-mediated glucose transport. An S. typhimurium mutant containing uncoupled enzyme IIGlc as the sole system for glucose uptake was grown in glucose-limited chemostat cultures. Selective pressure during growth in the chemostat resulted in adaptation to the glucose-limiting conditions in two different ways. At first, mutations appeared that led to a decrease in Km value of uncoupled enzyme IIGlc. These results suggested that uncoupled enzyme IIGlc had significant control on the growth rate under glucose-limiting conditions. More efficient glucose uptake enabled a mutant to outgrow its parent and caused a decrease in the steady-state glucose concentration in the chemostat. At very low glucose concentrations (10 microM), mutants arose that contained a constitutively synthesized methyl-beta-galactoside permease. Apparently, further changes in the uncoupled enzyme IIGlc did not lead to a substantial increase in growth rate at very low glucose concentrations.     

Specific Lysogenicity in Streptomyces azureus

Slope (or plate) cultures of thiostrepton-producing Streptomyces azureus (ATCC 14921) often showed spontaneously developing plaques. Plaques increased in number during serial subcultures. The production of aerial mycelia and sporulating aerial hyphae was interrupted by the overlapping plaques, whereas the growth of substrate mycelia continued in the plaques. These abnormal (eroded) cultures were easily restored to their normal conditions once they were passed through liquid cultures under shaking conditions. A few phage particles were found in the plaques, together with some headless tails and numerous tail tips which formed a hexagonal crystal or a large crystal mass when viewed in an electron microscope. No lytic phenomenon and no phage production were found in the liquid cultures, although all mycelia and spores harbored phage-producing abilities. It was also found that the propagation of phages was successful in solid culture, but not in liquid culture. The whole phage was named SAt2, which belongs to group B of Bradley's morphological classification. From these results, it is considered that S. azureus is lysogenic with temperate phage SAt2, of which virulent mutants are able to infect the aerial mycelia and sporulating hyphae of their lysogenic host.     

Effect of factors in the cultivation of lysogenic Escherichia coli K12 (lambda) on the spontaneous level of phage

The content of free phage in the culture of E. coli K12 (lambda) under different conditions of their cultivation, not affecting the M-concentration of these bacteria, may differ by several times. These differences are well reproduced in consecutive experiments and little affected by the fluctuations of the absolute values of the phage titer.     

Comparative study of the growth of Listeria monocytogenes in defined media and demonstration of growth in continuous culture

C.E. JONES, G. SHAMA, P.W. ANDREW, I.S. ROBERTS AND D.JONES. 1995 A basic requirement for physiological studies with Listeria monocytogenes is a chemically defined medium that supports growth of the bacterium in batch and continuous culture. A number of such media have been devised but comparative studies of their efficiency are few and none has been used in continuous culture. Six of the media were compared for their ability to sustain sequential growth of L. monocytogenes in static and aerated batch culture with glucose as sole carbon source. The most suitable, judged on the basis of ease of preparation, growth rate and yield, was that of Trivett and Meyer (1971). This medium was shown to support growth of L. monocytogenes NCTC 7973 in continuous culture in a chemostat. A lytic phenomenon, noted with the same strain under anaerobic conditions and in batch culture in the chemostat, is discussed.