Loss of the acetate switch in Vibrio vulnificus enhances predation defence against Tetrahymena pyriformis

Vibrio vulnificus is an opportunistic human pathogen and autochthonous inhabitant of coastal marine environments, where the bacterium is under constant predation by heterotrophic protists or protozoans. As a result of this selection pressure, genetic variants with anti-predation mechanisms are selected for and persist in the environment. Such natural variants may also be pathogenic to animal or human hosts, making it important to understand these defence mechanisms. To identify anti-predator strategies, thirteen V. vulnificus strains of different genotypes isolated from diverse environments were exposed to predation by the ciliated protozoan, Tetrahymena pyriformis , and only strain ENV1 was resistant to predation. Further investigation of the cell-free supernatant showed that ENV1 acidifies the environment by the excretion of organic acids, which is toxic to T. pyriformis . As this predation resistance was dependent on the availability of iron, transcriptomes of V. vulnificus in iron-replete and iron-deplete conditions were compared. This analysis revealed that ENV1 ferments pyruvate and the resultant acetyl-CoA leads to acetate synthesis under aerobic conditions, a hallmark of overflow metabolism. The anaerobic respiration global regulator, arcA , was upregulated when iron was available. An Δ arcA deletion mutant of ENV1 accumulated less acetate and importantly, was sensitive to grazing by T. pyriformis . Based on the transcriptome response and quantification of metabolites, we conclude that ENV1 has adapted to overflow metabolism and has lost a control switch that shifts metabolism from acetate excretion to acetate assimilation, enabling it to excrete acetate continuously. We show that overflow metabolism and the acetate switch contribute to prey-predator interactions. Importance Bacteria in the environment, including Vibrio spp., interact with protozoan predators. To defend against predation, bacteria evolve anti-predator mechanisms ranging from changing morphology, biofilm formation and secretion of toxins or virulence factors. Some of these adaptations may result in strains that are pathogenic to humans. Therefore, it is important to study predator defence strategies of environmental bacteria. V. vulnificus thrives in coastal waters and infects humans. Very little is know about the defence mechanisms V. vulnificus expresses against predation. Here we show that a V. vulnificus strain (ENV1) has rewired the central carbon metabolism enabling the production of excess organic acid that is toxic to the protozoan predator, T. pyriformis . This is a previously unknown mechanism of predation defence that protects against protozoan predators.

Acetate excretion by a methanotroph, Methylocaldum marinum S8, under aerobic conditions

ABSTRACT Methane-oxidizing bacteria (methanotrophs) often coexist with methylotrophs that utilize methanol excreted by methanotrophs. Recently, we found that a facultative methylotroph, Methyloceanibacter caenitepidi Gela4T, possibly utilizes acetate rather than methanol in the coculture with a methanotroph, Methylocaldum marinum S8. Here, we examined the effects of oxygen concentrations on growth of and acetate excretion by M. marinum S8 in pure culture and the coculture with M. caenitepidi Gela4T. M. marinum S8 excreted acetate during the exponential growth phase not only under microaerobic (O2 concentrations of 3.5%-6%) but also under aerobic (O2 concentrations of 20%-31%) conditions. RNA-Seq analyses of M. marinum S8 cells grown under aerobic conditions suggested that phosphoketolase and acetate kinase were candidate genes involved in acetate production. Nonmethylotrophic bacteria, Cupriavidus necator NBRC 102504, could grow when cocultured with M. marinum S8, also supporting the existence of methanol-independent cross-feeding from M. marinum S8 under aerobic conditions.

Contrasting effects of isocitrate dehydrogenase deletion on fluxes through enzymes of central metabolism in Escherichia coli

ABSTRACT Flux analysis is central to understanding cellular metabolism and successful manipulation of metabolic fluxes in microbial cell-factories. Isocitrate dehydrogenase (ICDH) deletion conferred contrasting effects on fluxes through substrate-level phosphorylation (SLP) reactions. While significantly increasing flux through pyruvate kinase, it diminishes flux through succinyl CoA synthetase and upregulates phosphotransacetylase (PTA) and acetate kinase (AK). In addition to acetate, the ICDH-less strain excretes pyruvate, citrate and isocitrate. While efflux to acetate excretion by the Escherichia coli parental strain and its ICDH-less derivative is a reflection of high throughput of glycolytic intermediates, excretion of pyruvate is a reflection of high throughput via pyruvate kinase. On the other hand, citrate and isocitrate excretion is a reflection of truncating the Krebs cycle at the level of ICDH. Furthermore, another striking finding is the inability of the ICDH-less cultures to utilize acetate as a source of carbon despite the availability of an adequate supply of extracellular glutamate (for biosynthesis) and elevated levels of AK and PTA (for acetate uptake). This striking observation is now explicable in the light of the newly proposed hypothesis that the expression of the ace operon enzymes is controlled in response to a minimum threshold signal (ATP), which could not be achieved in the ICDH-less strain.

Hierarchy in Pentose Sugar Metabolism in Clostridium acetobutylicum

ABSTRACTBacterial metabolism of polysaccharides from plant detritus into acids and solvents is an essential component of the terrestrial carbon cycle. Understanding the underlying metabolic pathways can also contribute to improved production of biofuels. Using a metabolomics approach involving liquid chromatography-mass spectrometry, we investigated the metabolism of mixtures of the cellulosic hexose sugar (glucose) and hemicellulosic pentose sugars (xylose and arabinose) in the anaerobic soil bacteriumClostridium acetobutylicum. Simultaneous feeding of stable isotope-labeled glucose and unlabeled xylose or arabinose revealed that, as expected, glucose was preferentially used as the carbon source. Assimilated pentose sugars accumulated in pentose phosphate pathway (PPP) intermediates with minimal flux into glycolysis. Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among the pentose sugars, with arabinose utilized preferentially over xylose. The phosphoketolase pathway (PKP) provides an alternative route of pentose catabolism inC. acetobutylicumthat directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate, bypassing most of the PPP. When feeding the mixture of pentose sugars, the labeling patterns of lower glycolytic intermediates indicated more flux through the PKP than through the PPP and upper glycolysis, and this was confirmed by quantitative flux modeling. Consistent with direct acetyl-phosphate production from the PKP, growth on the pentose mixture resulted in enhanced acetate excretion. Taken collectively, these findings reveal two hierarchies in clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP.

Metabolite and transcriptome analysis of Campylobacter jejuni in vitro growth reveals a stationary-phase physiological switch

Campylobacter jejuni is a prevalent cause of food-borne diarrhoeal illness in humans. Understanding of the physiological and metabolic capabilities of the organism is limited. We report a detailed analysis of the C. jejuni growth cycle in batch culture. Combined transcriptomic, phenotypic and metabolic analysis demonstrates a highly dynamic ‘stationary phase’, characterized by a peak in motility, numerous gene expression changes and substrate switching, despite transcript changes that indicate a metabolic downshift upon the onset of stationary phase. Video tracking of bacterial motility identifies peak activity during stationary phase. Amino acid analysis of culture supernatants shows a preferential order of amino acid utilization. Proton NMR (1H-NMR) highlights an acetate switch mechanism whereby bacteria change from acetate excretion to acetate uptake, most probably in response to depletion of other substrates. Acetate production requires pta (Cj0688) and ackA (Cj0689), although the acs homologue (Cj1537c) is not required. Insertion mutants in Cj0688 and Cj0689 maintain viability less well during the stationary and decline phases of the growth cycle than wild-type C. jejuni, suggesting that these genes, and the acetate pathway, are important for survival.

Acetate excretion during growth of Salmonella enterica on ethanolamine requires phosphotransacetylase (EutD) activity, and acetate recapture requires acetyl-CoA synthetase (Acs) and phosphotransacetylase (Pta) activities

This report shows that Salmonella enterica catabolizes ethanolamine to acetyl-CoA (Ac-CoA), which enters the glyoxylate bypass and tricarboxylic acid cycle for the generation of energy and central metabolites. During growth on ethanolamine, S. enterica excreted acetate, whose recapture depended on Ac-CoA synthetase (Acs) and the housekeeping phosphotransacetylase (Pta) enzyme activities. The Pta enzyme did not play a role in acetate excretion during growth of S. enterica on ethanolamine. It is proposed that during growth on ethanolamine, acetate excretion is necessary to maintain a pool of free CoA. Acetate excretion requires the eut operon-encoded phosphotransacetylase (EutD) and acetate kinase (Ack) enzymes. EutD function was not required for growth on ethanolamine, and an eutD strain showed only a slight reduction in growth rate. The existence of an as-yet-unidentified system that releases acetate was revealed during growth of a strain lacking Acs, the housekeeping phosphotransacetylase (Pta), and EutD. The functions of pyruvate oxidase (PoxB), Ack and STM3118 protein [a homologue of the Saccharomyces cerevisiae Ac-CoA hydrolase (Ach1p) enzyme] were not involved in the release of acetate by the acs pta eutD strain.