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The consequences of reciprocally exchanging the genomic sites of Integration Host Factor (IHF) subunit production for subunit stoichiometry and bacterial physiology in Salmonella enterica serovar Typhimurium

ABSTRACT: Integration host factor (IHF) is a heterodimeric nucleoid-associated protein that plays roles in bacterial nucleoid architecture and genome-wide gene regulation. The ihfA and ihfB genes that encode the subunits are located 350 kilobase pairs apart, in the Right replichore of the Salmonella chromosome. IHF is composed of one IhfA and one IhfB subunit. Despite this 1:1 stoichiometry, mass spectrometry revealed that IhfB is produced in 2-fold excess over IhfA. Results from several studies suggest that a gene's location on the bacterial chromosome is an important factor in establishing the appropriate spatiotemporal expression pattern of that gene and its influence on physiology. We re-engineered Salmonella to exchange reciprocally the protein-coding regions of ihfA and ihfB, such that each relocated protein-encoding region was driven by the expression signals of the other's gene. Mass spectrometry showed that in this 'rewired' strain, IhfA is produced in excess over IhfB, correlating with enhanced stability of the hybrid ihfB-ihfA mRNA that was expressed from the ihfB promoter. Nevertheless, the rewired strain grew at a similar rate to the wild type, had identical cell morphology, and was similar in competitive fitness. However, it was less motile than the wild type, had growth-phase-specific reductions in SPI-1 and SPI-2 gene expression and was engulfed at a higher rate by RAW macrophage. Our data show that while exchanging the physical locations of its ihf genes and the rewiring of their regulatory circuitry are well tolerated in Salmonella, genes involved in the production of type 3 secretion systems exhibit dysregulation that accompanies altered phenotypes.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Salmonella Enterica Subsp. Enterica Serovar Typhimurium Str. Sl1344

SUBMITTER: German Pozdeev

LAB HEAD: Charles James Dorman

PROVIDER: PXD027465 | Pride | 2021-11-02

REPOSITORIES: pride

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			Action	DRS
	2020_07_01_German_SWAP01.raw	Raw
	2020_07_01_German_SWAP02.raw	Raw
	2020_07_01_German_SWAP03.raw	Raw
	2020_07_23_German_WT01.raw	Raw
	2020_07_23_German_WT02.raw	Raw

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Project description:Salmonella Typhimurium is an enteric food-borne pathogen responsible for causing salmonellosis associated with acute diarrhea. The bacterial invasion initially requires flagellar motility to reach the intestinal lumen and to cross the mucus layer of the intestinal epithelia and subsequent adhesion to the host cell, mainly mediated by fimbriae .The key genes involved in the pathogenic process are encoded within highly conserved regions of the bacterial genome called Salmonella Pathogenicity Islands (SPIs). The key regulator HilA, which is positively regulated by HilC and HilD, is located in SPI-1;however, other virulence-related regulators, such as RtsA, are encoded outside this island . During the invasion, proteins encoded by the prgHIJK, spaMNOPQRS, and invABCEFGH SPI-1 operons constitute a Type-3 Secretion System. Effector proteins,such as SipA and SipC (encoded in SPI-1) and SigD/SopB (encoded in SPI-5), translocate to the host cytosol through this system causing intracellular changes and inducing the immune response . The survival and replication of intracellular Salmonella inside Salmonella-containing vacuoles (SCVs) is mainly mediated by the genes located in SPI-2. The immune response is activated through the recognition of pathogen-associated molecular patterns (PAMPs) by specific receptors expressed on the surface and/or inside different cell types. The most important PAMPs are flagellin (monomers comprising the flagella filaments), particularly the FliC subunit, which is highly expressed on the bacterial surface, and lipopolysaccharide (LPS).Oxygen availability is limited in the inflamed gut whereas other compounds, such as hydrogen sulfide (H2S) and H2 are abundantly produced by the colonic bacteria. In addition, nutrients are also limited inside the SCV, while reactive oxygen as wellas nitrogen species may be found . In anaerobic conditions, Salmonella is able to survive by using alternative energy sources, such as nitrate or fumarate, through the use of specific enzymes: nitrate and nitrite reductases, fumarate reductase , DMSO reductase and respiratory hydrogenases.In a previous study, we compared transcriptomes of a clinical isolate of S. Typhimurium (strain 50-wt) and its derivative-multidrug-resistant mutant (strain 50-64) using microarrays . In addition to showing antimicrobial resistance, strain was also less invasive. In the present study, we focused on the genes of unknown function that showed impaired expression. We further selected those genes with a putative role in the acquired resistance phenotype or in the observed repressed virulence observed. Here, 4 putative membrane proteins and the yedF of unknown function were investigated to evaluate their involvement inthese two phenotypes.

Dataset Information

The consequences of reciprocally exchanging the genomic sites of Integration Host Factor (IHF) subunit production for subunit stoichiometry and bacterial physiology in Salmonella enterica serovar Typhimurium

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