The Requirements of Pseudomonas aeruginosa Dissociants for Carbon,Nitrogen, and Phosphorus

Quantitative data on the nutritional requirements of microorganisms are necessary to predict the behavior of bacterial populations and to control their cultivation. The requirements of the R, S, and M dissociants of P. aeruginosa for carbon, nitrogen, and phosphorus were derived from the results of 88 cultivation experiments. For each of the dissociants, we derived a coefficient that relates the optical density and the number of cells in the dissociant culture, determined the time when the cultures entered the stationary growth phase, studied cultural changes induced by transfer to the stationary phase, and determined what nutrients limit the growth of particular dissociants. The nutritional requirements of the dissociants are discussed in relation to our earlier data.     

Dynamics of the growth and population composition of the mixed cultures of R, S, and M dissociants of Pseudomonas aeruginosa

The population composition of polycultures of Pseudomonas aeruginosa dissociants (R + M and R + S + M) developing on media with various contents and ratios of nitrogen and phosphorus has been studied. Irrespective of its proportion (10 to 90%) in the inoculum, the R variant accounted for 65 to 84% of the whole population of linear-phase and stationary-phase binary cultures of R and M dissociants, which differ in terms of energy metabolism and nutritional requirements. After prolonged cultivation, the population in the binary culture contained only R cells (100%), which are characterized by minimum requirements with respect to the main biogenic elements. These data agree with the predictive data of model studies and can be attributed to regulation of the population composition of bacterial cultures by trophic factors. It was established that the proportion of M cells, which are distinguished by maximum nutrient requirements and enhanced stability, increased during two developmental stages of the Ps. aeruginosa polycultures (R + M and R + S + M): the lag phase and the decay stage. This result cannot be due to the influence of trophic factors and presumably results from changes in the levels of autoregulatory factors (anabiosis autoinducers) involved in stress resistance and plausibly in the adaptive interconversion of dissociants upon transfer to a new medium (during the lag phase) and under starvation conditions (at the onset of the decay phase).     

Quantitative patterns of development of community of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> dissociants

The stationary phase of batch culture of Pseudomonas aeruginosa dissociants has been described by a variational model of consumption and growth. The generalized entropy functional was used as the objective function. The model parameters include the requirements of the dissociants for the main nutrients: carbon, nitrogen, and phosphorus. The variational model was used to calculate the limiting regions and microbial community composition during stationary growth for different initial combinations of the resources as a function of the limiting resources. A correspondence between the experimental data and model calculations has been demonstrated. A possibility to control the community structure is discussed.     

Development and population structure of mixed (S + M) Pseudomonas aeruginosa cultures in the late stationary growth phase

Population growth, the ratio between dissociants, pH, and levels of reducing sugars in the medium were monitored during prolonged (375 h) batch cultivation of Pseudomonas aeruginosa S and M dissociants on mineral medium with glucose. During the stationary growth phase (100-375 h), two scenarios were possible. The first one included extensive cell autolysis coupled to alkalinization of the medium and an increased ratio of the M dissociant. In the second case, acidification of the medium was coupled to the oscillating secondary growth, mostly of the M dissociant; the dynamics of cell numbers of this dissociant correlated with the dynamics of the culture optical density. In this scenario, periodical appearance of reducing sugars in the medium was detected; it was in the opposite phase with the changes of the M dissociant cell numbers. The differences between scenarios of P. aeruginosa growth in the late stationary phase were probably due to the physiological and biochemical characteristics of the S and M dissociants, including different pathways of glucose utilization (respiration or fermentation), resistance to acidification, synthetic (proteolytic) activity, and productivity of autoinducers.     

Dynamics of the Growth and Population Composition of Mixed Cultures of R,S, and M Dissociants of Pseudomonas aeruginosa

The population composition of polycultures of Pseudomonas aeruginosa dissociants (R + M and R + S + M) developing on media with various contents and ratios of nitrogen and phosphorus has been studied. Irrespective of its proportion (10 to 90%) in the inoculum, the R variant accounted for 65 to 84% of the whole population of linear-phase and stationary-phase binary cultures of R and M dissociants, which differ in terms of energy metabolism and nutritional requirements. After prolonged cultivation, the population in the binary culture contained only R cells (100%), which are characterized by minimum requirements with respect to the main biogenic elements. These data agree with the predictive data of model studies and can be attributed to regulation of the population composition of bacterial cultures by trophic factors. It was established that the proportion of M cells, which are distinguished by maximum nutrient requirements and enhanced stability, increased during two developmental stages of the Ps. aeruginosa polycultures (R + M and R + S + M): the lag phase and the decay stage. This result cannot be due to the influence of trophic factors and presumably results from changes in the levels of autoregulatory factors (anabiosis autoinducers) involved in stress resistance and plausibly in the adaptive interconversion of dissociants upon transfer to a new medium (during the lag phase) and under starvation conditions (at the onset of the decay phase).__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 475–482.Original Russian Text Copyright © 2005 by Mil’ko, Khabibullin, Nikolaev, Kozlova, El’-Registan.     

Development and population structure of mixed (S + M) <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> cultures in the late stationary growth phase

Population growth, the ratio between dissociants, pH, and levels of reducing sugars in the medium were monitored during prolonged (375 h) batch cultivation of Pseudomonas aeruginosa S and M dissociants on mineral medium with glucose. During the stationary growth phase (100–375 h), two scenarios were possible. The first one included extensive cell autolysis coupled to alkalinization of the medium and an increased ratio of the M dissociant. In the second case, acidification of the medium was coupled to the oscillating secondary growth, mostly of the M dissociant; the dynamics of cell numbers of this dissociant correlated with the dynamics of the culture optical density. In this scenario, periodical appearance of reducing sugars in the medium was detected; it was in the opposite phase with the changes of the M dissociant cell numbers. The differences between scenarios of P. aeruginosa growth in the late stationary phase were probably due to the physiological and biochemical characteristics of the S and M dissociants, including different pathways of glucose utilization (respiration or fermentation), resistance to acidification, as well as synthetic (proteolytic) activity and productivity of autoinducers.     

Effect of major nutrient elements on the growth and population homogeneity of the R, S and M dissociants of Pseudomonas aeruginosa and the glucose oxidation and fermentation pathways

The population homogeneity of the stationary-phase monocultures of Pseudomonas aeruginosa dissociants was studied as a function of the initial content of major nutrient elements (C, N, and P) in the cultivation medium. The monocultures of the dissociants remained homogeneous during cultivation if the initial concentrations of the major nutrient elements were either sufficiently high or, conversely, very low, but became heterogeneous during cultivation in unbalanced (with respect to the major nutrient elements) media. At the initial concentration of nitrate in the medium equal to 0.07% or phosphate equal to 0.004-0.014%, the initially homogeneous population of R dissociant cultivated to the stationary growth phase turned out to contain 30-40% of S-type cells, whereas the initially homogeneous population of S dissociant was found to contain 50-80% of M-type cells. The population of M dissociant remained homogeneous throughout the cultivation period. R dissociant grew better at sufficiently high concentrations of glucose, nitrate, and phosphate in the medium, whereas M dissociant grew better when the initial concentrations of these nutrients were low. During the cultivation of R dissociant, the pH of the medium changed insignificantly, and the C/P ratio (the ratio of the carbon and phosphorus consumed during growth) was minimal (among the three dissociants), indicating that the R dissociant accomplishes the oxidative pathway of glucose metabolism. During the cultivation of the M dissociant, the pH of the medium dropped to 3.4-3.9, and the C/P ratio was maximal, indicating that this dissociant accomplishes the fermentative pathway of glucose metabolism. During the cultivation of the S dissociant, the pH of the medium and the C/P ratio exhibited variations, indicating that this dissociant triggers its pathways of glucose metabolism.     

The Effect of Major Nutrient Elements on the Growth and Population Homogeneity of the R,S, and M Dissociants of Pseudomonas aeruginosa and the Glucose Oxidation and Fermentation Pathways

The population homogeneity of the stationary-phase monocultures of Pseudomonas aeruginosa dissociants was studied as a function of the initial content of major nutrient elements (C, N, and P) in the cultivation medium. The monocultures of the dissociants remained homogeneous during cultivation if the initial concentrations of the major nutrient elements were either sufficiently high or, conversely, very low, but became heterogeneous during cultivation in unbalanced (with respect to the major nutrient elements) media. At an initial concentration of nitrate in the medium equal to 0.07% or phosphate equal to 0.004–0.014%, the initially homogeneous population of R dissociant cultivated to the stationary growth phase turned out to contain 30–40% of S-type cells, whereas the initially homogeneous population of S dissociant was found to contain 50–80% of M-type cells. The population of M dissociant remained homogeneous throughout the cultivation period. R dissociant grew better at sufficiently high concentrations of glucose, nitrate, and phosphate in the medium, whereas M dissociant grew better when the initial concentrations of these nutrients were low. During the cultivation of R dissociant, the pH of the medium changed insignificantly, and the C/P ratio (the ratio of the carbon and phosphorus consumed during growth) was minimal (among the three dissociants), indicating that the R dissociant accomplishes the oxidative pathway of glucose metabolism. During the cultivation of the M dissociant, the pH of the medium dropped to 3.4–3.9, and the C/P ratio was maximal, indicating that this dissociant accomplishes the fermentative pathway of glucose metabolism. During the cultivation of the S dissociant, the pH of the medium and the C/P ratio exhibited variations, indicating that this dissociant triggers its pathways of glucose metabolism.     

Effect of reduced concentrations of carbon, nitrogen, and phosphorus in a medium on growth of three dissociants of Pseudomonas aeruginosa]

The effect of lowered concentrations of carbon, nitrogen, and phosphorus sources in the medium on the specific growth rate mu of the R, S, and M dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosa K-2, culture pH, and the population composition was studied. Within the first 16 hours of cultivation in all of the four media tested, the R, S, and M dissociants had virtually identical mu. The maximal values of mu were reached by the 20th h of growth in the basal medium (R and S dissociants) and in the carbon-deficient medium containing 0.4% glucose (M dissociant). The R and M dissociants showed the most rapid decrease in mu in the nitrogen-deficient medium containing 0.55% NaNO3. By the end of cultivation in the basal medium, the pH of the R, S, and M cultures decreased to 6.3, 5.3, and 3.3, respectively. In the case of the carbon-deficient medium, the drop in the culture pH was lower. After a 2.5-day incubation of the S dissociant in the phosphorus-deficient medium containing 0.028% NaH2PO4.2H2O and of the M dissociant in the basal medium supplemented with chalk powder, these dissociants were completely displaced from the media.     

Characteristics of carbohydrate metabolism of R-, S- i M-dissociants of Pseudomonas aeruginosa]

R and S dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosa K-2 differed but little in their growth in a minimal defined medium with glucose as the source of carbon and energy. At the same time, the number of cells of M dissociant in the late exponential phase was five orders of magnitude less than that of R and S dissociants. The growth of M dissociant was accompanied by the accumulation of formate in the culture liquid and a concurrent decrease in pH. All three dissociants contained the key enzymes of the Entner-Doudoroff pathway of glucose oxidation; however, the activities of these enzymes, especially 6-phosphogluconate dehydrogenase, were low in M dissociant. Conversely, the activity of formate dehydrogenase in cells of M dissociant was higher than in other dissociants. The activity of 6-phosphogluconate dehydrogenase, a key enzyme of the pentosephosphate pathway of glucose oxidation, was detected only in S dissociant. The peculiarities of the carbohydrate metabolism of M dissociant are probably responsible for its poor growth on glucose and determine the more pronounced anaerobic type of its metabolism.     

The process of bacterial population splitting into dissociants and long-term batch cultivation of bacteria

The growth and composition of a population were studied during long-term (up to 50 days) batch cultivation of mono and mixed cultures of Pseudomonas aeruginosa S- and M-dissociants and Rhodobacter sphaeroides R- and M-dissociants without the addition of nutrients. During the cultivation of P. aeruginosa on a glucose-containing mineral medium, periodic lysis followed by polyculture growth resumption in the late stationary phase occurred on account of the M-dissociant: the change in its cell number corresponded to the change in the total cell number of the association. It was shown that the periodic occurrence of reducing sugars in the medium preceded the resumption of polyculture growth. Periodic secondary growth of the mixed culture of R. sphaeroides photosynthesizing bacteria occurred because of fast growing R-cells after the lysis of some part of the R-dissociant population. In the monoculture of the R. sphaeroides M-dissociant, R-cells were found during the whole period of cultivation, making up to 1–10% of the population irrespective of its size, which probably corresponded to the frequency of occurrence of this dissociant. In the R-dissociant monoculture, M-cells were found only after 26 days, and their number gradually decreased to half of population by the end of cultivation period. The joint growth of dissociants was characterized by the biomass increment and bacterial growth acceleration compared to monocultures, which is important for the fast development of new habitats under natural conditions. The cells of both bacterial species were lysed during long-term cultivation by exoproteinases secreted by the thin-wall cells of M-dissociants.     

Biosynthesis of Bacillus intermedius glutamyl endopeptidase in recombinant Bacillus subtilis cells during the stationary growth phase

We studied the biosynthesis of Bacillus intermedius glutamyl endopeptidase in the recombinant Bacillus subtilis strain AJ73 delta58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase, and found that the nutritional requirements for glutamyl endopeptidase synthesis were different in the stationary phase and growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of glutamyl endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different.     

The Effect of Lowered Concentrations of Carbon,Nitrogen and Phosphorus Sources on the Growth Dynamics of the R,S, and M Dissociants of Pseudomonas aeruginosa

The effect of lowered concentrations of carbon, nitrogen, and phosphorus sources in the medium on the specific growth rate of the R, S, and M dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosaK-2, culture pH, and the population composition was studied. Within the first 16 hours of cultivation in all of the four media tested, the R, S, and M dissociants have virtually identical . The maximal values of were reached by the 20th h of growth in the basal medium (R and S dissociants) and in the carbon-deficient medium containing 0.4% glucose (M dissociant). The R and M dissociants showed the most rapid decrease in in the nitrogen-deficient medium containing 0.55% NaNO₃. By the end of cultivation in the basal medium, the pH of the R, S, and M cultures decreased to 6.3, 5.3, and 3.3, respectively. In the case of the carbon-deficient medium, the drop in the culture pH was lower. After a 2.5-day incubation of the S dissociant in the phosphorus- deficient medium containing 0.028% NaH₂PO₄· 2H₂O and of the M dissociant in the basal medium supplemented with chalk powder, these dissociants were completely displaced from the media.     

Transient self-inhibition of the growth of Lactobacillus delbrueckii subsp. bulgaricus in a pH-regulated fermentor

An industrial strain of Lactobacillus delbrueckii subsp. bulgaricus was grown in a synthetic medium on lactose as carbon substrate, in a pH-regulated fermentor. Growth proceeded in two distinct phases separated by a transient stationary phase. Various experimental approaches were used to identify the cause of this growth arrest. Growth experiments in L. bulgaricus culture supernatant fluids collected at different cultivation times in fermentor, and supplemented or not with various nutritional solutions, enabled us to discard the possibility of a nutritional limitation. Tube cultures of L. bulgaricus in medium supplemented with various lactic acid concentrations showed a potential inhibition by this metabolic end product but confirmed that this inhibition was not responsible for the cessation of growth. It was concluded that at least one inhibitory compound was produced during the growth phase of the strain, and this compound disappeared from the medium in the transient stationary phase, enabling the growth to start again later in the culture. Indeed, the stoichiometric analysis of the culture showed, firstly, that unidentified carbon compounds were produced from lactose during growth, which were probably converted in lactic acid during the transient stationary phase and, secondly, that part of the amino acids consumed gave catabolic end products. Finally, bacteriocin-like compounds were not considered to be responsible for this growth arrest.     

Effect of carbon, nitrogen and phosphorus nutrition on the R, S, and M dissociants of Pseudomonas aeruginosa in mixed cultures

The effect of glucose, nitrate, and phosphate on the stationary-phase growth characteristics of R, S, and M dissociants of the hydrocarbon-oxidizing strain P. aeruginosa K-2 was studied. The optimal concentrations of glucose and phosphate providing for at least 90% of the maximal culture density were found to be 2-7% glucose and 0.02-0.12% phosphate. The main factor that determined the proportion of dissociants in bacterial populations was the initial concentration of phosphate. The fraction of R dissociant in populations increased linearly with the concentration of glucose and varied nonlinearly with the concentration of phosphate in the growth medium. The fraction of M dissociant depended solely on the concentration of phosphate in a manner inverse to that typical of R dissociant. In glucose-deficient media containing sufficient amounts of phosphorus, S dissociant prevailed over R dissociant.     

Changes in the intracellular pool of free amino acids of Streptococcus lactis under different cultivation conditions]

The intracellular pool of free amino of Streptococcus lactis--lysine producer contains a good number of amino acids when cultivated on the corn medium. Glutamic acid, proline, alanine, lysine, leucine, histidine and arginine are in predominance. An almost complete amino acid pool develops at an early exponential phase of Str. lactis growth under stationary cultivation conditions. The content of free amino acids increases 4-fold during the transition from the early exponential phase to the stationary phases under submerged cultivation conditions. This can be attributed to a more intensive amino acid exchange during the medium stirring than during stationary cultivation.     

Obtaining of intrapopulational dissociants of some bacilli and the use of DIR-PCR for their identification

The paper is the first to suggest methods for rapid obtaining and genotypic identification of phenotypic (colonial-morphological) dissociants of bacterial cultures. For revelation of the potential dissociation ability and obtaining of dissociants, the use of bacterial cyst-like refractile cells (CRC) is recommended. These cells are characterized by enhanced variability; upon their first passage, an abrupt increase in the dissociation index is observed as a result of the emergence of cells that form morphologically different types of colonies. The approaches elaborated were tested with Bacillus cereus, B. subtilis, and B. licheniformis, for which colonial-morphological dissociants of various types were obtained after the first passage of CRC (both of those formed in the developmental cycle of bacteria and of those arising as a result of artificial increase of the concentration of anabiosis autoinducers in the cultivation medium). The genomic distinctions between dissociants of B. cereus and B. subtilis were estimated using polymerase chain reaction with a primer system designed based on the analysis of nucleotide sequences of complete prokaryotic genomes available in the GenBank database (DIR-PCR). The application of the suggested method allowed distinctions between the genomes of dissociants of Bacillus cereus and B. subtilis to be revealed, which is in agreement with the hypothesis that suggests reversible intragenomic rearrangements to be the basis of bacterial dissociation into subpopulations.     

Effect of carbon, nitrogen, and phosphorus sources of nutrition on the growth of R-, S- and M-dissociants of Pseudomonas aeruginosa]

The effect of glucose, nitrate, and phosphate on the stationary-phase growth parameters of the R, S, and M dissociants of the hydrocarbon-oxidizing bacterium Pseudomonas aeruginosa K-2 was studied. The data obtained were analyzed in terms of the Mitscherlich equation. S dissociant required less glucose than other dissociants, whereas M dissociant required less nitrogen and phosphorus. These findings were confirmed by the results of investigation of the combined action of glucose, nitrate, and phosphate in a 3 x 3 x 3 factor experiment. It is anticipated that M dissociant must prevail under conditions of nitrogen and phosphorus deficiency, and S dissociant must be dominant in the case of optimally chosen proportions between the biogenic elements studied.     

Peculiarities of the biosynthesis of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase in recombinant <Emphasis Type="Italic">Bacillus subtilis</Emphasis> cells during the stationary growth phase

We studied the biosynthesis of bacillus intermedius glutamyl endopeptidase in the recombinant bacillus subtilis strain AJ73 58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase and found that the nutritional requirements for glutamyl endopeptidase synthesis were different in the stationary phase and the growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During the final stages of the culture growth, the enzyme production was stimulated by Ca²⁺, Mn²⁺, and Co²⁺ and inhibited by Zn²⁺, Fe²⁺, and Cu²⁺. The synthesis of glutamyl endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 39–47.Original Russian Text Copyright © 2005 by Chastukhina, Sharipova, Gabdrakhmanova, Balaban, Kostrov, Rudenskaya, Leshchinskaya.