The Integration Host Factor (IHF) is a heterodimeric nucleoid-associated protein that plays roles in bacterial nucleoid architecture and genome-wide gene regulation. The and genes encode the subunits and are located 350 kbp apart, in the Right replichore of the 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 to exchange reciprocally the protein-coding regions of and 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 mRNA that was expressed from the 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 genes and the rewiring of their regulatory circuitry are well tolerated in , genes involved in the production of type 3 secretion systems exhibit dysregulation accompanied by altered phenotypes.