Project description:Abiotic stresses disturb and limit nitrogen-fixing symbioses between rhizobia and their host legumes. In particular, the effect of extracellular acidity on rhizobia has been taken as a model example for analysis because of the economic impact and worldwide distribution of these symbionts in agricultural countries. Except for valuable molecular-biological studies on different rhizobia, no consolidated models have been formulated to describe the central physiologic changes that occur in acid-stressed bacteria. We present here an integrated analysis entailing the main cultural, metabolic, and molecular responses of the model bacterium Sinorhizobium meliloti growing under controlled acid stress in a chemostat. A stepwise extracellular acidification of the culture medium had indicated that S. meliloti stopped growing at ca. pH 6.0–6.1. Under such a limiting acid stress the rhizobia increased the O2 consumption per cell by more than 5-fold. This phenotype, together with an increase in the transcripts for several membrane cytochromes, entails a remarkably higher aerobic-respiration rate in acid-stressed rhizobia. Changes in the transcripts encoding enzymes for lipid biosynthesis were also observed, consistent with previous data on rhizobial pH-dependent membrane remodelling. Together with increased energy demands under acidity, proteomic and transcriptomic data revealed that while at pH 7.0 the transport and biosynthesis of cellular compounds were quite active processes, under acid stress most overexpressed markers were associated with protein biosynthesis, macromolecular degradation and/or recycling, and energy metabolism. Within this context, the pentose-phosphate pathway exhibited increases in several transcripts, enzymes, and metabolites. Moreover, multivariate analysis of global metabolome data (more than 60 compounds) served to unequivocally correlate the specific-metabolite profiles with the extracellular pH for growth, with strikingly sensitive variations being observed in the rhizobial metabolomes upon extracellular-pH changes of less than 0.5 units. Except for a ca. 120-kb DNA stretch within the pSymA no specific genomic regions were associated with the observed acid-stress responses. Further practical analyses should be focussed on the phenotypic impact and time course of the observed changes during the acid-stress perception and on the search for common responses during the previously described sublethal acid-adaptive processes in rhizobia

Project description:While significant advances have been made in EHEC pathogenesis, we still do not fully understand the impact of environmental stress on EHEC virulence. During the course of infection, EHEC must evade or overcome several biological barriers, the first of which is the gastric acidity encountered during passage through the stomach. EHEC is remarkable in its ability to tolerate this acidity. There are four different acid resistance systems that provide E. coli O157:H7 protection against exposure to low pH (2-2.5). Interestingly, EHEC uses these acid resistance systems differentially for survival in foods versus the bovine intestinal tract. The glutamate-dependent acid-resistance system is thought to offer the best protection below pH 3. Since the infectious dose of EHEC is so low (50-100 organisms), acid resistance becomes an important virulence trait. Studies of EHEC response to acid stress have focused primarily on levels of acid tolerance and the molecular basis of tolerance. However, the impact of acid stress on EHEC virulence is less well understood. In the related pathogen, EPEC, the plasmid-encoded regulator, Per, that regulates expression of many EPEC virulence factors, is regulated negatively at pH 5.5 and positively at pH 8.0, suggesting that virulence gene expression is repressed during mild acid stress and enhanced in alkaline pH typical of the small intestine. Expression of EPEC type III secreted factors involved in A/E lesion formation has been shown to be influenced by factors including culture media, iron and calcium levels. Protein secretion was inhibited at pH 6 and 8. In a third study, a gadE (encoding acid resistance regulator) mutation resulted in increased adhesion of E.coli O157:H7 to colonic epithelial cells, suggesting negative regulation of one or more adhesins. Other studies have reported that shiga toxin production is sensitive to culture conditions including pH. However, there are no studies of EHEC virulence changes after more severe acid stress nor studies linking stressed EHEC virulence phenotype with transcriptional changes. The goal of this study was to determine how acid stress affects EHEC virulence properties and through microarray analysis, define the genetic basis for these changes. Understanding how acid stress modulates the virulence potential of this pathogen is essential for delineating the pathogenesis of disease caused by EHEC infection and may offer novel approaches to prevent and treat EHEC infections.

Project description:While significant advances have been made in EHEC pathogenesis, we still do not fully understand the impact of environmental stress on EHEC virulence. During the course of infection, EHEC must evade or overcome several biological barriers, the first of which is the gastric acidity encountered during passage through the stomach. EHEC is remarkable in its ability to tolerate this acidity. There are four different acid resistance systems that provide E. coli O157:H7 protection against exposure to low pH (2-2.5). Interestingly, EHEC uses these acid resistance systems differentially for survival in foods versus the bovine intestinal tract. The glutamate-dependent acid-resistance system is thought to offer the best protection below pH 3. Since the infectious dose of EHEC is so low (50-100 organisms), acid resistance becomes an important virulence trait. Studies of EHEC response to acid stress have focused primarily on levels of acid tolerance and the molecular basis of tolerance. However, the impact of acid stress on EHEC virulence is less well understood. In the related pathogen, EPEC, the plasmid-encoded regulator, Per, that regulates expression of many EPEC virulence factors, is regulated negatively at pH 5.5 and positively at pH 8.0, suggesting that virulence gene expression is repressed during mild acid stress and enhanced in alkaline pH typical of the small intestine. Expression of EPEC type III secreted factors involved in A/E lesion formation has been shown to be influenced by factors including culture media, iron and calcium levels. Protein secretion was inhibited at pH 6 and 8. In a third study, a gadE (encoding acid resistance regulator) mutation resulted in increased adhesion of E.coli O157:H7 to colonic epithelial cells, suggesting negative regulation of one or more adhesins. Other studies have reported that shiga toxin production is sensitive to culture conditions including pH. However, there are no studies of EHEC virulence changes after more severe acid stress nor studies linking stressed EHEC virulence phenotype with transcriptional changes. The goal of this study was to determine how acid stress affects EHEC virulence properties and through microarray analysis, define the genetic basis for these changes. Understanding how acid stress modulates the virulence potential of this pathogen is essential for delineating the pathogenesis of disease caused by EHEC infection and may offer novel approaches to prevent and treat EHEC infections. Bacteria were grown in LB broth overnight, then subcultured into DMEM and grown at 37C, 5%Co2. Bacteria were then subjected to one of three acid stress protocols: 1) UA30: growth in DMEM pH 7.4 followed by growth in DMEM pH 3.0 for 30 minutes; 2) AA30: growth in DMEM pH 5.0 (adaptation) followed by growth in DMEM pH 3.0; 3) UA15: growth in DMEM pH 7.4 followed by growth in DMEM pH 3.0 for 15 minutes. DMEM was supplemented with 25 mM MES (pH 5.0) and in the case of the control (unadapted, unshocked) 25 mM MOPS (pH 7.4) and the adaptation step was again carried out at 37C and 5% CO2. Acid shocking was done at pH 3.0 (unbuffered) at room temperature for all treatments

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A consolidated analysis of the physiological and molecular responses induced under acid stress in the legume symbiont model soil bacterium Sinorhizobium meliloti

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