Ketogulonicigenium vulgare cells: Acid-stressed vs. Control
ABSTRACT: Transcriptional profiling of K. vulgare cells, co-cultured with Bacillus megaterium, comparing control untreated cells with cells treated with pH 4.0 for 2 h. Differentially expressed genes in acid-stressed cells were analyzed. The aim was to investigate the mechanisms of K. vulgare in response to acid stress on global gene expression. Two-condition experiment, acid-stressed (pH 4.0) cells vs. control cells. Biological replicates: 3 control replicates, 3 acid-stressed replicates.
Project description:Transcriptional profiling of K. vulgare cells co-cultured with Bacillus megaterium compared to K. vulgare mono-cultured cells. Differentially expressed genes in co-cultured and mono-cultured K. vulgare cells were analyzed. The aim was to investigate the mechanisms of B. megaterium stimulating K. vulgare propagation on global gene expression. Two-condition experiment: co-cultured K. vulgare and B. megaterium cells vs. mono-cultured K. vulgare cells. Biological replicates: 3 co-cultured replicates, 3 mono-cultured replicates.
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 pH 6.1 vs pH 7
Project description:The metabolic state of microflora (mixed microbial cultures) in microbial fuel cells (MFCs) is currently unclear. Metabolomic analyses were conducted of microflora growing on the anodic electrodes of MFCs operated at pH 7.0, 5.5, or 4.0 and utilizing starch as the major carbon substrate. A much higher current was produced at pH 7.0 than at pH 5.5 and 4.0, correlating with an increased population ratio of Geobacter species to the total bacteria growing on the electrode. Most intracellular metabolites related to the tricarboxylic acid (TCA) cycle were present at a higher level at pH 7.0 than at pH 5.5 and 4.0, and the levels of metabolites correlated well with the obtained current densities. A high intracellular adenosine triphosphate (ATP)/adenosine diphosphate (ADP) ratio at pH 7.0, compared to at pH 5.5 and 4.0, likewise supported current production. Overall, the metabolomic analyses demonstrated that activation of the TCA cycle and increased ATP generation are critical parameters for electricity generation by microflora.
Project description:Sourdough is a very competitive and challenging environment for microorganisms. Usually, a stable microbiota composed of lactic acid bacteria (LAB) and yeasts comes to dominate this ecosystem. Although rich in carbohydrates, thus providing an ideal environment to grow, the low pH presents a particular challenge. The nature of the adaptation to this low pH was investigated for Lactobacillus plantarum IMDO 130201, an isolate from a laboratory wheat sourdough fermentation. Batch fermentations were carried out in wheat sourdough simulation medium, and total RNA was isolated from mid-exponential growth phase cultures, followed by differential gene expression analysis using a LAB functional gene microarray. At low pH values, an increased expression of genes involved in peptide and amino acid metabolism was observed as well as of genes involved in plantaricin production and lipoteichoic acid synthesis. The results highlight cellular mechanisms that allow L. plantarum to function at a low environmental pH. The labeled aRNA samples were hybridized using a loop design, i.e. two consecutive samples (e.g., pH 3.5 and pH 4.0, pH 4.0 and pH 4.5, etc.) were hybridized on the same microarray slide, each labeled with another fluorescent dye (Cy3 or Cy5), and the loop was closed by hybridizing sample pH 5.5 together with sample pH 3.5.
Project description:The advent of ‘omics’ techniques bear significant potential for the assessment of the microbiological stability of foods. This requires the integration of molecular data with their implication for cellular physiology. Here we performed a comparative physiological and transcriptional analysis of Bacillus subtilis stressed with three different weak organic acids: the commonly used food preservatives sorbic- and acetic- acid, plus the well-known uncoupler carbonyl cyanide-m-chlorophenyl hydrazone (CCCP). The concentration of each compound needed to cause a similar reduction of the growth rate negatively correlated with their membrane solubility, and positively with the concentration of undissociated acid. Intracellular acidification was demonstrated by expressing a pH-sensitive GFP derivative. The largest drop in intracellular pH was observed in CCCP-stressed cells and was accompanied by the transcriptional induction of the general stress response (GSR) and SigM regulon, responses known to be induced by acidification. The GSR was induced by acetate, but not by sorbate in mildly-stressed cells. Microarray analysis further revealed that all three acids activate transcriptional programs normally seen upon nutrient limitation and cause diverse responses indicative of an adaptation of the cell envelope. Based on the responses observed and the utilized pH measurements, the inhibitory effect of sorbic acid seems to be more focused on the cell membrane than that of acetic acid or CCCP. Time-series of B. subtilis response to 25 mM potassium acetate and 0.85 µM CCCP during controlled growth in batch-fermentors in defined minimal medium (pH 6.4). Samples for microarrays were taken from both the treated and control cultures at 0, 10, 20, 30, 40, and 50 min after addition of acetate and CCCP. Two biologically independent experiments were performed.
Project description:Sourdough is a very competitive and challenging environment for microorganisms. Usually, a stable microbiota composed of lactic acid bacteria (LAB) and yeasts comes to dominate this ecosystem. Although rich in carbohydrates, thus providing an ideal environment to grow, the low pH presents a particular challenge. The nature of the adaptation to this low pH was investigated for Lactobacillus plantarum IMDO 130201, an isolate from a laboratory wheat sourdough fermentation. Batch fermentations were carried out in wheat sourdough simulation medium, and total RNA was isolated from mid-exponential growth phase cultures, followed by differential gene expression analysis using a LAB functional gene microarray. At low pH values, an increased expression of genes involved in peptide and amino acid metabolism was observed as well as of genes involved in plantaricin production and lipoteichoic acid synthesis. The results highlight cellular mechanisms that allow L. plantarum to function at a low environmental pH. Overall design: The labeled aRNA samples were hybridized using a loop design, i.e. two consecutive samples (e.g., pH 3.5 and pH 4.0, pH 4.0 and pH 4.5, etc.) were hybridized on the same microarray slide, each labeled with another fluorescent dye (Cy3 or Cy5), and the loop was closed by hybridizing sample pH 5.5 together with sample pH 3.5.
Project description:Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl(-)/H(+) antiport current (I(ClC-3)) in HEK293 cells. The outward rectification and time dependence of I(ClC-3) closely resemble an endogenous HEK293 cell acid-activated Cl(-) current (ICl(acid)) seen at extracellular pH <or= 5.5. ICl(acid) was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. ICl(acid) was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (E(rev)) of I(ClC-3) between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl(-) and H(+) transport become uncoupled at low pH. At pH 4.0 E(rev) was completely Cl(-) dependent (55.8 +/- 2.3 mV/decade). Several findings linked ClC-3 with native ICl(acid); 1) RNA interference directed at ClC-3 message reduced native ICl(acid); 2) removal of the extracellular "fast gate" (E224A) produced large currents that were pH-insensitive; and 3) wild-type I(ClC-3) and ICl(acid) were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10-500 microm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type I(ClC-3) and from native ICl(acid) by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native ICl(acid). Low pH uncouples Cl(-)/H(+) transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl(-)/H(+) antiporters and perhaps for pH regulation in highly acidic intracellular compartments.
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
Project description:The ability of Escherichia coli and Bacillus subtilis to regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. The E. coli cytoplasmic pH fell to a value that varied among individual cells (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In an E. coli biofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells. B. subtilis cells were acid shifted from pH 7.5 to pH 6.0. In B. subtilis, unlike the case with E. coli, cytoplasmic pH showed no "overshoot" but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individual B. subtilis cells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.