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Random minitransposon mutagenesis was used to identify genes involved in the survival ofBordetella bronchiseptica within eukaryotic cells. One of the mutants which exhibited a reduced ability to survive intracellularly harbored a minitransposon insertion in a locus (ris ) which displays a high degree of homology to two-component regulatory systems. This system exhibited less than 25% amino acid sequence homology to the only other two-component regulatory system described inBordetella spp., thebvg locus. ArisA′-′lacZ translational fusion was constructed and integrated into the chromosome ofB. bronchiseptica . Determination of β-galactosidase activity under different environmental conditions suggested thatris is regulated independently ofbvg and is optimally expressed at 37°C, in the absence of Mg2+ , and when bacteria are in the intracellular niche. This novel regulatory locus, present in allBordetella spp., is required for the expression of acid phosphatase byB. bronchiseptica . Although catalase and superoxide dismutase production were unaffected, theris mutant was more sensitive to oxidative stress than the wild-type strain. Complementation ofbvg -positive andbvg -negativeris mutants with the intactris operon incorporated as a single copy into the chromosome resulted in the reestablishment of the ability of the bacterium to produce acid phosphatase and to resist oxidative stress. Mouse colonization studies demonstrated that theris mutant is cleared by the host much earlier than the wild-type strain, suggesting thatris -regulated products play a significant role in natural infections. The identification of a second two-component system inB. bronchiseptica highlights the complexity of the regulatory network needed for organisms with a life cycle requiring adaptation to both the external environment and a mammalian host.

infection and immunity

A Second Two-Component Regulatory System of <i>Bordetella bronchiseptica</i> Required for Bacterial Resistance to Oxidative Stress, Production of Acid Phosphatase, and In Vivo Persistence

A Second Two-Component Regulatory System of Bordetella bronchiseptica Required for Bacterial Resistance to Oxidative Stress, Production of Acid Phosphatase, and In Vivo Persistence