The small RNA RyhB homologs from Salmonella typhimurium participate in the response to S-nitrosoglutathione-induced stress.

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 Salmonellatyphimurium paralogous 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 screen to identify novel RyhB-1 and RyhB-2 regulated targets. A subset of genes was filtered based on their potential role in the response to nitrosative stress and their expression was analyzed by quantitative RT-PCR in 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 bo oxidase), cydB (cytochrome bd oxidase), 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 oxidases and the nitrate reductase system, allowing the cell to cope with GSNO-induced stress.

Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq.

A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.

Differences in gene content between Salmonella enterica serovar Enteritidis isolates and comparison to closely related serovars Gallinarum and Dublin.

Salmonella enterica serovar Enteritidis is often transmitted into the human food supply through eggs of hens that appear healthy. This pathogen became far more prevalent in poultry following eradication of the fowl pathogen S. enterica serovar Gallinarum in the mid-20th century. To investigate whether changes in serovar Enteritidis gene content contributed to this increased prevalence, and to evaluate genetic heterogeneity within the serovar, comparative genomic hybridization was performed on eight 60-year-old and nineteen 10- to 20-year-old serovar Enteritidis strains from various hosts, using a Salmonella-specific microarray. Overall, almost all the serovar Enteritidis genomes were very similar to each other. Excluding two rare strains classified as serovar Enteritidis in the Salmonella reference collection B, only eleven regions of the serovar Enteritidis phage type 4 (PT4) chromosome (sequenced at the Sanger Center) were absent or divergent in any of the other serovar Enteritidis strains tested. The more recent isolates did not have consistent differences from 60-year-old field isolates, suggesting that no large genomic additions on a whole-gene scale were needed for serovar Enteritidis to become more prevalent in domestic fowl. Cross-hybridization of phage genes on the array with related genes in the examined genomes grouped the serovar Enteritidis isolates into two major lineages. Microarray comparisons of the sequenced serovar Enteritidis PT4 to isolates of the closely related serovars Dublin and Gallinarum (biovars Gallinarum and Pullorum) revealed several genomic areas that distinguished them from serovar Enteritidis and from each other. These differences in gene content could be useful in DNA-based typing and in understanding the different phenotypes of these related serovars.

Genomic comparisons of Salmonella enterica serovar Dublin, Agona, and Typhimurium strains recently isolated from milk filters and bovine samples from Ireland, using a Salmonella microarray.

Salmonella-induced enterocolitis is the leading food-borne illness with a lethal outcome and causes millions of cases of gastroenteritis each year. We examined genomic variation among 12 environmental, veterinary, and clinical Salmonella enterica serovar Dublin, Agona, and Typhimurium strains isolated in Ireland between 2000 and 2003, as well as two clinical isolates from Canada and four archival isolates, which belonged to serovars Dublin and Agona. Using DNA-DNA hybridization to a microarray consisting of most of the predicted protein-encoding sequences of the S. enterica serovar Typhimurium LT2 genome, we identified a number of genomic regions that were absent in one or more serovars. The 34 genomic regions encoded proteins involved in sugar metabolism, transport, fimbrial and phage biogenesis, and transcriptional regulation, as well as inner and outer membrane-associated proteins. Two of the four prophages identified in strain LT2, prophages Gifsy-1 and Gifsy-2, were present in all six serovar Typhimurium strains examined. Prophage Fels-1 was absent from all 18 isolates examined, and Fels-2 was completely absent from the serovar Typhimurium isolates and the Salmonella Reference Collection B serovar Dublin strain Du2. All five Salmonella pathogenicity islands were present in all isolates. Plasmid pSLT was absent from all serovar Agona isolates, and only homologues of the spv genes were present in eight of the nine serovar Dublin strains. Only limited intraserovar diversity was found among the nine serovar Dublin, three serovar Agona, and six serovar Typhimurium isolates examined even though these isolates had extensive geographic, temporal, and source differences.

Characterization of Salmonella enterica subspecies I genovars by use of microarrays.

Subspecies 1 of Salmonella enterica is responsible for almost all Salmonella infections of warm-blooded animals. Within subspecies 1 there are over 2,300 known serovars that differ in their prevalence and the diseases that they cause in different hosts. Only a few of these serovars are responsible for most Salmonella infections in humans and domestic animals. The gene contents of 79 strains from the most prevalent serovars were profiled by microarray analysis. Strains within the same serovar often differed by the presence and absence of hundreds of genes. Gene contents sometimes differed more within a serovar than between serovars. Groups of strains that share a distinct profile of gene content can be referred to as "genovars" to distinguish them from serovars. Several misassignments within the Salmonella reference B collection were detected by genovar typing and were subsequently confirmed serologically. Just as serology has proved useful for understanding the host range and pathogenic manifestations of Salmonella, genovars are likely to further define previously unrecognized specific features of Salmonella infections.

Differences in gene content among Salmonella enterica serovar typhi isolates.

We used a nonredundant microarray of the Salmonella enterica serovar Typhimurium LT2 and Typhi CT18 genomes to assess the genomic content of a diverse set of isolates of serovar Typhi. Comparative genomic hybridization revealed 13 regions of absent or divergent gene content in the eight Typhi strains examined compared to Typhi CT18. In particular, two Typhi CT18 prophage regions, STY1048 to STY1077 and STY2038 to STY2077, as well as a five-gene islet (STY3188 to STY3193) were absent or divergent in all other Typhi strains examined. Seven Typhi strains lacked most or all of the IS1 elements present in strain CT18, and three Typhi strains lacked a P4-like phage (STY4821 to STY4834). One strain was devoid of a 149-gene region (STY4521 to STY4680), which encodes numerous phage genes and the Vi antigen biosynthesis and export gene cluster, a type IV pilus, and numerous phage genes. In Typhi strain 26T25, an amplification of an entire inter-ribosomal region encompassing 31 genes has occurred. Furthermore, a 257-gene region (STY1360 to STY1639) showed an aberrant replication pattern in three Typhi isolates. Overall, these differences in gene content indicate that even within a highly clonal bacterial population the genomic reservoir is unstable.

The DeltauvrB mutations in the Ames strains of Salmonella span 15 to 119 genes.

The DeltauvrB mutations present in strains of Salmonella enterica Typhimurium used commonly in the Salmonella (Ames) mutagenicity assay were isolated independently for at least five different his mutants. These deletions all involved the galactose operon, biotin operon, nucleotide-excision-repair uvrB gene, and chlorate-resistance genes. Beyond this, the size of the deletions and the number and type of genes deleted have remained unknown for nearly 30 years. Here, we have used genomic hybridization to a Typhimurium microarray to characterize these five DeltauvrB deletions. The number of genes (and amount of DNA) deleted due to the DeltauvrB mutations are 15 (16kb) each in TA97 and TA104, 47 (50kb) in TA100, 87 (96kb) in TA1537, and 119 (125kb) in TA98, accounting for 0.3, 0.3, 1.0, 1.9, and 2.6% of the genome, respectively. In addition, TA97 and TA104 contain an identical three-gene deletion elsewhere in their genomes, and, most remarkably, TA104 contains a 282-gene amplification, representing 7% of the genome. Missing genes include mfdA and mdaA, encoding a multi-drug translocase and a major nitroreductase, respectively, both absent in TA98; dps, encoding a DNA-binding protein absent in TA1537 and TA98; and dinG, encoding a lexA-regulated repair enzyme, absent in three DeltauvrB lineages. Genes involved in molybdenum cofactor biosynthesis and a number of ORFs of unknown functions are missing in all DeltauvrB strains investigated. Studies in DeltauvrB strains of Escherichia coli have found that the enhanced mutagenesis of some base analogues was due to the deletion of genes involved in molybdenum cofactor biosynthesis rather than to deletion of uvrB. These discoveries do not diminish the value of the data generated in the Ames strains. However, absence of genes other than uvrB may account for the enhanced mutagenicity of some compounds in DeltauvrB Ames strains. In general, microarrays will be useful for characterizing the extent and nature of deletion and amplification mutations.

Complete genome sequence of Salmonella enterica serovar Typhimurium LT2.

Salmonella enterica subspecies I, serovar Typhimurium (S. typhimurium), is a leading cause of human gastroenteritis, and is used as a mouse model of human typhoid fever. The incidence of non-typhoid salmonellosis is increasing worldwide, causing millions of infections and many deaths in the human population each year. Here we sequenced the 4,857-kilobase (kb) chromosome and 94-kb virulence plasmid of S. typhimurium strain LT2. The distribution of close homologues of S. typhimurium LT2 genes in eight related enterobacteria was determined using previously completed genomes of three related bacteria, sample sequencing of both S. enterica serovar Paratyphi A (S. paratyphi A) and Klebsiella pneumoniae, and hybridization of three unsequenced genomes to a microarray of S. typhimurium LT2 genes. Lateral transfer of genes is frequent, with 11% of the S. typhimurium LT2 genes missing from S. enterica serovar Typhi (S. typhi), and 29% missing from Escherichia coli K12. The 352 gene homologues of S. typhimurium LT2 confined to subspecies I of S. enterica-containing most mammalian and bird pathogens-are useful for studies of epidemiology, host specificity and pathogenesis. Most of these homologues were previously unknown, and 50 may be exported to the periplasm or outer membrane, rendering them accessible as therapeutic or vaccine targets.

Helicobacter pylori motility.

Motility is essential for Helicobacter pylori colonization. This review discusses the biochemistry, genetics and genomics of the H. pylori flagellum, and compares these features with well-characterized bacteria.

Molecular characterization of a flagellar export locus of Helicobacter pylori.

Motility of Helicobacter species has been shown to be essential for successful colonization of the host. We have investigated the organization of a flagellar export locus in Helicobacter pylori. A 7-kb fragment of the H. pylori CCUG 17874 genome was cloned and sequenced, revealing an operon comprising an open reading frame of unknown function (ORF03), essential housekeeping genes (ileS and murB), flagellar export genes (fliI and fliQ), and a homolog to a gene implicated in virulence factor transport in other pathogens (virB11). A promoter for this operon, showing similarity to the Escherichia coli sigma70 consensus, was identified by primer extension. Cotranscription of the genes in the operon was demonstrated by reverse transcription-PCR, and transcription of virB11, fliI, fliQ, and murB was detected in human or mouse biopsies obtained from infected hosts. The genetic organization of this locus was conserved in a panel of H. pylori clinical isolates. Engineered fliI and fliQ mutant strains were completely aflagellate and nonmotile, whereas a virB11 mutant still produced flagella. The fliI and fliQ mutant strains produced reduced levels of flagellin and the hook protein FlgE. Production of OMP4, a member of the outer membrane protein family identified in H. pylori 26695, was reduced in both the virB11 mutant and the fliI mutant, suggesting related functions of the virulence factor export protein (VirB11) and the flagellar export component (FliI).

The 52 degrees-55 degrees segment of the Bacillus subtilis chromosome: a region devoted to purine uptake and metabolism, and containing the genes cotA, gabP and guaA and the pur gene cluster within a 34960 bp nucleotide sequence.

Within the framework of the international project for sequencing the entire Bacillus subtilis genome, we have determined the complete sequence of the segment flanking the purE-D gene cluster (55 degrees) as far as cotA (52 degrees). This segment (34960 bp) contains, as well as 12 genes already identified as part of the pur operon, 17 putative ORFs and one partial one. Two of them (gabP and guaA) are known B. subtilis genes. The gene product of cotA (formerly pig) shows significant similarity to oxidoreductases (phenoxazine synthase and bilirubin oxidase). The putative products of ORFs yeaB (Czd protein), yeaC (MoxR), yebA (CNG-channel and cGMP-channel proteins from eukaryotes), yebB (hypothetical 32.9 kDa protein of Escherichia coli), yecA (amino acid permease) and yecB (adenine deaminase) were similar to proteins in data banks.