Different biomolecules have been identified in bacterial pathogens that sense changes in temperature and trigger expression of virulence programs upon host entry. However, the dynamics and quantitative outcome of this response in individual cells of a population, and how this influences pathogenicity are unknown. Here, we address these questions using a thermosensing virulence regulator of an intestinal pathogen (RovA of Yersinia pseudotuberculosis) as a model. We reveal that this regulator is part of a novel thermoresponsive bistable switch, which leads to high- and low-invasive subpopulations within a narrow temperature range. The temperature range in which bistability is observed is defined by the degradation and synthesis rate of the regulator, and is further adjustable via a nutrient-responsive regulator. The thermoresponsive switch is also characterized by a hysteretic behavior in which activation and deactivation occurred on vastly different time scales. Mathematical modeling accurately mirrored the experimental behavior and predicted that the thermoresponsiveness of this sophisticated bistable switch is mainly determined by the thermo-triggered increase of RovA proteolysis. We further observed RovA ON and OFF subpopulations of Y. pseudotuberculosis in the Peyer’s patches and caecum of infected mice, and that changes in the RovA ON/OFF cell ratio reduce tissue colonization and overall virulence. This points to a bet-hedging strategy in which the thermoresponsive bistable switch plays a key role in adapting the bacteria to the fluctuating conditions encountered as they pass through the host’s intestinal epithelium and suggests novel strategies for the development of antimicrobial therapies.

ABSTRACT Mycobacterium tuberculosis persists inside granulomas in the human lung. Analysis of the metabolic composition of granulomas from guinea pigs revealed that one of the organic acids accumulating in the course of infection is acetate (B. S. Somashekar, A. G. Amin, C. D. Rithner, J. Troudt, R. Basaraba, A. Izzo, D. C. Crick, and D. Chatterjee, J Proteome Res 10:4186–4195, 2011, doi: http://dx.doi.org/10.1021/pr2003352 ), which might result either from metabolism of the pathogen or might be provided by the host itself. Our studies characterize a metabolic pathway by which M. tuberculosis generates acetate in the cause of fatty acid catabolism. The acetate formation depends on the enzymatic activities of Pta and AckA. Using actyl coenzyme A (acetyl-CoA) as a substrate, acetyl-phosphate is generated and finally dephosphorylated to acetate, which is secreted into the medium. Knockout mutants lacking either the pta or ackA gene showed significantly reduced acetate production when grown on fatty acids. This effect is even more pronounced when the glyoxylate shunt is blocked, resulting in higher acetate levels released to the medium. The secretion of acetate was followed by an assimilation of the metabolite when other carbon substrates became limiting. Our data indicate that during acetate assimilation, the Pta-AckA pathway acts in concert with another enzymatic reaction, namely, the acetyl-CoA synthetase (Acs) reaction. Thus, acetate metabolism might possess a dual function, mediating an overflow reaction to release excess carbon units and resumption of acetate as a carbon substrate. IMPORTANCE During infection, host-derived lipid components present the major carbon source at the infection site. β-Oxidation of fatty acids results in the formation of acetyl-CoA. In this study, we demonstrate that consumption of fatty acids by Mycobacterium tuberculosis activates an overflow mechanism, causing the pathogen to release excess carbon intermediates as acetate. The Pta-AckA pathway mediating acetate formation proved to be reversible, enabling M. tuberculosis to reutilize the previously secreted acetate as a carbon substrate for metabolism.

Yersinia pseudotuberculosis is a human pathogen that causes acute intestinal and systemic diseases. This study investigated the link between pathogenic traits and the metabolic core machinery of Y. pseudotuberculosis using a systems biology approach: the integration of gene expression profiles with metabolic pathway fluxes in the wild type and virulence regulator mutants. The absence of specific virulence regulators particularly perturbed fluxes and gene expression of pyruvate metabolism and tricarboxylic acid cycle, suggesting an involvement of this metabolic node in the virulence management system. Mutants, genetically perturbed in regulators of this metabolic branch point and one of its central enzymes, showed a significant reduction of virulence in an oral mouse infection model. This revealed the pyruvate- TCA cycle node as a focal point for controlling host colonization. Flux rerouting was also identified as response to applied antibiotic therapies. The examination of Yersinia’s fine-tuned adaptation was expanded to temperature, an important infection parameter, using a continuous culture with advanced temperature control to mimic the infection process. The virulence regulator RovA, known to respond to temperature and to control metabolic, stress, and virulence genes, was quantified by Western blot analysis and fluorescence-activated cell sorting. RovA showed a bistable behavior that generally maximizes survival by heterogeneity. Das humanpathogene Bakterium Yersinia pseudotuberculosis verursacht ernsthafte intestinale und systemische Erkrankungen. In dieser Arbeit wurde die Verknupfung von Pathogenitat und Metabolismus in Yersinia pseudotuberculosis unter Verwendung von Transkriptom- und metabolischen 13C-Stoffflussanalysen untersucht. Virulenz-Regulator-Mutanten wiesen im Vergleich zum Wildtyp starke Veranderungen im Pyruvatmetabolismus und im Zitratzyklus auf. Die gezielte genetische Deregulation dieses Stoffwechselknotenpunktes fuhrte zu Mutanten mit stark reduzierter Virulenz im Mausmodell. Der Pyruvat-Zitronensaure-Knoten konnte demnach als ein zentraler Punkt der Virulenzregulation identifiziert werden. Der Einfluss verschiedener Antibiotika auf die Stoffflussverteilung wies daruber hinaus auf einen komplexen Zusammenhang von Metabolismus und inharenter Resistenz hin. Die feinregulierte Anpassung des Organismus wahrend der Infektion wurde weiterfuhrend anhand des temperaturabhangigen Transkriptionsregulators RovA untersucht, der Gene des Metabolismus, der Stressantwort und des Virulenzprogramms der Zelle kontrolliert. Der Infektionsprozess wurde dazu in einer kontinuierlichen Kultur mit praziser Temperaturfuhrung simuliert. Western Blot Analysen und fluoreszenzgestutzte Durchflusszytometrie zeigten bistabiles Verhalten des Proteins RovA. Die daraus hervorgehende Heterogenitat der Population kann die Uberlebenswahrscheinlichkeit der Gesamtpopulation erhohen.

Background: a the Y. . Results: The pyruvate-tricarboxylic acid cycle-node in the carbon core metabolism of Y. pseudotuberculosis is a focal point of its virulence control system . Conclusion: Mutants genetically perturbed at this metabolic control point are less virulent in mice infection studies. Significance: Learning, how pathogenic traits are controlled is crucial to find novel drug targets against the pathogen. ABSTRACT Despite our increasing knowledge of the specific pathogenicity factors in bacteria, the contribution of metabolic processes to virulence is largely unknown. Here, we elucidate a tight connection between pathogenicity and core metabolism in the enteric pathogen Yersinia pseudotuberculosis

Background:Yersinia pseudotuberculosis is a human pathogen and the ancestor of Y. pestis. Results: The pyruvate-tricarboxylic acid cycle node in the carbon core metabolism of Y. pseudotuberculosis is a focal point of its virulence control system. Conclusion: Mutants genetically perturbed at this metabolic control point are less virulent in mouse infection studies. Significance: Learning how pathogenic traits are controlled is crucial for finding novel drug targets against the pathogen.Despite our increasing knowledge of the specific pathogenicity factors in bacteria, the contribution of metabolic processes to virulence is largely unknown. Here, we elucidate a tight connection between pathogenicity and core metabolism in the enteric pathogen Yersinia pseudotuberculosis by integrated transcriptome and [C-13]fluxome analysis of the wild type and virulence-regulator mutants. During aerobic growth on glucose, Y. pseudotuberculosis reveals an unusual flux distribution with a high level of secreted pyruvate. The absence of the transcriptional and post-transcriptional regulators RovA, CsrA, and Crp strongly perturbs the fluxes of carbon core metabolism at the level of pyruvate metabolism and the tricarboxylic acid (TCA) cycle, and these perturbations are accompanied by transcriptional changes in the corresponding enzymes. Knock-outs of regulators of this metabolic branch point and of its central enzyme, pyruvate kinase (pykF), result in mutants with significantly reduced virulence in an oral mouse infection model. In summary, our work identifies the pyruvate-TCA cycle node as a focal point for controlling the host colonization and virulence of Yersinia.

BACKGROUND:Genome scale metabolic reconstructions are developed to efficiently engineer biocatalysts and bioprocesses based on a rational approach. However, in most reconstructions, due to the lack of appropriate measurements, experimentally determined growth parameters are simply taken from literature including other organisms, which reduces the usefulness and suitability of these models. Pseudomonas putida KT2440 is an outstanding biocatalyst given its versatile metabolism, its ability to generate sufficient energy and turnover of NADH and NAD. To apply this strain optimally in industrial production, a previously developed genome-scale metabolic model (iJP815) was experimentally assessed and streamlined to enable accurate predictions of the outcome of metabolic engineering approaches.RESULTS:To substantially improve the accuracy of the genome scale model (iJP815), continuous bioreactor cultures on a mineral medium with glucose as a sole carbon source were carried out at different dilution rates, which covered pulling analysis of the macromolecular composition of the biomass. Besides, the maximum biomass yield (on substrate) of 0.397 gDCW · gglc-1, the maintenance coefficient of 0.037 gglc · gDCW-1 · h-1 and the maximum specific growth rate of 0.59 h-1 were determined. Only the DNA fraction increased with the specific growth rate. This resulted in reliable estimation for the Growth-Associated Maintenance (GAM) of 85 mmolATP · gDCW-1 and the Non Growth-Associated Maintenance (NGAM) of 3.96 mmolATP · gDCW-1 · h-1. Both values were found significantly different from previous assignment as a consequence of a lower yield and higher maintenance coefficient than originally assumed. Contrasting already published 13C flux measurements and the improved model allowed for constraining the solution space, by eliminating futile cycles. Furthermore, the model predictions were compared with transcriptomic data at overall good consistency, which helped to identify missing links.CONCLUSIONS:By careful interpretation of growth stoichiometry and kinetics when grown in the presence of glucose, this work reports on an accurate genome scale metabolic model of Pseudomonas putida, providing a solid basis for its use in designing superior strains for biocatalysis. By consideration of substrate specific variation in stoichiometry and kinetics, it can be extended to other substrates and new mutants.

In this study, we replaced the natural start codons of different enzymes in the central carbon metabolism of Corynebacterium glutamicum to influence their activity toward improved production of the feed amino acid lysine. It was found that the translational start codon directly affects the intracellular activity of the encoded enzyme, whereby the common ATG generally led to higher values as compared with the rare variant GTG. This could be exploited to specifically amplify or attenuate enzyme activities in order to redirect carbon flux from undesired, competing pathways toward reactions supporting lysine formation. Replacement of the natural ATG codon by GTG reduced the specific enzyme activity of pyruvate dehydrogenase (PDH) and phosphoglucoisomerase by 60 and 40%, respectively. Vice versa , the activity of glucose 6‐phosphate dehydrogenase was increased by 40% by the substitution GTG‐>ATG. Implementation of the attenuated pyruvate dehydrogenase in the background of lysine producing C. glutamicum increased product yield by 17%. This was related to a redirection of the metabolic flux toward the supply of the lysine precursor oxaloacetate. The amplified expression of glucose 6‐phosphate dehydrogenase by the start codon exchange increased lysine yield by 10%, linked to an increased flux toward NADPH supply in the pentose phosphate pathway.