Similar effects of an NO challenge and an lpdA knockout on transcription of Salmonella enterica sv Typhimurium 14028
ABSTRACT: Transcriptional profiling of Salmonella enterica sv Typhimurium under NO stress and comparison of profiles between wild type and lpdA mutant cultures. We investigated the bacteriostatic nature of NO·. S. Typhmurium 14028s is prototrophic for all amino acids but cannot synthesize Methionine or Lysine during nitrosative stress. This MK auxotrophy results from reduced availability of succinyl-CoA, a consequence of NO· targeting lipoamide-dependent LpdA activity. LpdA is an essential component of the pyruvate and α-ketoglutarate dehydrogenase complexes. Additional effects of NO· on gene regulation prevent compensatory pathways of succinyl-CoA production. By microarray analysis, more than 50% of the transcriptional response of S. Typhimurium to nitrosative stress is attributable to LpdA inhibition. Two separate two-condition experiment: NO treated versus untreated, wild type versus lpdA mutant. Three biological replicates. Dye swaps performed on two of these.
Project description:Typically, the expression of sRNAs is activated in response to environmental stimuli in order to regulate gene expression through post-transcriptional mechanisms. In the present work we show that the Salmonella Typhimurium paralog sRNAs RyhB-1 and RyhB-2 are induced in response to the nitrosating agent S-nitrosoglutathione (GSNO). Inactivation of these sRNAs decreased S. Typhimurium resistance to GSNO and increased the levels of nitrosylated proteins. These results prompted us to evaluate a possible role of these sRNAs in nitrosative stress resistance. RNA profiling was used as a screening to identify novel RyhB-1 and RyhB-2 regulated targets, and a subset of genes was filtered based on their potential role in the response to nitrosative stress. To confirm our observation, expression of the candidate targets was analyzed by quantitative RT-PCR in a wild type, single and double mutant strains (ΔryhB1, ΔryhB2 and ΔryhB1 ΔryhB2) treated with GSNO. In response to GSNO RyhB-1 and RyhB-2 negatively regulate the expression of the genes cyoABC (cytochrome o oxidase complex), cydB (cytochrome d oxidase complex), cybC (cytochrome b-562), and positively regulate the nirBCD operon (nitrite reductase system). Together, these results suggest that RyhB-1 and RyhB-2 finely tune the expression of genes coding for cytochrome and the nitrate reductase system, allowing the cell to cope with GSNO-induced stress. Total RNA was harvested from two biological replicates of wt S. Typhimurium overexpressing the sRNA RyhB-2 grown in LB medium, in order to identify RhyB-2 targets that might be implicated in reactive nitrogen stress resistance.
Project description:RamA, a 113-amino-acid regulatory protein, belongs to the AraC-XylS transcriptional activator family. Here, the network of genes regulated by RamA was investigated by determining changes in transcript profiles when ramA gene was knocked out from wild type LTL strain, which expresses ramA constitutively. Analysis used aerobic log-phase state RNA from S. Typhimurium strain LTL (ciprofloxacin MIC: 4 µg/ml) derived from S. Typhimurium LT2 by in vitro-induction using ciprofloxacin as control samples for comparison to the experimental samples taken from its mutant strain, LTLvramA with ramA-deletion. Six biological replicates were used for the experiment.Two replicates for each sample. One replicate per array.
Project description:This study provides comprehensive information about plant responses to nitrosative stress at transcript level and would prove helpful in understanding and incorporating mechanisms associated with nitrosative stress responses in plants. 1mM CysNO Treatment to Leaf disc, Transcriptome profiling
Project description:The adaptation strategies and regulatory networks behind the adaptation of D. shibae DFL12T to nitrosative stress under anaerobic starvation was investigated. Furthermore, the role of the oxygen sensing regulator FnrL and the heme coordinating regulators DnrD and DnrF were determined under nitrosative stress conditions. Therefore transcriptional analyses were performed using regulator knock out strains. Overall design: Dinoroseobacter shibae DFL12T (DSM 16493T) and corresponding fnrL, dnrD and dnrF depletion strains (DS001 (DfnrL), DS002 (DdnrD) and DS004 (DdnrF)) were grown in artificial saltwater minimal medium (SWM) supplemented with 16.9 mM succinate in baffled flasks shaking at 200 rpm for aerobic growth and for anaerobic cultivation 25 mM pyruvate was added and incubation was performed in serum flasks with rubber stoppers shaking a 100 rpm. For nitrosative stress conditions 50 nM of NO saturated water was added. D. shibae wild type and mutant strains were grown under aerobic conditions up to the mid exponential growth phase (OD578 nM 0.5) and shifted to anaerobiosis to mimicke the physiologic conditions in the marine habitat. The samples were taken before (as reference) NO injection and after 30 minutes of nitrosative stress. Three biological replicas were analyzed. Comparison: Identification of genes induced or repressed in the Dinoroseobacter shibae DSM 16493T Crp/ Fnr regulator mutant strains and wild type strain the under nitrosative stress conditions.
Project description:Salmonella enterica serovar Typhimurium is a Gram-negative bacterium, facultative anaerobe and intracellular pathogen that causes enteric fever in mice. Once orally ingested, Salmonella invades and traverse the mucosal intestinal epithelia, where it is phagocytized by specialized cells including macrophages, dendritic cells and neutrophils. Within these cells, the bacterium is kept in a compartment termed Salmonella containing vacuole where it is exposed to different adverse conditions including nutrient deprivation, acid pH, reactive oxygen (ROS) as well as nitrogen (RNS) species and low oxygen levels. Among the signals encountered by the bacteria, oxidative stress is one of the main challenges that it has to overcome in order to survive. In this context, the OxyR and SoxRS proteins are the most studied regulators involved in response to ROS. However, in the past years growing evidence suggests that the ArcAB two-component system might play a key role in modulating gene expression in response to ROS. Furthermore, the global regulator ArcA is required for the resistance of Escherichia coli, S. Enteritidis and Typhimurium to hydrogen peroxide (H2O2), however, the ArcA regulon under oxidative stress conditions remains elusive. Therefore, the aim of this work was to demonstrate that ArcAB regulates the expression of genes in response to hydrogen peroxide and determine the ArcA regulon under this condition. To achieve this, we evaluated transcriptomic changes in strain 14028s, ∆arcA in response to H2O2. Total RNA was harvested from three biological replicates of wt and arcA mutant cultures exposed or unexposed to 1.5 mM hydrogen peroxide for 20 min in LB medium.
Project description:Transcriptional profiling of P. gingivalis cells comparing cells grown in anaerobic conditions to cells grown in anaerobic conditions and exposed to nitrite and to GSNO. Two-condition experiment, anaerobically vs. anaerobically exposed to nitrosative stress cells. Biological replicates: 1 independently grown and harvested bacterial cells for each nitrosative exposure tested. Four technical replicates for each experiment.
Project description:RNA sequencing was performed on Candida albicans wild type cells (JC50) grown to exponential phase on YPD , YPD plus Nitrosative Stress 2.5mM DPTA NONOate, and compared to exponential Candida albicans hog1 deletion mutant cells grown on on YPD , YPD plus Nitrosative Stress 2.5mM DPTA NONOate. Three independent experiments were performed.
Project description:The aim of this study is to investigate the transcriptional response of S. Typhimurium to heat, osmotic, oxidative and acid stress under anoxic and oxic conditions and to non-stressed anoxic conditions.
Project description:Transcriptional regulation mediates adaptation of pathogens to environmental stimuli and is important for host colonisation. The Campylobacter jejuni genome sequence reveals a surprisingly small set of regulators, mostly of unknown function, suggesting an intricate regulatory network. Interestingly, C. jejuni lacks the homologues of ubiquitous regulators involved in stress response found in many other Gram-negative bacteria. Nonetheless, cj1000 is predicted to code for the sole LysR-type regulator in the C. jejuni genome, and thus may be involved in major adaptation pathways. A cj1000 mutant strain was constructed and found to be attenuated in its ability to colonise 1-day old chicks. Complementation of cj1000 mutation restored the colonisation ability to that of wild type levels. The mutant strain was also outcompeted in a competitive colonisation assay of the piglet intestine. High resolution oxygraphy was carried out for the first time on C. jejuni and revealed a role for Cj1000 in controlling O2 consumption. Furthermore, microarray analysis of the cj1000 mutant revealed both direct and indirect regulatory targets, including genes involved in energy metabolism and oxidative stress defences. These results highlight the importance of Cj1000 regulation in host colonisation and in major physiological pathways. Microarray data was collected from three independent biological replicates and 3-9 technical replicates for each biological replicate.
Project description:The antimicrobial action of the curing agent NaNO2, which is added as a preservative to raw meat products, depends on its conversion to nitric oxide and other reactive nitrogen species under acidic conditions. In this study, we applied RNA-sequencing to analyze the acidified NaNO2 shock and adaptive response of Salmonella Typhimurium, a frequent contaminant in raw meat. Upon a 10 minute exposure to 150 mg/l NaNO2 in LB pH 5.5 acidified with lactic acid, genes involved in nitrosative stress protection together with several other stress related genes were induced. To the contrary, genes involved in translation, transcription, replication and motility were down-regulated. Induction of stress tolerance and reduction of cell proliferation obviously promote survival under harsh acidified NaNO2 stress. The subsequent adaptive response was characterized by up-regulation of NsrR-regulated genes and iron-uptake systems and down-regulation of genes involved in anaerobic respiratory pathways. Strikingly, amino acid decarboxylase systems, which contribute to acid tolerance, displayed increased transcript levels in response to acidified NaNO2. The induction of systems known to be involved in acid resistance indicates a nitrite mediated increase of acid stress. Transcriptome of Salmonella Typhimurium 14028, treated at OD600 = 0.80-0.85 with 150 mg/l NaNO2 (acidified by lactic acid in the growth medium) for 10 min (shock response) or until an OD600 = 1.45-1.55 is reached (1.5 - 2.0 h, adaptation response), was compared to respective control cultures without NaNO2. Amplified cDNA libraries for sequencing on the SOLiD 5500xl system were prepared from one culture per condition.