ABSTRACT: We performed transcriptome abundance analysis of Salmonella Typhimurium strain SL1344 swap which has been genetically engineered to express the hns open-reading frame from the stpA promoter and the stpA open reading frame from the hns promoter. This strain is designated SL1344(swap). Transcript abundance was compared with that of wild-type SL1344. This comparison was performed to determine the effect of chromosome location of the expression of two related global regulators and how alterations to their expression patterns would impact on their regulons. Three independent RNA samples were harvested from wild-type SL1344 and SL1344(swap) grown to exponential phase (OD600nm = 0.3) and hybridized to a microarray containing multiple probes for each of the SL1344 open reading frames. Please note that the original raw data file for the 'SL1344_wildtype_BR2' sample is unavailable, however, the 'wildtype SL1344 BR2.gpr' contains the raw data missing only the header information.
Project description:Biodegradative arginine decarboxylase is inducible by acid and is derepressed in an hns mutant. Several plasmids from an Escherichia coli library that could complement the hns phenotype were characterized and placed into groups. One group includes plasmids that contain the hns gene and are considered true complements. Another group was found to carry the hfq gene, which encodes the host factor HF-1 for bacteriophage Q beta replication. Plasmids of the third group contain inserts that mapped at 60.2 min on the E. coli chromosome. We identified an open reading frame (stpA) with a deduced amino acid sequence showing more than 60% identity with the sequences of H-NS proteins from several species as being responsible for the hns complementing phenotype of the third group.
Project description:Knowledge of global regulatory networks has been exploited to rewire the gene control programmes of the model bacterium Salmonella enterica serovar Typhimurium. The product is an organism with competitive fitness that is superior to that of the wild type but tuneable under specific growth conditions. The paralogous hns and stpA global regulatory genes are located in distinct regions of the chromosome and control hundreds of target genes, many of which contribute to stress resistance. The locations of the hns and stpA open reading frames were exchanged reciprocally, each acquiring the transcription control signals of the other. The new strain had none of the compensatory mutations normally associated with alterations to hns expression in Salmonella; instead it displayed rescheduled expression of the stress and stationary phase sigma factor RpoS and its regulon. Thus the expression patterns of global regulators can be adjusted artificially to manipulate microbial physiology, creating a new and resilient organism.
Project description:We performed chromatin immunoprecipitation-microarray analysis to investigate differences in function between StpA and H-NS in Escherichia coli cells. StpA binding regions essentially overlap those of H-NS in wild-type cells, while they are reduced to one-third in the hns mutant. The H-NS binding profile was unaffected by stpA inactivation.
Project description:The nucleoid-associated protein H-NS is important for gene regulation in Escherichia coli. We have studied H-NS interaction with StpA and an uncharacterized H-NS-like protein, Hfp, in the uropathogenic E. coli isolate 536 that expresses all three nucleoid-associated proteins. We found distinct interactions of the three proteins at the protein level, resulting in the formation of heteromers, as well as differences in their gene expression at the transcriptional level. Mutants lacking either StpA or Hfp alone did not exhibit a phenotype at 37 degrees C, which is consistent with a low level of expression at that temperature. Expression of the hfp and stpA genes was found to be induced by apparently diametrical conditions, and StpA and Hfp levels could be correlated to modulatory effects on the expression of different H-NS targets, the bgl operon and operons for virulence factors such as fimbriae and capsular polysaccharide. The hns/hfp and hns/stpA double mutants displayed severe growth defects at low and high temperatures respectively. Our findings demonstrated different requirements for the alternative H-NS/Hfp/StpA combinations under these growth conditions. We propose that Hfp and StpA have distinct functions and roles in a dynamic pool of nucleoid-associated proteins that is adapting to requirements in a particular environment.
Project description:The galU gene of Escherichia coli, thought to encode the enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase, had previously been mapped to the 27-min region of the chromosome (J. A. Shapiro, J. Bacteriol. 92:518-520, 1966). By complementation of the membrane-derived oligosaccharide biosynthetic defect of strains with a galU mutation, we have now identified a plasmid containing the galU gene and have determined the nucleotide sequence of this gene. The galU gene is located immediately downstream of the hns gene, and its open reading frame would be transcribed in the direction opposite that of the hns gene (i.e., clockwise on the E. coli chromosome). The nucleotide sequences of five galU mutations were also determined. The enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase was purified from a strain containing the galU gene on a multicopy plasmid. The amino-terminal amino acid sequence (10 residues) of the purified enzyme was identical to the predicted amino acid sequence (after the initiating methionine) of the galU-encoded open reading frame. The functional enzyme appears to be a tetramer of the galU gene product.
Project description:The global regulator H-NS represses transcription in gram negative bacteria. Sfh is a homologue of H-NS and is encoded by plasmid pSfR27. Sfh provides a 'stealth' function that allows pSfR27 to be transmitted to a new host without disrupting the competitive fitness of the new host We used ChIP-on-chip to profile Sfh (3xFLAG-tagged) and H-NS binding sites in Salmonella Typhimurium strain SL1344 and found that Sfh provides its 'stealth' function by targeting a sub-set of H-NS bound genes that display reduced levels of H-NS occupancy with the SL1344 chromosome upon acquisition of plasmid pSfR27 Identification of Sfh binding sites in strains SL1344 (pSfR27) and SL1344 hns (pSfR27) as well as identification of H-NS binding sites in strains SL1344, SL1344 (pSfR27), and SL1344 (pSfR27) sfh
Project description:The bacterial H-NS protein silences expression from sequences with higher AT-content than the host genome and is believed to buffer the fitness consequences associated with foreign gene acquisition. Loss of H-NS results in severe growth defects in Salmonella, but the underlying reasons were unclear. An experimental evolution approach was employed to determine which secondary mutations could compensate for the loss of H-NS in Salmonella. Six independently derived S. Typhimurium hns mutant strains were serially passaged for 300 generations prior to whole genome sequencing. Growth rates of all lineages dramatically improved during the course of the experiment. Each of the hns mutant lineages acquired missense mutations in the gene encoding the H-NS paralog StpA encoding a poorly understood H-NS paralog, while 5 of the mutant lineages acquired deletions in the genes encoding the Salmonella Pathogenicity Island-1 (SPI-1) Type 3 secretion system critical to invoke inflammation. We further demonstrate that SPI-1 misregulation is a primary contributor to the decreased fitness in Salmonella hns mutants. Three of the lineages acquired additional loss of function mutations in the PhoPQ virulence regulatory system. Similarly passaged wild type Salmonella lineages did not acquire these mutations. The stpA missense mutations arose in the oligomerization domain and generated proteins that could compensate for the loss of H-NS to varying degrees. StpA variants most able to functionally substitute for H-NS displayed altered DNA binding and oligomerization properties that resembled those of H-NS. These findings indicate that H-NS was central to the evolution of the Salmonellae by buffering the negative fitness consequences caused by the secretion system that is the defining characteristic of the species.
Project description:Members of the H-NS protein family play a role both in the chromosome architecture and in the regulation of gene expression in bacteria. The genomes of the enterobacteria encode an H-NS paralogue, the StpA protein. StpA displays specific regulatory properties and provides a molecular backup for H-NS. Some enterobacteria also encode third H-NS paralogues. This is the case of the enteroaggregative E. coli (EAEC) strain 042, which encodes the hns, stpA and hns2 genes. We provide in this paper novel information about the role of the H-NS2 protein in strain 042. A C?>?T transition in the hns2 promoter leading to increased H-NS2 expression is readily selected in hns mutants. Increased H-NS2 expression partially compensates for H-NS loss. H-NS2 levels are critical for the strain 042 fitness. Under some circumstances, high H-NS2 expression levels dictated by the mutant hns2 promoter can be deleterious. The selection of T?>?C revertants or of clones harboring insertional inactivations of the hns2 gene can then occur. Temperature also plays a relevant role in the H-NS2 regulatory activity. At 37 °C, H-NS2 targets a subset of the H-NS repressed genes contributing to their silencing. When temperature drops to 25 °C, the repressory ability of H-NS2 is significantly reduced. At low temperature, H-NS plays the main repressory role.
Project description:Bacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the family Enterobacteriaceae, cells express the global regulator H-NS and its paralogue StpA. In Escherichia coli, out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregative E. coli strain 042 carries, in addition to the hns and stpA genes, a third gene encoding an hns paralogue (hns2). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within the Enterobacteriaceae. IMPORTANCE Global regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregative E. coli strain 042 carries a new hitherto uncharacterized copy of the hns gene. We decided to investigate why this pathogenic E. coli strain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool.
Project description:Transcriptional profiling of Salmonella Typhimurium strains SL1344 (pSfR27) and SL1344 (pSfR27) delta sfh to identify 'Sfh-dependent' transcripts Two condition experiment in which the transcriptomes of wild-type SL1344 (pSfR27) and SL1344 (pSfR27) delta sfh were compared to identify 'Sfh-dependent' transcripts