Transcriptomic analysis identifies growth rate modulation as a component of the adaptation of mycobacteria to survival inside the macrophage.

The adaptation of the tubercle bacillus to the host environment is likely to involve a complex set of gene regulatory events and physiological switches in response to environmental signals. In order to deconstruct the physiological state of Mycobacterium tuberculosis in vivo, we used a chemostat model to study a single aspect of the organism's in vivo state, slow growth. Mycobacterium bovis BCG was cultivated at high and low growth rates in a carbon-limited chemostat, and transcriptomic analysis was performed to identify the gene regulation events associated with slow growth. The results demonstrated that slow growth was associated with the induction of expression of several genes of the dormancy survival regulon. There was also a striking overlap between the transcriptomic profile of BCG in the chemostat model and the response of M. tuberculosis to growth in the macrophage, implying that a significant component of the response of the pathogen to the macrophage environment is the response to slow growth in carbon-limited conditions. This demonstrated the importance of adaptation to a low growth rate to the virulence strategy of M. tuberculosis and also the value of the chemostat model for deconstructing components of the in vivo state of this important pathogen.

Compiling a molecular inventory for Mycobacterium bovis BCG at two growth rates: evidence for growth rate-mediated regulation of ribosome biosynthesis and lipid metabolism.

An experimental system of Mycobacterium tuberculosis growth in a carbon-limited chemostat has been established by the use of Mycobacterium bovis BCG as a model organism. For this model, carbon-limited chemostats with low concentrations of glycerol were used to simulate possible growth rates during different stages of tuberculosis. A doubling time of 23 h (D = 0.03 h(-1)) was adopted to represent cells during the acute phase of infection, whereas a lower dilution rate equivalent to a doubling time of 69 h (D = 0.01 h(-1)) was used to model mycobacterial persistence. This chemostat model allowed the specific response of the mycobacterial cell to carbon limitation at different growth rates to be elucidated. The macromolecular (RNA, DNA, carbohydrate, and lipid) and elemental (C, H, and N) compositions of the biomass were determined for steady-state cultures, revealing that carbohydrates and lipids comprised more than half of the dry mass of the BCG cell, with only a quarter of the dry weight consisting of protein and RNA. Consistent with studies of other bacteria, the specific growth rate impacts on the macromolecular content of BCG and the proportions of lipid, RNA, and protein increased significantly with the growth rate. The correlation of RNA content with the growth rate indicates that ribosome production in carbon-limited M. bovis BCG cells is subject to growth rate-dependent control. The results also clearly show that the proportion of lipids in the mycobacterial cell is very sensitive to changes in the growth rate, probably reflecting changes in the amounts of storage lipids. Finally, this study demonstrates the utility of the chemostat model of mycobacterial growth for functional genomic, physiology, and systems biology studies.

Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence.

The presence of a plasmid, containing gene sequences for DNA immunotherapy that are not expressed in microbial culture, imposed a degradation in bioreactor performance in cultures of the host E. coli strain. Significant decreases in growth rate (24%) and biomass yield (7%) and a corresponding increase in overflow metabolism were observed in a strain containing a therapeutic sequence (a hepatitis B antigen under the control of a CMV promotor). The observed increase in overflow metabolism was incorporated into a Metabolic Flux Analysis (MFA) model (as acetate secretion). Metabolic flux analysis revealed an increase in TCA cycle flux, consistent with an increased respiration rate observed in plasmid-containing cells. These effects are thought to result from increased ATP synthesis requirements (24%) arising from the expression of the Kanr plasmid marker gene whose product accounted for 18% of the cell protein of the plasmid-containing strain. These factors will necessitate significantly higher aeration and agitation rates or lower nutrient feed rates in high-density cultures than would be expected for plasmid-free cultures.

Cyclic fed-batch culture for production of human serum albumin in Pichia pastoris.

Simple cyclic fed-batch culture (cfbc), consisting of a constant medium feed with periodic withdrawals of culture, resulted in a product yield (13.4 mg protein per gram biomass) similar to that obtained using the complex multiphase industrial production strategy (13.7 mg protein per gram biomass). In cfbc, productivity was ultimately limited by the rate at which the cells could assimilate methanol. Glycerol was inhibitory to growth at high concentrations. However, product yield continued to increase as the glycerol concentration was increased. In chemostat culture, dissolved oxygen concentration influenced product yield independently of any detectable influence on cell growth.

Decreasing the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338 leads to increased resistance to breakage and increased antibiotic production.

Mutation and selection for increased resistance to cell-wall synthesis inhibitors led to alterations in the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338. Mutants with decreased branching frequency exhibited increased hyphal strength (estimated by in vitro micromanipulation). As the hyphal strength was increased, this led to a greater proportion of hyphal particles in liquid culture with a hyphal fragment diameter of greater than 88 microm. This, in turn, coincided with proportionately increased antibiotic production.

Sporulation at minimum specific growth rate in Aspergillus nidulans chemostat culture predicted using protein synthesis efficiency estimations.

Ribosomal efficiency (RE) estimates provide a quantitative descriptor of intrinsic growth rate of cell populations using readily-obtainable experimental data. In Aspergillus nidulans chemostat cultures, RE increased linearly with growth rate over the range 25-60% of maximum growth rate (mu max), consistent with increasing ribosomal usage with increased growth rate. Above 60%, RE did not increase significantly, suggesting that all ribosomes were functional at 60% of mu max, further increases in growth rate, presumably resulting from increased polypeptide chain elongation rate. Extrapolating the linear part of the RE/growth rate curve predicted zero RE at a growth rate of 0.04 h-1. Chemostat steady state cultures at 0.04 h-1 contained spores (conidia), apparently undergoing a continuous sporulation/germination cycle. We propose that the RE estimates provide a means of predicting the value of minimum specific growth rate (mu min) below which net growth cannot take place.

A physiological model for the control of erythromycin production in batch and cyclic fed batch culture.

Reported differences in antibiotic production dynamics resulting from altering the growth-limiting nutrient (growth-dissociated production in carbon-limited culture and apparent growth-associated production in nitrogen-limited culture) are due to the different effects on growth kinetics. The substrate affinity for nitrate is significantly lower than that for glucose, resulting in nitrogen limitation effectively occurring throughout the culture. Glucose limitation occurs later in the culture, coinciding with the induction of antibiotic production. Induction occurs at the start of nitrogen-limited culture so that production appears to be growth-associated. Evidence that this hypothesis is consistent with production kinetics in cyclic fed batch culture was also obtained.

The physiology of erythromycin biosynthesis in cyclic fed batch culture.

Antibiotic production in Saccharopolyspora erythraea was significantly enhanced in cyclic fed batch culture (c.f.b.c) compared to batch culture, whereas chemostat culture resulted in reduced production. C.f.b.c. allowed the specific growth rate to be varied, with time, according to an asymptotically decreasing trajectory without the necessity for nutrient exhaustion. It was, therefore, possible to increase productivity by increasing the growth-limiting substrate concentration. It was necessary to apply the c.f.b.c. regime to early-exponential-phase cultures in order to obtain a stable, nutrient-limited, c.f.b.c. The antibiotic production rate during any c.f.b.c. cycle was dependent on the relationship between the specific growth rate at the time and the growth rate at the start of the cycle.

Genetic engineering of hybridoma glutamine metabolism.

The murine hybridoma PQXB1/2 cannot be adapted to grow in culture media containing < 0.5 mM glutamine. Transformants selected following electroporation of PQXB1/2 cells with vectors containing a Chinese hamster glutamine synthetase (GS) cDNA under the control of the SV40 early promoter also failed to grow in the absence of glutamine in the culture medium. PQXB1/2 cells have, however, been transformed to glutamine independence following electroporation with a vector containing this glutamine synthetase cDNA under the control of the human cytomegalovirus immediate early promoter. In these cells, sufficient active glutamine synthetase was expressed from one vector per cell to enable growth in glutamine-free media. The specific activity of glutamine synthetase in two transformed cell lines producing parental levels of antibody was increased by 128 and 152%, respectively (0.57 and 0.63 mumol min-1 per 10(6) cells in transformants compared with parental levels of 0.25 mumol min-1 per 10(6) cells). This reprogramming of glutamine synthetase expression and glutamine metabolism is important for developing strategies to deal with ammonia toxicity and the production of cell lines with improved metabolic processes.

Enhancement of monoclonal antibody yield by hybridoma fed-batch culture, resulting in extended maintenance of viable cell population.

Hybridoma batch cultures were extended using feed formulations based on nutrient consumption measured during different batch culture phases when (a) growth but negligible antibody production was taking place; (b) maximum antibody production rate and declining viable cell growth rate were observed. Strategy (a) was the more successful (2.8-fold compared with 1.8-fold antibody titer increase) and maintained cell viability for longer. Analysis of the effects of omitting individual amino acids yielded results which were consistent with those from the feeding experiment.

Estimation of the kinetic constants and elucidation of trends in growth and erythromycin production in batch and continuous cultures of Saccharopolyspora erythraea using curve-fitting techniques.

The kinetics of erythromycin production were dependent on the identity of the growth rate-limiting nutrient during batch cultures of Saccharopolyspora erythraea. Semilogarithmic linear regression provided a single estimate of growth rate during the exponential phase, but partial cubic spline curve fit derivatives provided time-dependent specific growth and production rate profile. Non-growth-linked product formation was observed when the medium was glucose- or phosphate-limited. However, growth-linked product formation was observed in a nitrate-limited medium. The kinetics observed in nitrate-limited chemostat culture provided evidence that Saccharopolyspora erythraea may be subject to noncompetitive inhibition by a growth-linked product under these conditions. A mathematical model was used to test this theory. The model simulation fitted the observed data very closely and was used to calculate estimates of the kinetic parameters involved: [formula; see text]

A three-phase pattern in growth, monoclonal antibody production, and metabolite exchange in a hybridoma bioreactor culture.

The use of partial cubic spline data interpolation for the calculation of volumetric metabolite exchange rates suggested the existence of three distinct metabolic phases during bioreactor culture of a hybridoma cell line. During phase 1, a rapid amino acid uptake rate and ammonia release rate were observed. The growth rate was low and glutamine synthetase activity fell. In phase 2, maximum growth rate and minimum glutamine assimilation and ammonium production rates were observed. Attempts to corroborate the apparent ammonia assimilation in this phase using (15)NH(4)Cl resulted in low incorporation rates into alanine and glutamine. Maximum glutamine synthetase activity took place during this period. Maximum antibody production rate was observed during phase 3 during which peaks in glutamine assimilation, ammonia release, and glutamine synthetase activity were observed. The apparent existence of the three phases prompted us to carry out Northern blot analysis of glutamine synthetase RNA at appropriate times during the process. This revealed a pattern of appearance and dis-appearance of mRNA consistent with the three phases indicated by the fermentation parameters.

Positive selection of antibiotic-producing soil isolates.

Stepwise discriminant analysis was used to identify the most powerful selective substrates which could be used to formulate media capable of enriching for antibiotic-producing soil isolates. This was achieved by characterizing a collection of 74 soil bacteria, including eubacteria and actinomycetes, according to their ability to produce antibacterial antibiotics and their growth responses to 43 physiological and nutritional tests. The characters which were selective for actinomycetes relative to eubacteria included growth on proline (1%, w/v) and humic acid (0.1%) as sole sources of both carbon and nitrogen, growth on nitrate as a nitrogen source, and growth at pH 7.7-8.0. Growth on proline (1%) and humic acid (0.1%) as sole carbon/nitrogen sources, growth on asparagine as a nitrogen source, and growth in the presence of vitamins were among the characteristics which allowed antibiotic-producing actinomycetes to be differentiated from non-antibiotic-producing strains. Several simple isolation media which incorporated the selective substrates identified by discriminant analysis succeeded in increasing the proportion of actinomycetes isolated from soil samples. Furthermore, the percentage of isolates capable of antibiotic production was considerably increased.

The application of materials balancing to the characterization of sequential secondary metabolite formation in Streptomyces cattleya NRRL 8057.

The high substrate yield factor (0.73 g biomass g glucose-1) and low R.Q. (respiratory quotient, i.e. mol CO2 evolved per mol O2 consumed) value (0.8) measured during growth-phase batch cultures of Streptomyces cattleya could be rationalized in terms of the fermentation mass balance when the oxidized elemental composition of biomass was considered. R.Q. was also indicative of the sequence of secondary metabolite formation, the value rising in steps as each new product was formed. The period of maximum respiratory activity and phosphate uptake preceded maximum growth and glucose uptake. At the end of the lytic phase, a cyclopentenedione cobalt chelator was produced. The termination of lysis coincided with melanin production. Sequential cephamycin C and thienamycin production then took place. Specific hyphal protein content (per unit RNA) peaked before the production of each new metabolite. Melanin, cephamycin C and thienamycin production were initiated when glucose, ammonia and phosphate, respectively, became growth-limiting.

Anaplerotic metabolism of Aspergillus nidulans and its effect on biomass synthesis in carbon limited chemostats.

Anaplerotic fixation of carbon dioxide by the fungus Aspergillus nidulans when grown under carbon-limited conditions was mediated by pyruvate carboxylase and a phosphoenol pyruvate (PEP)-metabolising enzyme which has been tentatively designated as PEP carboxylase. The activities of both enzymes were growth rate dependent and measurements of H14CO3 incorporation by growing mycelium indicated that they were responsible for almost all the assimilated carbon dioxide. In carbon-limited chemostats, the maximum rate of bicarbonate assimilation occurred at a dilution rate of 0.11 h-1, equivalent to 1/2 micromax. The affinity of the pyruvate carboxylase for bicarbonate was twice that of the PEP carboxylase under the conditions of growth used. The effect of changing the bicarbonate concentration in carbon-limited chemostats was substantial: increasing the HCO-3 concentration over the range 0.7 - 2.8 mM enhanced biomass synthesis by 22%. Over-shoots in bicarbonate assimilation and carboxylase activity occurred when steady state chemostat cultures were subjected to a step down in dilution rate.