Unknown,Transcriptomics,Genomics,Proteomics

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The nucleoid-associated protein HU controls three regulons that coordinate virulence and general physiology in Salmonella enterica serovar Typhimurium

ABSTRACT: The regulon of genes controlled by the HU nucleoid-associated proteins was examined in Salmonella enterica serovar Typhimurium (S. Typhimurium). DNA microarrays were used to detect the effect on the bacterial transcriptome of knockout mutations in the hupA, the hupB and in both the hupA and hupB genes. Distinct but overlapping patterns of gene expression were detected in the transcriptome at each time point for each of the three mutants, pointing to the existence of not one but three regulons of genes controlled by the HU proteins. Mutations in the hup genes altered the expression of regulatory and structural genes in both the SPI1 and the SPI2 pathogenicity islands. The hupA hupB double mutant was found to be defective in invasion of CACO2 and CHO epithelial cell lines and in its ability to survive in J774A.1 macrophage-like cells. The double mutant also had defective swarming activity and had a competitive fitness disadvantage compared to the wild type. In contrast, inactivation of just the hupB gene resulted in increased fitness and correlated with the up-regulation of members of the RpoS regulon in exponential phase cultures. Elimination of both the hupA and hupB genes resulted in strong down-regulation of the genes coding for the integration host factor, IHF, at all three time points. The effect of hupA and hupB single-deletions and hupA and hupB double-deletion on S. Typhimurium gene expression during growth in LB was analysed at 3 different time points (1, 4 and 6 hours post-inoculation). Between two and three biological replicates, as well as technical replicates, were performed for each mutant and time point. For this study, we used Salmonella genomic DNA as the comparator which also acted as the control for spot quality.

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium

SUBMITTER: Sacha Lucchini

PROVIDER: E-GEOD-22860 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Nucleoid-associated protein HU controls three regulons that coordinate virulence, response to stress and general physiology in Salmonella enterica serovar Typhimurium.

Mangan Michael W MW Lucchini Sacha S Ó Cróinín Tadhg T Fitzgerald Stephen S Hinton Jay C D JCD Dorman Charles J CJ

Microbiology (Reading, England) 20110106 Pt 4

The role of the HU nucleoid-associated proteins in gene regulation was examined in Salmonella enterica serovar Typhimurium. The dimeric HU protein consists of different combinations of its α and β subunits. Transcriptomic analysis was performed with cultures growing at 37 °C at 1, 4 and 6 h after inoculation with mutants that lack combinations of HU α and HU β. Distinct but overlapping patterns of gene expression were detected at each time point for each of the three mutants, revealing not one b ...[more]

PMID: 21212121

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Project description:Background: Biofilm formation by Salmonella species enhances the capacity of these pathogenic bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir to contaminate food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most transcriptomic studies of Salmonella have focused on the effect of infection-relevant stressors on virulence and on comparing mutant and wild-type (WT). However little is known about the physiological responses taking place inside a Salmonella biofilm. Results: We have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 of detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% (433 genes) of S. Typhimurium genes showed a biofilm-specific pattern of transcription (i.e. a 2-fold or more change in the biofilm compared with planktonic cells). The genes that were significantly up-regulated implicated specific cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2), but that a functional SPI2 secretion system regulator (ssrA) was required for WT biofilm formation. We identified STM0341 as a gene of unknown function that was needed for WT biofilm growth. Genes involved in tryptophan (trp) biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to a decrease in bacterial attachment and we show that this biofilm defect is restored by exogenous tryptophan or indole. Conclusion: Biofilm growth of S. Typhimurium causes distinct changes in gene and protein expression. Our results show that aromatic amino acids make an important contribution to biofilm formation, and reveal that SPI2 genes, required for virulence in host cells, show opposing patterns of expression to biofilm-specific genes in S. Typhimurium. Overnight cultures of SL1344 grown in CFA broth were standardized to achieve an initial concentration of 10^6 CFU ml-1 and injected into a stirring influent flask containing 5 L of pre-warmed (25C) sterile CFA medium. The inoculated influent was then pumped through silicon tubing (5 mm internal diameter, Samco Silicon Products Ltd.) at 60 ml h-1 using a peristaltic pump (Minipulse 3, Gilson). The bacteria were allowed to flow through the closed system and either attach to a vertical piece of silicon tubing (1 meter length, 16 mm internal diameter, Samco Silicon Products Ltd.) or flow out into a waste reservoir. The tubing was positioned vertically to collect biofilm cells adherent to the tubing and minimise collecting bacteria that had simply sedimented. Possible backflow of media or bacteria into the influent from the silicon tubing was eliminated using media breaks. Samples taken to determine the pH of the medium or bacterial cell counts were removed via a media port located near the effluent and influent vessel, respectively. Planktonic cells were removed after 72 h of growth from the influent vessel to avoid any contamination with biofilm cells. The entire system was kept at a constant temperature of 25C and biofilm cells were isolated after 72 h of growth. All planktonic and biofilm samples were isolated from the same experimental system and used for both RNA and protein isolation. For this study, four biological replicates were performed at four different dates (Jun25, Jul5, Jul16, Oct11; as indicated in the sample title). For the dual-colour microarray hybridisations, we used Salmonella genomic DNA as the comparator which also acted as the control for spot quality. Individual labelled cDNA samples (RNA) were hybridised up to 4 times (technical replicates).

Dataset Information

The nucleoid-associated protein HU controls three regulons that coordinate virulence and general physiology in Salmonella enterica serovar Typhimurium

Publications

Nucleoid-associated protein HU controls three regulons that coordinate virulence, response to stress and general physiology in Salmonella enterica serovar Typhimurium.

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