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.