Reciprocally rewiring and repositioning the Integration Host Factor (IHF) subunit genes in Salmonella enterica serovar Typhimurium: impacts on physiology and virulence

Abstract

The 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 encode the subunits and are located 350 kbp apart, in the Right replichore of the Salmonella chromosome. IHF is composed of one IhfA and one IhfB subunit. Despite this 1 : 1 stoichiometry, MS revealed that IhfB is produced in 2-fold excess over IhfA. 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. MS 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 and was similar in competitive fitness. However, compared to the wild-type, it was less motile, 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 accompanied by altered phenotypes.

Regulation of flagellar motility and biosynthesis in enterohemorrhagic Escherichia coli O157:H7

“…IHF is a DNA-binding protein that binds to specific consensus sites and bends the DNA to form nucleoprotein complexes. 81 IHF is a heterodimer composed of IHF-α and IHF-β subunits that are encoded by the ihfA and ihfB genes, respectively. 81 IHF is involved in various cellular processes, including site-specific recombination, DNA replication, transposition, packaging, and gene regulation.…”

“… 81 IHF is a heterodimer composed of IHF-α and IHF-β subunits that are encoded by the ihfA and ihfB genes, respectively. 81 IHF is involved in various cellular processes, including site-specific recombination, DNA replication, transposition, packaging, and gene regulation. 81 In EHEC O157:H7 and EPEC O127:H6, the ihfA::kan mutant was highly motile, and the motility of the strains expressing IHF, either genomic or in trans from a plasmid, was attenuated.…”

“… 81 IHF is involved in various cellular processes, including site-specific recombination, DNA replication, transposition, packaging, and gene regulation. 81 In EHEC O157:H7 and EPEC O127:H6, the ihfA::kan mutant was highly motile, and the motility of the strains expressing IHF, either genomic or in trans from a plasmid, was attenuated. 36 Electron micrographs of the wild-type cells and the ihfA::kan mutant grown in Dulbecco’s modified Eagle medium (DMEM) showed that the wild-type cells either completely lack flagella or rarely possess (approximately 5% of bacteria) a single flagellum.…”

Physiological Robustness of Model Gram-Negative Bacteria in Response to Genome Rewiring

“…Sequence-specific DNA binding and bending protein [Mangan et al, 2006;Kasho et al, 2021;Pozdeev et al, 2022] StpA stpA Homodimer; can form heterodimers with H-NS and other H-NS-like proteins Similar properties to H-NS, but with a distinct production pattern [Leonard et al, 2009;Fitzgerald et al, 2015Fitzgerald et al, , 2020 Genome Rewiring and Bacterial Physiology…”

“…Reciprocal exchange of the protein-coding regions of the ihfA and ihfB genes in Salmonella IhfA is produced in excess over IhfB (reversing the wild-type production pattern); altered production of the type 3 secretion systems involved in Salmonella virulence (SPI1 and SPI2) and motility; reduced SPI2-dependent survival rates in cultured macrophage [Pozdeev et al, 2022] rrn Ribosome components Moved from an oriC-proximal position to a ter-proximal location on the large chromosome of Vibrio cholerae Relocation imposed a fitness penalty, even during slow growth, consistent with the natural position of the gene being of authentic physiological significance [Soler-Bistué et al, 2017] Microb Physiol 2022;32:158-176 DOI: 10.1159/000526651 duced in late stationary phase [Nair and Finkel, 2004;Karas et al, 2015;Janissen et al, 2018]. The genes that encode these proteins are also found in opposite replichores, at diametrically opposite locations on the chromosome, with fis being in the NS-Left region and dps in the Right macrodomain (shown in Fig.…”