Effect of growth conditions on levels of components of the phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus mutans and Streptococcus sobrinus grown in continuous culture.

The membrane-bound, sugar-specific enzyme II (EII) component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Streptococcus mutans Ingbritt is repressed by growth on glucose under various conditions in continuous culture. Compared with optimal PTS conditions (i.e., glucose limitation, dilution rate [D] of 0.1 h-1, and pH 7.0), EII activity for glucose (EIIGlc) and mannose (EIIMan) in cells grown at a D of 0.4 h-1 and pH 5.5 with the same glucose concentration was reduced 24- to 27-fold. EII activity with methyl alpha-glucoside and 2-deoxyglucose was reduced 6- and 26-fold, respectively. Growth with excess glucose (i.e., nitrogen limitation) resulted in 26- to 88-fold repression of EII activity with these substrates. The above conditions of low pH, high dilution rate, and excess glucose also repressed EII activity for fructose (EIIFru), but to a lesser extent (two- to fivefold). Conversely, growth of S. mutans DR0001 at a D of 0.2 h-1 and pH 5.5 resulted in increased EIIGlc and EIIMan activity. Unlike the EII component, the HPr concentration in S. mutans Ingbritt varied only twofold (5.5 to 11.4 nmol/mg of protein) despite growth at pH 5.5 with limiting and excess glucose. The HPr concentrations in S. mutans DR0001 and the glucose-PTS-defective mutant DR0001/6 were similar. In a companion study, the soluble components of the PTS (i.e., HPr, EI, and EIIILac) in Streptococcus sobrinus grown on limiting lactose in a chemostat were not influenced significantly by growth at various pHs (7.0 and 5.0) and growth rates (D of 0.1, 0.54, and 0.8 h-1). However, growth on lactose resulted in repression of both EIIGlc and EIIFru, confirming earlier results with batch-grown cells. Thus, the glucose-PTS in some strains of S. mutans is regulated at the level of EII synthesis by certain environmental conditions.     

Nitrogenase activity and growth of Frankia in batch and continuous culture.

Frankia grown in batch culture was unable to maintain a high rate of nitrogenase activity and, once a peak level was reached, activity rapidly declined. Addition of 5 mM carbon source of cultures or transfer to fresh medium was followed by brief recovery of nitrogenase activity. The extent of recovery decreased as additions or transfers were made to progressively older cultures. Daily addition of fresh medium (dilution rate = 0.125 day-1) allowed Frankia to be maintained in continuous, derepressed culture with stable rates of growth and nitrogenase activity for more than 30 days. The proportion of active, mature vesicles also remained constant in continuous culture but decreased with time in batch culture.     

Effect of ionophores and pH on growth of Streptococcus bovis in batch and continuous culture.

Batch cultures (pH 6.7) of Streptococcus bovis JB1 were severely inhibited by 1.25 and 5 microM lasalocid and monensin, respectively, even though large amounts of glucose remained in the medium. However, continuous cultures tolerated as much as 10 and 20 microM, respectively, and used virtually all of the glucose. Although continuous cultures grew with high concentrations of ionophore, the yield of bacterial protein decreased approximately 10-fold. When pH was decreased from 6.7 to 5.7, the potency of both ionophores increased, but lasalocid always caused a larger decrease in yield. The increased activity of lasalocid at pH 5.7 could largely be explained by an increased binding of the ionophore to the cell membrane. Because monensin did not show an increased binding at low pH, some other factor (e.g., ion turnover) must have been influencing its activity. There was a linear increase in lasalocid binding as the concentration increased, but monensin binding increased markedly at high concentrations. Based on the observations that (i) S. bovis cells bound significant amounts of ionophore (the ratio of ionophore to cell material was more important than the absolute concentration), (ii) batch cultures responded differently from continuous cultures, and (iii) pH can have a marked effect on ionophore activity, it appears that the term "minimum inhibitory concentration" may not provide an accurate assessment of microbial growth inhibition in vivo.     

Application of a competition model to the growth of Streptococcus mutans and Streptococcus sanguis in binary continuous culture.

Streptococcus mutans 6715-15 and Streptococcus sanguis 10558 were grown together in continuous culture with glucose as the limiting carbon source. The relationship of growth rate to substrate concentration was determined for pure cultures of each organism in continuous and batch cultures. A model based on competition for a growth-limiting substrate (glucose) was used to predict the proportions of each organism when grown in binary cultures. The results indicate that interactions other than competition for glucose carbon exist between S. mutans and S. sanguis grown under these conditions.     

Application of a competition model to the growth of Streptococcus mutans and Streptococcus sanguis in binary continuous culture

Streptococcus mutans 6715-15 and Streptococcus sanguis 10558 were grown together in continuous culture with glucose as the limiting carbon source. The relationship of growth rate to substrate concentration was determined for pure cultures of each organism in continuous and batch cultures. A model based on competition for a growth-limiting substrate (glucose) was used to predict the proportions of each organism when grown in binary cultures. The results indicate that interactions other than competition for glucose carbon exist between S. mutans and S. sanguis grown under these conditions.     

Inducible and constitutive formation of fructanase in batch and continuous cultures of Streptococcus mutans

The production of extracellular beta-D-fructanase by several strains of Streptococcus mutans was studied in continuous culture. When glucose was the limiting nutrient, S. mutans K1-R and OMZ176 accumulated fructanase to maximum levels at low growth rates (dilution rate 0.05-0.10 h-1), due to the longer residence times of the bacteria in the culture vessel under these conditions. Extracellular fructanase activity was greater than has been previously reported for batch cultures. The rate of fructanase production for both S. mutans strains K1-R and OMZ176 increased with increasing growth rate when glucose was limiting. Under conditions of glucose sufficiency, the rate of fructanase production was always lower than in cultures where glucose was limiting, irrespective of the growth rate. Cultures of S. mutans Ingbritt (serotype c) grown with sorbitol- or glucose-limitation synthesized fructanase at a very low basal rate. When fructose was the limiting carbohydrate the enzyme was induced with a maximum rate of production occurring at a dilution rate of 0.40 h-1. Strains of S. mutans from other serotypes (a, d, d/g) were either not affected by changing the limiting sugar from glucose to fructose or else fructanase activity was slightly decreased in the fructose-limited medium. Fructanases from various strains of S. mutans readily hydrolysed (2----6)-beta-D-fructans, but all possessed the ability to hydrolyse (2----1)-beta-D-fructans to varying degrees.     

Adaptation of Streptococcus mutans and Enterococcus hirae to acid stress in continuous culture.

Streptococcus mutans GS-5 and IB1600 adapted to growth in acidic environments in continuous culture at slow (generation time = 8.3 h) or fast (generation time = 2.4 h) rates of growth in complex medium with a restricted glucose supply. The extent of adaptation was indicated by changes in minimum pH values attained by harvested cells suspended in dense suspensions with excess glucose and by increased levels of ATPase activity assayed in permeabilized cells. Also, adapted cells better withstood potentially lethal acidification. Cells harvested from cultures growing at pH values close to 5 reduced suspension pH to lower values than cells from cultures maintained at pH 7. Cells from pH 6 cultures were intermediate. The IB1600 strain had a higher level of constitutive acid resistance than the GS-5 strain and also was better able to adapt to growth in acidified media. Both had less adaptive capacity than Enterococcus hirae ATCC 9790. Adaptation occurred rapidly, mainly within a single generation in continuous culture, while deadaptation occurred more slowly over multiple generations. The capacity of S. mutans to adapt to acid conditions is likely to be important in the ecology of dental plaque and also for the cariogenicity of the organism.     

Adaptation of Streptococcus mutans and Enterococcus hirae to acid stress in continuous culture.

Streptococcus mutans GS-5 and IB1600 adapted to growth in acidic environments in continuous culture at slow (generation time = 8.3 h) or fast (generation time = 2.4 h) rates of growth in complex medium with a restricted glucose supply. The extent of adaptation was indicated by changes in minimum pH values attained by harvested cells suspended in dense suspensions with excess glucose and by increased levels of ATPase activity assayed in permeabilized cells. Also, adapted cells better withstood potentially lethal acidification. Cells harvested from cultures growing at pH values close to 5 reduced suspension pH to lower values than cells from cultures maintained at pH 7. Cells from pH 6 cultures were intermediate. The IB1600 strain had a higher level of constitutive acid resistance than the GS-5 strain and also was better able to adapt to growth in acidified media. Both had less adaptive capacity than Enterococcus hirae ATCC 9790. Adaptation occurred rapidly, mainly within a single generation in continuous culture, while deadaptation occurred more slowly over multiple generations. The capacity of S. mutans to adapt to acid conditions is likely to be important in the ecology of dental plaque and also for the cariogenicity of the organism.     

Kinetic analysis of batch and continuous culture of Streptococcus cremoris HP1.

The growth of Streptococcus cremoris on a semidefined medium was studied at initial lactose concentrations of 0.2-5.0% in batch culture, and in lactose-limited chemostat cultures at 0.5% lactose. Kinetic analysis of the batch data, using statisitcal techniques, indicated the importance of lactose limitation and lactic acid inhibition of the growth of S. cremoris. A model for the biomass production, lactose utilization, and lactic acid production in batch culture was proposed. In continuous culture, it was found that steady state populations were maintained at higher dilution rates (D = 0.6-0.7 h-1) than the maximum predicted by batch culture (0.56h-1). No evidence for a selection of fast growing mutants was obtained. Copious growth adhering to the walls of the fermentor (i.e. wall growth) occurred very rapidly at higher dilution rates and this undoubtedly affected steady-state growth and wash-out and, as a consequence, the apparent maximum dilution rate.     

Reproduction of batch growth curves in continuous culture

Summary The authors have experimentally verified the theoretically deduced relation for the concentration of bacteria in the individual vessels of a multistage continuous system arranged so as to enable—at suitably chosen retention times in the individual vessels—the study of even rapidly occuring changes in the bacterial culture. Moreover, such system permits to compare the development of the batch and continuous culture. It has been demonstrated that according to the chosen retention times any section of the growth curve, the individual phases of which are spacially separated in the individual vessels can be reproduced in the continuous system.     

Hydrocarbon fermentations using Candida lipolytica. I. Basic growth parameters for batch and continuous culture conditions

Candida lipolytica (strain ATCC 8661) was grown on a simple defined medium with n-dodecane as sole carbon source under batch and continuous fermentation conditions. The composition of cellular material recovered from the fermentations, the oxygen demand of the cells, and the effect of operating conditions on cell growth were evaluated experimentally. These basic data are presented and discussed.     

The influence of culture conditions on carotenogenesis in Streptococcus faecium UNH564P.

The growth of Streptococcus faecium UNH564P and its production of triterpenoid carotenoids under a variety of culture conditions were examined. Total extractable cell lipid and carotenoid levels increased with culture age and paralleled the growth curve of the bacterium. Variations of the medium glucose concentration produced significant changes in both cell growth and carotenoid production, with the xanthophyll content decreasing at high glucose concentrations. Carotenoid degradation products were found in highly aerated cultures although a high glucose concentration appeared to have a sparing effect on oxidative degradation. Culture age appeared to have little effect on carotene:xanthophyll ratios. The significance of the production of total and individual carotenoids under the various culture conditions is discussed and related to a postulated scheme of triterpenoid carotenoid biosynthesis in the organism.     

The influence of HtrA expression on the growth of Streptococcus mutans during acid stress

When proteins are damaged under stresses conditions, these proteins are either refolded or degraded by quality control system of molecular chaperones and protease. High-temperature requirement A (htrA) is of particular interest because it can perform the roles of both protease and a chaperone. HtrA plays an important role in maintaining the physiological homeostasis of bacteri against environmental stress such as elevated temperature, oxidative and osmotic stress. Inactivation of htrA genes can thus restrict the survival ability of bacteria. These observations suggested that htrA might be responsible for acid tolerance of Streptococcus mutans. In this study, we have generated an htrA mutant and an htrA-complemented strain of S. mutans K7 isolated from a Korean in order to investigate the role of htrA in growth under acidic conditions. In terms of growth under cidic conditions, the htrA mutant exhibited 20% to 23% lower growth than the control group. In ddition, glucosyltransferaseB nd glucosyltransferaseC expression levels significantly decreased. When the htrA expression level was restored by adding the htrA gene to the htrA mutant strain, the normal growth phenotype was restored under acid stress. Further, similar results were obtained for S. mutans UA159. Thus, htrA in S. mutans K7, as well as S. mutans UA159, can be concluded to play an important role during acid stress.     

Electrochemical proton gradient and lactate concentration gradient in Streptococcus cremoris cells grown in batch culture.

The lactate concentration gradient and the components of the electrochemical proton gradient (delta micro H+) were determined in cells of Streptococcus cremoris growing in batch culture. The membrane potential (delta psi) and the pH gradient (delta pH) were determined from the accumulation of the lipophilic cation tetraphenylphosphonium and the weak acid benzoate, respectively. During growth the external pH decreased from 6.8 to 5.3 due to the production of lactate. Delta pH increased from 0 to -35 mV, inside alkaline (at an external pH of 5.7), and fell to zero directly after growth stopped. Delta psi was nearly constant at -90 mV during growth and also dissipated within 40 min after termination of growth. The internal lactate concentration decreased from 200 mM at the beginning of growth (at pH 6.8) to 30 mM at the end of growth (at pH 5.3); the external lactate concentration increased from 8 to 30 mM due to the fermentation of lactose. Thus, the lactate gradient decreased from 80 mV to zero as growth proceeded and the external pH decreased. From the data obtained on delta psi, delta pH, and the lactate concentration gradient, the H+/lactate stoichiometry (n) was calculated. The value of n varied with the external pH from 1.9 (at pH 6.8) to 0.9 (at pH values below 6). This implies that especially at high pH values the carrier-mediated efflux of lactate supplies a significant quantity of metabolic energy to S. cremoris cells. At pH 6.8 this energy gain was almost two ATP equivalents per molecule of lactose consumed if the H+/ATP stoichiometry equals 2. These results supply strong experimental evidence for the energy recycling model postulated by Michels et al.     

Analysis of Streptococcus mutans proteins modulated by culture under acidic conditions

Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.     

The effect of pH on the growth and metabolism of Streptococcus bovis in continuous culture

Streptococcus bovis H13/1 was grown anaerobically at pHs between 5.0 and 6.5 in a glucose-limited chemostat at a dilution rate of 0.05/h. The growth yield and the production of acetate, ethanol and formate decreased at pHs less than 6.5 whereas the production of lactate increased at the lower pH values. When a culture was subjected to sequential pH changes, growth yield and fermentation products were influenced not only by the pH existing in the culture medium but also by the metabolic activity of the cells at the preceding pHs in the sequence. The results are discussed in relation to the mechanisms available for the maintenance of pH homeo-stasis and for the metabolic control of fermentation pathways in Strep. bovis.     

The effect of pH on the growth and metabolism of Streptococcus bovis in continuous culture

Streptococcus bovis H13/1 was grown anaerobically at pHs between 5.0 and 6.5 in a glucose-limited chemostat at a dilution rate of 0.05/h. The growth yield and the production of acetate, ethanol and formate decreased at pHs less than 6.5 whereas the production of lactate increased at the lower pH values. When a culture was subjected to sequential pH changes, growth yield and fermentation products were influenced not only by the pH existing in the culture medium but also by the metabolic activity of the cells at the preceding pHs in the sequence. The results are discussed in relation to the mechanisms available for the maintenance of pH homeostasis and for the metabolic control of fermentation pathways in Strep. bovis.     

Protoheme,a dispensable growth factor forBacteroides fragilis grown by batch and continuous culture in a basal medium

The growth yields of 10 strains ofBacteroides fragilis isolated from a variety of clinical sites were determined in (a) basal medium, (b) basal medium plus heme, and (c) basal medium plus heme and menadione. The molar growth yield values, expressed as a function of glucose (Y_G) and ATP produced (Y_ATP) for 24 h and 48 h were used for a comparison of different strains. Considerable variation occurred among strains, but in general only the results from 24-h grown cells were reproducible. After this period, the microscopic appearance of cells changed dramatically from well-formed, intact cells to large collections of extracellular vesicles and lysed cells. All strains were stimulated by heme, but marked differences occurred among strains. The addition of heme and menadione to the basal medium increased the Y_G values of some strains, whereas others were unaffected. Heme-cultured cells produced acetate, propionate, and succinate as major metabolic end products and possessed cytochrome b, menaquinone-10, and fumarate reductase activity. Strain NCTC 9343 grown without added heme by continuous culture or batch culture produced cells that were morphologically and biochemically similar. Under both conditions these cells lacked cytochromes, menaquinones, and fumarate reductase activity, but produced high levels of lactate and fumarate together with lower levels of acetate, propionate, and succinate.     

Persistence of Streptococcus mutans in stationary-phase batch cultures and biofilms

Streptococcus mutans is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of S. mutans strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation; BacLight staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (+/-8) days, and an average of 2.7 x 10(5) CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.