Biomedical subjects

Thomas Ferenci

Publications and source records attributed to Thomas Ferenci.

18 recordsLinked to original sources

PubMed2006

Divergence and redundancy of transport and metabolic rate-yield strategies in a single Escherichia coli population.

The energetic efficiency of nutrient uptake and conversion into biomass is a key factor in the ecological behavior of microorganisms. The constraints shaping the metabolic rate-yield trade-off in bacteria are not well understood. To examine whether metabolic rate-yield settings and physiological strategies evolve toward a particular optimum in a constant environment, we studied multiple Escherichia coli isolates evolving in a glucose-limited chemostat population. A major divergence in transport and metabolic strategies was observed, and the isolates included inefficient rate strategists (polluters or cheaters) and yield strategists (conservationists), as well as various hybrid rate-yield strategists and alternative ecotypes (dropouts). Sugar transport assays, strain comparisons based on metabolomics, and Biolog profiling revealed variance to the point of individuality within an evolving population. Only 68 of 177 metabolites assayed were not affected in 10 clonally related strains. The parallel enrichment of rate and yield strategists and the divergence in metabolic phylogenies indicate that bacteria do not converge on a particular rate-yield balance or unique evolutionary solutions. Redundancies in transport and metabolic pathways are proposed to have laid the framework for the multiplicity of bacterial adaptations.

Adaptation, Physiological↗

PubMed2006

Clonal adaptive radiation in a constant environment.

The evolution of new combinations of bacterial properties contributes to biodiversity and the emergence of new diseases. We investigated the capacity for bacterial divergence with a chemostat culture of Escherichia coli. A clonal population radiated into more than five phenotypic clusters within 26 days, with multiple variations in global regulation, metabolic strategies, surface properties, and nutrient permeability pathways. Most isolates belonged to a single ecotype, and neither periodic selection events nor ecological competition for a single niche prevented an adaptive radiation with a single resource. The multidirectional exploration of fitness space is an underestimated ingredient to bacterial success even in unstructured environments.

Adaptation, Physiological↗

PubMed2006

Genotype-by-environment interactions influencing the emergence of rpoS mutations in Escherichia coli populations.

Polymorphisms in rpoS are common in Escherichia coli. rpoS status influences a trade-off between nutrition and stress resistance and hence fitness across different environments. To analyze the selective pressures acting on rpoS, measurement of glucose transport rates in rpoS+ and rpoS bacteria was used to estimate the role of F(nc), the fitness gain due to improved nutrient uptake, in the emergence of rpoS mutations in nutrient-limited chemostat cultures. Chemostats with set atmospheres, temperatures, pH's, antibiotics, and levels of osmotic stress were followed. F(nc) was reduced under anaerobiosis, high osmolarity, and with chloramphenicol, consistent with a reduced rate of rpoS enrichment in these conditions. F(nc) remained high, however, with alkaline pH and low temperature but rpoS sweeps were diminished. Under these conditions, F(sp), the fitness reduction due to lowered stress protection, became significant. We also estimated whether the fitness need for the gene was related to its regulation. No consistent pattern emerged between the level of RpoS and the loss of rpoS function in particular environments. This dissection allows an unprecedented view of the genotype-by-environment interactions controlling a mutational sweep and shows that both F(nc) and F(sp) are influenced by individual stresses and that additional factors contribute to selection pressure in some environments.

Bacterial Proteins↗

PubMed2005

Divergent roles of RpoS in Escherichia coli under aerobic and anaerobic conditions.

Escherichia coli exhibited different levels of rpoS expression and general stress resistance under aerobiosis and anaerobiosis. Expression measured using reporter gene fusions and protein levels was lower under anaerobic conditions. Consistent with earlier findings, rpoS mutants were selected in aerobic nutrient-limited cultures but rpoS mutants were not enriched under anaerobiosis. This result suggested that, despite its decreased level, RpoS had a function under anaerobic conditions not essential under aerobiosis. Competition experiments between rpoS(+) and rpoS bacteria confirmed the advantage conferred by RpoS under anaerobiosis. In contrast, stress resistance assays suggested RpoS made a greater contribution to general stress resistance under aerobiosis than anaerobiosis. These results indicate a significant, but different role of RpoS in aerobic and anaerobic environments.

Aerobiosis↗

PubMed2005

Maintaining a healthy SPANC balance through regulatory and mutational adaptation.

Stress protection is an important but costly contributor to bacterial survival. Two distinct forms of environmental protection share a common cost and a significant species-wide variability. Porin-mediated outer membrane permeability and the RpoS-controlled general stress response both involve a trade-off between self-preservation and nutritional competence, called the SPANC balance. Interestingly, different Escherichia coli strains exhibit distinct settings of the SPANC balance. It is tilted towards high stress resistance and a restricted diet in some isolates whereas others have broader nutritional capability and better nutrient affinity but lower levels of resistance. Growth- or stress-related selective pressures working in opposite directions (antagonistic pleiotropy) result in polymorphisms affecting porins and RpoS. Consequently, these important cellular components are present at distinct concentrations in different isolates. A generalized hypothesis to explain bacterial adaptation, based on the SPANC investigations, is offered. A holistic approach to bacterial adaptation, involving a gamut of regulation and mutation, is likely to be the norm in broadening the capabilities of a species. Indeed, there is unlikely to be a standard regulatory setting typical for all members of a species. Gene regulation provides a limited fine control for maintaining the right level of adaptation in a particular niche but mutational changes provide the coarse control for adaptation between the species-wide environments of free-living bacteria.

Adaptation, Physiological↗

PubMed2004

The role of isocitrate lyase and the glyoxylate cycle in Escherichia coli growing under glucose limitation.

Escherichia coli changes its metabolism in response to environmental circumstances, and metabolic adaptations are evident in hungry bacteria growing slowly in glucose-limited chemostats. The role of isocitrate lyase (AceA) was examined in E. coli growing under glucose limitation. AceA activity was elevated in a strain-dependent manner in the commonly used E. coli K-12 laboratory strains MG1655 and MC4100, but an aceA disruption surprisingly increased fitness under glucose limitation in both strains. However, in bacteria adapted to limiting glucose in long-term chemostats, mutations outside aceA changed its role from a negative to a positive influence. These results suggest that a recently proposed pathway of central metabolism involving the glyoxylate cycle enzymes is redundant in wild-type bacteria, but may take on a beneficial role after context adaptation. Interestingly, the aceA gene sequence did not alter during prolonged selection, so mutations in unidentified genes changed the metabolic context of unaltered AceA from a negative to a positive influence in bacteria highly adapted to limiting glucose.

Bacterial Proteins↗

PubMed2004

The multifactorial influences of RpoS, Mlc and cAMP on ptsG expression under glucose-limited and anaerobic conditions.

The ptsG gene encodes the high-affinity glucose receptor component of the PEP:glucose phosphotransferase system. PtsG is the major glucose transporter in Escherichia coli under glucose-excess conditions but its regulation under glucose limitation or anaerobiosis is poorly defined. Using a ptsG-lacZ transcriptional fusion, ptsG expression was found to peak with low (micromolar) external glucose levels in glucose-limited chemostats, so PtsG is primed to contribute to glucose scavenging under hunger response conditions. This regulatory pattern was confirmed using methyl- alpha-glucoside transport assays of PtsG-dependent transport. The regulation of ptsG by cAMP contributed to the optimal expression with micromolar glucose but ptsG was actually repressed to levels below that in glucose-excess batch cultures at very slow growth rates and submicromolar glucose concentrations. RpoS contributed to repression of ptsG in slow-growing bacteria but not under glucose-excess conditions. Also, Mlc increasingly contributed to the repression of ptsG at residual glucose concentrations too low to saturate PtsG. A similar pattern of ptsG regulation was observed in anaerobic cultures with either glucose-excess or glucose-limiting situations.

Anaerobiosis↗

PubMed2004

A regulatory trade-off as a source of strain variation in the species Escherichia coli.

There are few existing indications that strain variation in prokaryotic gene regulation is common or has evolutionary advantage. In this study, we report on isolates of Escherichia coli with distinct ratios of sigma factors (RpoD, sigmaD, or sigma70 and RpoS or sigmaS) that affect transcription initiated by RNA polymerase. Both laboratory E. coli K-12 lineages and nondomesticated isolates exhibit strain-specific endogenous levels of RpoS protein. We demonstrate that variation in genome usage underpins intraspecific variability in transcription patterns, resistance to external stresses, and the choice of beneficial mutations under nutrient limitation. Most unexpectedly, RpoS also controlled strain variation with respect to the metabolic capability of bacteria with more than a dozen carbon sources. Strains with higher sigmaS levels were more resistant to external stress but metabolized fewer substrates and poorly competed for low concentrations of nutrients. On the other hand, strains with lower sigmaS levels had broader nutritional capabilities and better competitive ability with low nutrient concentrations but low resistance to external stress. In other words, RpoS influenced both r and K strategist functions of bacteria simultaneously. The evolutionary principle driving strain variation is proposed to be a conceptually novel trade-off that we term SPANC (for "self-preservation and nutritional competence"). The availability of multiple SPANC settings potentially broadens the niche occupied by a species consisting of individuals with narrow specialization and reveals an evolutionary advantage offered by polymorphic regulation. Regulatory diversity is likely to be a significant contributor to complexity in a bacterial world in which multiple sigma factors are a universal feature.

Acids↗

PubMed2003

The effect of growth atmosphere on the ability of Listeria monocytogenes to survive exposure to acid, proteolytic enzymes and bile salts.

Four isolates of Listeria monocytogenes from food, human and environmental sources were grown separately in broth (pH 6.0 at 8 degrees C) under atmospheres of air, 100% N(2), 40% CO(2):60% N(2) or 100% CO(2). Exponential and stationary phase cells were harvested to determine if growth atmosphere and growth phase influenced this pathogen's ability to survive exposure to an acid environment coupled with proteolytic enzymes, and the activity of bile salts. In general, isolates were more resistant to the acid environment than the bile salts environment and stationary phase cells were significantly more resistant to both environments than exponential phase cells. Irrespective of prior growth atmosphere, none of the isolates when in exponential phase remained detectable following full exposure to the acid environment (110 min at 37 degrees C) or the bile environment (3 h at 37 degrees C). With the exception of one isolate grown under the atmosphere of 40% CO(2):60% N(2), all isolates when in stationary phase were detectable following full exposure to the acid environment but death rates varied significantly. Stationary phase cells of all isolates grown under 40% CO(2):60% N(2) and 100% CO(2) were highly susceptible to the bile salts environment: cells were not detectable after a 2-min exposure whereas stationary phase cells grown under air or 100% N(2) were recovered following full exposure to the bile environment. Survival curves were characterised by a population decline of at least 3 log(10)/ml (from an initial level of 7 log(10) CFU/ml) in the first 15 min; thereafter a constant population number of approximately 4 log(10)/ml was maintained over the remaining exposure period. No survival was observed when stationary phase cells of L. monocytogenes FRRB 2538 grown in air and 100% N(2) were subjected to the acid environment followed by immediate exposure to the bile salts environment. The results showed that growth atmosphere and growth phase could influence survival of this pathogen against conditions that imitate the extremes of the most important nonspecific defence mechanisms against microbial infection: the acid environment of the stomach coupled with the activity of proteolytic enzymes, and the activity of bile salts in the small intestine.

Acids↗

PubMed2003

The influence of cellular physiology on the initiation of mutational pathways in Escherichia coli populations.

The factors affecting the direction of evolutionary pathways and the reproducibility of adaptive responses were investigated under closely related but non-identical conditions. Replicate chemostat cultures of Escherichia coli were compared when adapting to partial or severe glucose limitation. Four independent populations used a reproducible sequence of early mutational changes under both conditions, with rpoS mutations always occurring first before mgl. However, there were interesting differences in the timing of mutational sweeps: rpoS mutations appeared in a clock-like fashion under both partial and severe glucose limitation, while mgl sweeps arose under both conditions but at different times. Interestingly, malT and mlc mutations appeared only under severe limitation. Even though the ancestors were genotypically identical, the semi-differentiated properties of bacteria growing with mild or severe glucose limitation sent the populations in characteristic directions. Mutation supply and the fitness contribution of mutations were estimated and demonstrated to be potential influences in the choice of particular adaptation pathways under severe and mild glucose limitation. Predicting all the mutations fixed in adapting populations is beyond our current understanding of evolutionary processes, but the interplay between ancestor physiology and the initiation of adaptation pathways is demonstrated and definable in bacterial populations.

Adaptation, Physiological↗

PubMed2003

Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli.

The global pool of all metabolites in a cell, or metabolome, is a reflection of all the metabolic functions of an organism under any particular growth condition. In the absence of in situ methods capable of universally measuring metabolite pools, intracellular metabolite measurements need to be performed in vitro after extraction. In the past, a variety of cell lysis methods were adopted for assays of individual metabolites or groups of intermediates in pathways. In this study, metabolites were extracted from Escherichia coli using six different commonly used procedures including acid or alkaline treatments, permeabilization by freezing with methanol, high-temperature extraction in the presence of ethanol or methanol, and by lysis with chloroform-methanol. Metabolites were extracted by the six methods from cells grown under identical conditions and labeled with [14C]glucose. The metabolomes were compared after 2-dimensional thin-layer chromatography of labeled compounds. For global analysis, extraction with cold (-40 degrees C) methanol showed the greatest promise, allowing simultaneous resolution of more than 95 metabolite spots. In contrast, 80 or less spots were obtained with other extraction methods. Extraction also influenced quantitative analysis of particular compounds. Metabolites such as adenosine exhibited up to 20-fold higher abundance after cold methanol extraction than after extraction with acid, alkali, or chloroform. The simplicity, rapidity, and universality of cold methanol extraction offer great promise if a single method of lysis is to be adopted in metabolome analysis.

Carbon Radioisotopes↗

PubMed2003

What is driving the acquisition of mutS and rpoS polymorphisms in Escherichia coli?

Pathogenic and commensal Escherichia coli isolates frequently contain defective alleles of the mutS and rpoS genes, located in a highly polymorphic segment of the chromosome. The environments leading to enrichment of rpoS mutations and the selective advantages of these mutants are becoming apparent. Unexpectedly, rpoS defects occur because of a basic design limitation in cellular regulation. Antagonistic pleiotropy results from the futile competition between different sigma factors associated with the RNA polymerase, and drives the elimination of RpoS (or sigma(S)) in environments requiring high levels of transcription that is dependent on RpoD (or sigma(D) or sigma(70)). Nutrient-limited environments provide an ideal breeding ground for rpoS mutations. By contrast, in other settings, increased stress resistance selects for restoration of rpoS function. Hence extensive polymorphism in the mutS-rpoS region is postulated to result from cycling between environments in which the functional or non-functional genes provide distinct fitness advantages.

Adenosine Triphosphatases↗

PubMed2003

The activator of GntII genes for gluconate metabolism, GntH, exerts negative control of GntR-regulated GntI genes in Escherichia coli.

Gluconate is one of the preferred carbon sources of Escherichia coli, and two sets of gnt genes (encoding the GntI and GntII systems) are involved in its transport and metabolism. GntR represses the GntI genes gntKU and gntT, whereas GntH was previously suggested to be an activator for the GntII genes gntV and idnDO-gntWH. The helix-turn-helix residues of the two regulators GntR and GntH exhibit extensive homologies. The similarity between the two regulators prompted analysis of the cross-regulation of the GntI genes by GntH. Repression of gntKU and gntT by GntH, as well as GntR, was indeed observed using transcriptional fusions and RNA analysis. High GntH expression, from cloned gntH or induced through 5-ketogluconate, was required to observe repression of GntI genes. Two GntR-binding elements were identified in the promoter-operator region of gntKU and were also shown to be the target sites of GntH by mutational analysis. However, the GntI genes were not induced by gluconate in the presence of enhanced amounts of GntH, whereas repression by GntR was relieved by gluconate. The repression of GntI genes by GntH is thus unusual in that it is not relieved by the availability of substrate. These results led us to propose that GntH activates GntII and represses the GntI genes in the presence of metabolites derived from gluconate, allowing the organism to switch from the GntI to the GntII system. This cross-regulation may explain the progressive changes in gnt gene expression along with phases of cell growth in the presence of gluconate.

Amino Acid Sequence↗

PubMed2002

Enrichment and elimination of mutY mutators in Escherichia coli populations.

The kinetics of mutator sweeps was followed in two independent populations of Escherichia coli grown for up to 350 generations in glucose-limited continuous culture. A rapid elevation of mutation rates was observed in both populations within 120-150 generations, as was apparent from major increases in the proportion of the populations with unselected mutations in fhuA. The increase in mutation rates was due to sweeps by mutY mutators. In both cultures, the enrichment of mutators resulted from hitchhiking with identified beneficial mutations increasing fitness under glucose limitation; mutY hitchhiked with mgl mutations in one culture and ptsG in the other. In both cases, mutators were enriched to constitute close to 100% of the population before a periodic selection event reduced the frequency of unselected mutations and mutators in the cultures. The high proportion of mutators persisted for 150 generations in one population but began to be eliminated within 50 generations in the other. The persistence of mutator, as well as experimental data showing that mutY bacteria were as fit as near-isogenic mutY(+) bacteria in competition experiments, suggest that mutator load by deleterious mutations did not explain the rapidly diminishing proportion of mutators in the populations. The nonmutators sweeping out mutators were also unlikely to have arisen by reversion or antimutator mutations; the mutY mutations were major deletions in each case and the bacteria sweeping out mutators contained intact mutY. By following mgl allele frequencies in one population, we discovered that mutators were outcompeted by bacteria that had rare mgl mutations previously as well as additional beneficial mutation(s). The pattern of appearance of mutY, but not its elimination, conforms to current models of mutator sweeps in bacterial populations. A mutator with a narrow mutational spectrum like mutY may be lost if the requirement for beneficial mutations is for changes other than GC --> TA transversions. Alternatively, epistatic interactions between mutator mutation and beneficial mutations need to be postulated to explain mutator elimination.

DNA Glycosylases↗

PubMed2002

The N terminus of the Escherichia coli transcription activator MalT is the domain of interaction with MalY.

The maltose system of Escherichia coli consists of a number of genes encoding proteins involved in the uptake and metabolism of maltose and maltodextrins. The system is positively regulated by MalT, its transcriptional activator. MalT activity is controlled by two regulatory circuits: a positive one with maltotriose as effector and a negative one involving several proteins. MalK, the ATP-hydrolyzing subunit of the cognate ABC transporter, MalY, an enzyme with the activity of a cystathionase, and Aes, an acetyl esterase, phenotypically act as repressors of MalT activity. By in vivo titration assays, we have shown that the N-terminal 250 amino acids of MalT contain the interaction site for MalY but not for MalK. This was confirmed by gel filtration analysis, where MalY was shown to coelute with the N-terminal MalT structural domain. Mutants in MalT causing elevated mal gene expression in the absence of exogenous maltodextrins were tested in their response to the three repressors. The different MalT mutations exhibited a various degree of sensitivity towards these repressors, but none was resistant to all of them. Some of them became nearly completely resistant to Aes while still being sensitive to MalY. These mutations are located at positions 38, 220, 243, and 359, most likely defining the interaction patch with Aes on the three-dimensional structure of MalT.

ATP-Binding Cassette Transporters↗

PubMed2002

Regulation of mutY and nature of mutator mutations in Escherichia coli populations under nutrient limitation.

Previous analysis of aerobic, glucose-limited continuous cultures of Escherichia coli revealed that G:C-to-T:A (G:C-->T:A) transversions were the most commonly occurring type of spontaneous mutation. One possible explanation for the preponderance of these mutations was that nutrient limitation repressed MutY-dependent DNA repair, resulting in increased proportions of G:C-->T:A transversions. The regulation of the mutY-dependent DNA repair system was therefore studied with a transcriptional mutY-lacZ fusion recombined into the chromosome. Expression from the mutY promoter was fourfold higher under aerobic conditions than under anaerobic conditions. But mutY expression was higher in glucose- or ammonia-limited chemostats than in nutrient-excess batch culture, so mutY was not downregulated by nutrient limitation. An alternative explanation for the frequency of G:C-->T:A transversions was the common appearance of mutY mutator mutations in the chemostat populations. Of 11 chemostat populations screened in detail, six contained mutators, and the mutator mutation in four cultures was located in the region of mutY at 66 min on the chromosome. The spectrum of mutations and rate of mutation in these isolates were fully consistent with a mutY-deficiency in each strain. Based on PCR analysis of the region within and around mutY, isolates from three individual populations contained deletions extending at least 2 kb upstream of mutY and more than 5 kb downstream. In the fourth population, the deletion was even longer, extending at least 5 kb upstream and 5 kb downstream of mutY. The isolation of mutY mutator strains from four independent populations with extensive chromosomal rearrangements suggests that mutY inactivation by deletion is a means of increasing mutation rates under nutrient limitation and explains the observed frequency of G:C-->T:A mutations in glucose-limited chemostats.

DNA Glycosylases↗

PubMed2002

rpoS mutations and loss of general stress resistance in Escherichia coli populations as a consequence of conflict between competing stress responses.

The general stress resistance of Escherichia coli is controlled by the RpoS sigma factor (phi(S)), but mutations in rpoS are surprisingly common in natural and laboratory populations. Evidence for the selective advantage of losing rpoS was obtained from experiments with nutrient-limited bacteria at different growth rates. Wild-type bacteria were rapidly displaced by rpoS mutants in both glucose- and nitrogen-limited chemostat populations. Nutrient limitation led to selection and sweeps of rpoS null mutations and loss of general stress resistance. The rate of takeover by rpoS mutants was most rapid (within 10 generations of culture) in slower-growing populations that initially express higher phi(S) levels. Competition for core RNA polymerase is the likeliest explanation for reduced expression from distinct promoters dependent on phi(70) and involved in the hunger response to nutrient limitation. Indeed, the mutation of rpoS led to significantly higher expression of genes contributing to the high-affinity glucose scavenging system required for the hunger response. Hence, rpoS polymorphism in E. coli populations may be viewed as the result of competition between the hunger response, which requires sigma factors other than phi(S) for expression, and the maintenance of the ability to withstand external stresses. The extent of external stress significantly influences the spread of rpoS mutations. When acid stress was simultaneously applied to glucose-limited cultures, both the phenotype and frequency of rpoS mutations were attenuated in line with the level of stress. The conflict between the hunger response and maintenance of stress resistance is a potential weakness in bacterial regulation.

Bacterial Proteins↗

PubMed1997

The relationship between external glucose concentration and cAMP levels inside Escherichia coli: implications for models of phosphotransferase-mediated regulation of adenylate cyclase.

The concentration of glucose in the medium influences the regulation of cAMP levels in Escherichia coli. Growth in minimal medium with micromolar glucose results in 8- to 10-fold higher intracellular cAMP concentrations than observed during growth with excess glucose. Current models would suggest that the difference in cAMP levels between glucose-rich and glucose-limited states is due to altered transport flux through the phosphoenolpyruvate: glucose phosphotransferase system (PTS), which in turn controls adenylate cyclase. A consequence of this model is that cAMP levels should be inversely related to the saturation of the PTS transporter. To test this hypothesis, the relationship between external glucose concentration and cAMP levels inside E. coli were investigated in detail, both through direct cAMP assay and indirectly through measurement of expression of cAMP-regulated genes. Responses were followed in batch, dialysis and glucose-limited continuous culture. A sharp rise in intracellular cAMP occurred when the nutrient concentration in minimal medium dropped to approximately 0.3 mM glucose. Likewise, addition of > 0.3 mM glucose, but not < 0.3 mM glucose, sharply reduced the intracellular cAMP level of starving bacteria. There was no striking shift in growth rate or [14C] glucose assimilation in bacteria passing through the 0.5 to 0.3 mM concentration threshold influencing cAMP levels, suggesting that neither metabolic flux nor transporter saturation influenced the sensing of nutrient levels. The (IIA/IIBC)Glc PTS is 96-97% saturated at 0.3 mM glucose so these results are not easily reconcilable with current models of cAMP regulation. Aside from the transition in cAMP levels initiated above 0.3 mM, a second shift occurred below 1 muM glucose. Approaching starvation, well below saturation of the PTS, cAMP levels either increased or decreased depending on unknown factors that differ between common E. coli K-12 strains.

Adenylyl Cyclases↗