Project description:Staphylococcus aureus is a leading cause of hospital- and community-associated infections. The organism’s ability to cause disease can, in part, be attributable to its ability to adapt to otherwise deleterious host-associated stresses. Like other bacterial species, the modulation of mRNA turnover appears to play an important role in S. aureus adaptation to certain environmental stresses. In the current study Affymetrix GeneChips® were used to examine the S. aureus responses to acid and alkaline shock-inducing conditions and to assess whether stress dependent changes in mRNA turnover are likely to facilitate the organism’s ability to tolerate extreme pH challenge. Results indicate that S. aureus adapts to pH shock by eliciting responses expected of organisms coping with pH alteration, including neutralizing cellular internal pH, DNA repair, amino acid biosynthesis and virulence factor expression. Further, it was found that the cellular response to alkaline conditions elicits a transcriptional profile that is similar to that of stringent response induced cells. Consistent with that observation, we show that the activator of the stringent response, (p)ppGpp, levels are profoundly elevated during alkaline shock conditions. We also show that the mRNA turnover properties of acid or alkaline shocked cells significantly differ from that of cells grown at neutral pH. A comparison of the mRNA degradation properties of transcripts whose titers either increased or decreased in response to sudden pH change revealed that alterations in mRNA degradation may, in part, account for the changes in the mRNA levels of factors predicted to mediate pH tolerance. Finally, a set of small stable RNA molecules were induced in response to acid or alkaline shock conditions. As in other organisms, these molecules may mediate mRNA stability and adaptation to otherwise deleterious growth conditions. Staphylococcus aureus strain UAMS-1 was grown to exponential phase and either mock treated (pH maintained at 7.4) or subjected to acid (pH 4)- or alkaline (pH 10) conditions for 30 min. Next, 200 micrograms per ml of rifampicin were added to arrest transcript synthesis. RNA was extracted from cell suspensions at 0, 2.5, 5, 15, and 30 min post-transcriptional arrest, labeled and hybridized to S. aureus GeneChips. A comparison of 0 min samples allowed assessment of acid and alkaline shock responses. A comparison of 0 min to that of various post-transcriptional arrest RNA samples allowed assessment of the mRNA turnover properties of mock vs acid or alkaline shocked cells. Duplicates of each experimental condition and corresponding post-transcriptional arrest time point were used (biological replicates).

Project description:Comparative phenotype and transcriptome analyses were performed with Bacillus cereus ATCC 14579 exposed to acid down-shock to pH 5.5 set with different acidulants. When acidified with hydrochloric acid (HCl), growth was diminished, whereas 2 mM undissociated lactic acid (HL) or acetic acid (HAc) stopped growth without inactivation (bacteriostatic condition), and 15 mM undissociated HAc caused growth arrest and, finally, cell death, as reflected by a 3 to 4 log inactivation (bactericidal condition). Within the first 60 min after pH down-shock, the intracellular ATP levels of cultures shocked with HCl were increased. The bacteriostatic pH shocks did not result in increased nor decreased intracellular ATP levels, indicating that the high energy status within the stressed aerobically grown B. cereus cells could be maintained. In contrast, exposure to 15 mM undissociated HAc resulted in significant lower ATP levels, which was in accordance with the observed inactivation. The transcriptomic responses pH down-shocked cultures were studied in the same time frame. The analyses revealed general and specific responses coupled to the different phenotypes and the acidulant used. The general acid stress response, shown in all different pH shocks, involves modulation of pyruvate metabolism and an oxidative stress response. The shifts in pyruvate metabolism include induction dehydrogenases of a butanediol fermentation pathway under non-lethal acid stress conditions and of lactate, formate, and ethanol fermentation pathways under 15 mM HAc stress. Other 15 mM HAc-specific responses were induction of the alternative electron-transport systems, including cydAB, and fatty acid biosynthesis genes. Differences in gene expression for the bacteriostatic organic acid stress conditions compared to the growth-retarded inorganic stress condition indicated a more stringent oxidative stress response, including induction of an additional catalase gene and a gene encoding a Dps-like protein. Moreover, modulations in amino acid and oligopeptide transport were also found for the 2 mM HAc and HL shocks. HL-specific and HAc-specific responses both involve amino acid metabolism. Our study on the genome-wide responses of aerobically grown B. cereus pH 5.5 shocks provides a unique overview of the different responses induced by three acidulants relevant for food preservation. Per acid down-shock three exposure times (i.e., 10, 30 and 60 min) were each compared with non-exposed cells (i.e., t0). In total 4 different pH 5.5 acid down-shocks were applied. pH 5.5 was reached by adding different acidulants i.e., hydrochloric acid (HCl), lactic acid (HL) resulting in 2 mM undissociated HL, acetic acid (HAc) resulting in 15 mM undissociated HAc, and a combination of acetic acid and hydrochloric acid (HAc/HCl) resulting in 2 mM undissociated HAc. The experiments were performed in duplicate and the duplicate samples were hybridised with a dye-swap.

Project description:Comparative phenotype and transcriptome analyses were performed with Bacillus cereus ATCC 14579 exposed to acid down-shock to pH 5.5 set with different acidulants. When acidified with hydrochloric acid (HCl), growth was diminished, whereas 2 mM undissociated lactic acid (HL) or acetic acid (HAc) stopped growth without inactivation (bacteriostatic condition), and 15 mM undissociated HAc caused growth arrest and, finally, cell death, as reflected by a 3 to 4 log inactivation (bactericidal condition). Within the first 60 min after pH down-shock, the intracellular ATP levels of cultures shocked with HCl were increased. The bacteriostatic pH shocks did not result in increased nor decreased intracellular ATP levels, indicating that the high energy status within the stressed aerobically grown B. cereus cells could be maintained. In contrast, exposure to 15 mM undissociated HAc resulted in significant lower ATP levels, which was in accordance with the observed inactivation. The transcriptomic responses pH down-shocked cultures were studied in the same time frame. The analyses revealed general and specific responses coupled to the different phenotypes and the acidulant used. The general acid stress response, shown in all different pH shocks, involves modulation of pyruvate metabolism and an oxidative stress response. The shifts in pyruvate metabolism include induction dehydrogenases of a butanediol fermentation pathway under non-lethal acid stress conditions and of lactate, formate, and ethanol fermentation pathways under 15 mM HAc stress. Other 15 mM HAc-specific responses were induction of the alternative electron-transport systems, including cydAB, and fatty acid biosynthesis genes. Differences in gene expression for the bacteriostatic organic acid stress conditions compared to the growth-retarded inorganic stress condition indicated a more stringent oxidative stress response, including induction of an additional catalase gene and a gene encoding a Dps-like protein. Moreover, modulations in amino acid and oligopeptide transport were also found for the 2 mM HAc and HL shocks. HL-specific and HAc-specific responses both involve amino acid metabolism. Our study on the genome-wide responses of aerobically grown B. cereus pH 5.5 shocks provides a unique overview of the different responses induced by three acidulants relevant for food preservation.

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 6.0â??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 pH 6.1 vs pH 7

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Acid shock responses in Staphylococcus aureus

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