Search

BackgroundComplete and accurate genome annotation is crucial for comprehensive and systematic studies of biological systems. However, determining protein-coding genes for most new genomes is almost completely performed by inference using computational predictions with significant documented error rates (> 15%). Furthermore, gene prediction programs provide no information on biologically important post-translational processing events critical for protein function.ResultsWe experimentally annotated the bacterial pathogen Salmonella Typhimurium 14028, using "shotgun" proteomics to accurately uncover the translational landscape and post-translational features. The data provide protein-level experimental validation for approximately half of the predicted protein-coding genes in Salmonella and suggest revisions to several genes that appear to have incorrectly assigned translational start sites, including a potential novel alternate start codon. Additionally, we uncovered 12 non-annotated genes missed by gene prediction programs, as well as evidence suggesting a role for one of these novel ORFs in Salmonella pathogenesis. We also characterized post-translational features in the Salmonella genome, including chemical modifications and proteolytic cleavages. We find that bacteria have a much larger and more complex repertoire of chemical modifications than previously thought including several novel modifications. Our in vivo proteolysis data identified more than 130 signal peptide and N-terminal methionine cleavage events critical for protein function.ConclusionThis work highlights several ways in which application of proteomics data can improve the quality of genome annotations to facilitate novel biological insights and provides a comprehensive proteome map of Salmonella as a resource for systems analysis.

To cause a systemic infection, Salmonella must respond to many environmental cues during mouse infection and express specific subsets of genes in a temporal and spatial manner, but the regulatory pathways are poorly established. To unravel how micro-environmental signals are processed and integrated into coordinated action, we constructed in-frame non-polar deletions of 83 regulators inferred to play a role in Salmonella enteriditis Typhimurium (STM) virulence and tested them in three virulence assays (intraperitoneal [i.p.], and intragastric [i.g.] infection in BALB/c mice, and persistence in 129X1/SvJ mice). Overall, 35 regulators were identified whose absence attenuated virulence in at least one assay, and of those, 14 regulators were required for systemic mouse infection, the most stringent virulence assay. As a first step towards understanding the interplay between a pathogen and its host from a systems biology standpoint, we focused on these 14 genes. Transcriptional profiles were obtained for deletions of each of these 14 regulators grown under four different environmental conditions. These results, as well as publicly available transcriptional profiles, were analyzed using both network inference and cluster analysis algorithms. The analysis predicts a regulatory network in which all 14 regulators control the same set of genes necessary for Salmonella to cause systemic infection. We tested the regulatory model by expressing a subset of the regulators in trans and monitoring transcription of 7 known virulence factors located within Salmonella pathogenicity island 2 (SPI-2). These experiments validated the regulatory model and showed that the response regulator SsrB and the MarR type regulator, SlyA, are the terminal regulators in a cascade that integrates multiple signals. Furthermore, experiments to demonstrate epistatic relationships showed that SsrB can replace SlyA and, in some cases, SlyA can replace SsrB for expression of SPI-2 encoded virulence factors.

Using sample-matched transcriptomics and proteomics measurements it is now possible to begin to understand the impact of post-transcriptional regulatory programs in Enterobacteria. In bacteria post-transcriptional regulation is mediated by relatively few identified RNA-binding protein factors including CsrA, Hfq and SmpB. A mutation in any one of these three genes, csrA, hfq, and smpB, in Salmonella is attenuated for mouse virulence and unable to survive in macrophages. CsrA has a clearly defined specificity based on binding to a specific mRNA sequence to inhibit translation. However, the proteins regulated by Hfq and SmpB are not as clearly defined. Previous work identified proteins regulated by hfq using purification of the RNA-protein complex with direct sequencing of the bound RNAs and found binding to a surprisingly large number of transcripts. In this report we have used global proteomics to directly identify proteins regulated by Hfq or SmpB by comparing protein abundance in the parent and isogenic hfq or smpB mutant. From these same samples we also prepared RNA for microarray analysis to determine if alteration of protein expression was mediated post-transcriptionally. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all possible Salmonella proteins, respectively, with limited correlation between transcription and protein expression. These proteins represent a broad spectrum of Salmonella proteins required for many biological processes including host cell invasion, motility, central metabolism, LPS biosynthesis, two-component regulatory systems, and fatty acid metabolism. Our results represent one of the first global analyses of post-transcriptional regulons in any organism and suggest that regulation at the translational level is widespread and plays an important role in virulence regulation and environmental adaptation for Salmonella.

Research has been conducted on the Salmonella enterica genome. The authors hypothesized that SdiA was used to detect the signals of the other bacterial species and the results of the test of this hypothesis are reported.

Research was conducted to examine the hypothesis that the virulence plasmid of Salmonella typhimurium strains, LT2, 14028 and SR-11, is self-transmissible while the plasmid of strain SL1344 is not. Filter matings on M9 minimal glucose plates with a recipient strain lacking the virulence plasmid is required in optimal conjugation frequency. Results indicate that matings reduce efficiency by up to 400-fold.

Escherichia and Salmonella diverged in their evolution from a common ancestor some 100 to 160 mil years ago but the relative order of the genes in their chromosomes has been conserved. The intergenic region between smpB and nrdE of the Salmonella enterica chromosome is analyzed using restriction fragment length polymorphism studies and Southern blotting techniques to determine the genetic architecture of this segment. Results indicate that the mosaic structure of this region was brought about by multiple insertions and deletions during the evolutionary development of the organism.

Research was conducted to describe IroN, a novel outer membrane siderophore receptor characteristic of Salmonella enterica. The gene is located downstream of the IroBC operon in the iroA locus of Salmonella enterica serotype Typhi. Mutational analysis of the gene in S. enterica and expression in E. coli identified a 78-kDa outer membrane protein as the iroN gene product. Results indicate that the iroA locus functions in iron acquisition in S. enterica.

The Salmonella typhimurium SdiA homolog was cloned and a systematic screen was implemented on the sdiA-controlled genes. Findings from the analysis of the sequencing of a 4.4-kb fragment, which encodes the S. typhimurium sdiA gene, revealed that the fragment encodes the 5' end UvrC, all of SdiA and SirA and the 3' end of YecC. The S. typhimurium sdiA gene was also found to weakly support the E. coli sdiA gene for suppression of filamentation and for activation of ftsQAZ at promoter 2 due to an ftsZ(Ts) allele.

The distribution of fimbrial operons among two Salmonella serotypes with distant phylogenetic relationships was analyzed to determine the role of horizontal gene transfer and deletion in the development of the fimbrial operons. Genetic analysis of Salmonella enterica serotypes indicated the limited phylogenetic expression of the sef and lbf operons which were characterized as recent acquisitions by the two distantly related serotypes. Furthermore, the acquisition of specific fimbrial operons served as one of the mechanisms that enabled Salmonella serotypes to expand their host range.

Transposon mutagenesis generated transcriptional fusions were isolated and Fur titration assays were performed on a plasmid gene bank to identify Fur regulon or iron-regulated genes in Salmonella typhimurium. The methods were both successful with the first finding seven Fur-regulated insertions and the second eight Fur-regulated promoters. The assay method was easier and allowed for immediate sequence analysis while mutagenesis allowed for the analysis of more loci. Eleven genes with known homologs were identified while three Fur regulon indicated areas that were not known to be Fur regulated.

A new fimbrial operon encoding a fimbrial operon and containing five genes named lpfABCDE, has been identified at 78 min on the Salmonella typhimurium chromosome by hybridization. The lpf operon is related to the fim operon encoding 1 fimbriae of Salmonella typhimurium in terms of conservation of the amino acid sequence and gene order. The lpf gene cluster does not contain any gene encoding regulator proteins.

A 63-kb nucleotide fragment of the Salmonella typhimurium ethanolamine utilization operon codes for six complete open reading frames eutJ, eutH, eutG, cchB, cchA and eutE. eutI codes for an enzyme that resembles phosphate acetyltransferase, while EutE and EutG proteins resemble the AdhE sequence of Escherichia coli alcohol dehydrogenase. Mutants resulting from eutE or eutG mutations are incapable of utilizing ethanolamine as a carbon source and exhibit reduced utilization of the same as a nitrogen source. CchA and CchB proteins contribute to the generation of intracellular microcompartments for ethanolamine metabolism.

Two-dimensional proton nuclear magnetic resonance spectroscopy was used to study the labile protons of two 32-base-pair, four-arm models of immobile Holliday junctions. Complete resonance assignments were obtained by slow exchange between imino and cytosineamino protons. All four arms of both Holliday junctions possessed base-pairing up to and including the junction point. Moreover, arm stacking geometries were opposite for the two junctions studied.

The phoP gene of Salmonella typhimurium is involved in the transcriptional regulation of genes involved in virulence and survival. By screening a library of Escherichia coli clones, the phoP gene of E. coli was isolated and charactrized. The E. coli phoP gene was mapped to 25 minutes in the E. coli chromosome adjacent to the purB locus. Sequence data showed that the E. col PhoP is 98% similar to the Salmonella PhoP. Studies on phoP mutants showed that the gene is involved in response to stress situations such as nutrient deprivation and exposure to toxins.

Abstract Background Complete and accurate genome annotation is crucial for comprehensive and systematic studies of biological systems. However, determining protein-coding genes for most new genomes is almost completely performed by inference using computational predictions with significant documented error rates (> 15%). Furthermore, gene prediction programs provide no information on biologically important post-translational processing events critical for protein function. Results We experimentally annotated the bacterial pathogen Salmonella Typhimurium 14028, using "shotgun" proteomics to accurately uncover the translational landscape and post-translational features. The data provide protein-level experimental validation for approximately half of the predicted protein-coding genes in Salmonella and suggest revisions to several genes that appear to have incorrectly assigned translational start sites, including a potential novel alternate start codon. Additionally, we uncovered 12 non-annotated genes missed by gene prediction programs, as well as evidence suggesting a role for one of these novel ORFs in Salmonella pathogenesis. We also characterized post-translational features in the Salmonella genome, including chemical modifications and proteolytic cleavages. We find that bacteria have a much larger and more complex repertoire of chemical modifications than previously thought including several novel modifications. Our in vivo proteolysis data identified more than 130 signal peptide and N-terminal methionine cleavage events critical for protein function. Conclusion This work highlights several ways in which application of proteomics data can improve the quality of genome annotations to facilitate novel biological insights and provides a comprehensive proteome map of Salmonella as a resource for systems analysis.