Repression of phase-variable cup gene expression by H-NS-like proteins in Pseudomonas aeruginosa.

Division of Infectious Diseases, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 09/2005; 102(31):11082-7. DOI: 10.1073/pnas.0502663102
Source: PubMed

ABSTRACT The cupA gene cluster of Pseudomonas aeruginosa encodes components of a putative fimbrial structure that enable this opportunistic human pathogen to form biofilms on abiotic surfaces. In P. aeruginosa, cupA gene expression is repressed by MvaT, a putative transcription regulator thought to belong to the H-NS family of nucleoid-associated proteins that typically function by repressing transcription. Here, we present evidence that MvaT controls phase-variable (ON/OFF) expression of the cupA fimbrial gene cluster. Using a directed proteomic approach, we show that MvaT associates with a related protein in P. aeruginosa called MvaU. Analysis with a bacterial two-hybrid system designed to facilitate the study of protein dimerization indicates that MvaT and MvaU can form both heteromeric and homomeric complexes, and that formation of these complexes is mediated through the N-terminal regions of MvaT and MvaU, both of which are predicted to adopt a coiled-coil conformation. We show further that, like MvaT, MvaU can repress phase-variable expression of the cupA gene cluster. Our findings suggest that fimbrial genes important for biofilm formation can be expressed in a phase-variable manner in P. aeruginosa, provide insight into the molecular mechanism of MvaT-dependent gene control, and lend further weight to the postulate that MvaT proteins are H-NS-like in nature.

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    ABSTRACT: The bacterial H-NS protein silences expression from sequences with higher AT-content than the host genome and is believed to buffer the fitness consequences associated with foreign gene acquisition. Loss of H-NS results in severe growth defects in Salmonella, but the underlying reasons were unclear. An experimental evolution approach was employed to determine which secondary mutations could compensate for the loss of H-NS in Salmonella. Six independently derived S. Typhimurium hns mutant strains were serially passaged for 300 generations prior to whole genome sequencing. Growth rates of all lineages dramatically improved during the course of the experiment. Each of the hns mutant lineages acquired missense mutations in the gene encoding the H-NS paralog StpA encoding a poorly understood H-NS paralog, while 5 of the mutant lineages acquired deletions in the genes encoding the Salmonella Pathogenicity Island-1 (SPI-1) Type 3 secretion system critical to invoke inflammation. We further demonstrate that SPI-1 misregulation is a primary contributor to the decreased fitness in Salmonella hns mutants. Three of the lineages acquired additional loss of function mutations in the PhoPQ virulence regulatory system. Similarly passaged wild type Salmonella lineages did not acquire these mutations. The stpA missense mutations arose in the oligomerization domain and generated proteins that could compensate for the loss of H-NS to varying degrees. StpA variants most able to functionally substitute for H-NS displayed altered DNA binding and oligomerization properties that resembled those of H-NS. These findings indicate that H-NS was central to the evolution of the Salmonellae by buffering the negative fitness consequences caused by the secretion system that is the defining characteristic of the species.
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