Article

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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    • "This explains why these proteins are members of “H-NS family proteins”, which include both H-NS-like proteins and its functional homologs [3], [19], [20], although it remains unknown whether the mechanisms for their function are the same among H-NS family proteins. In Pseudomonas aeruginosa PAO1, MvaT and its homologous protein MvaU were shown to interact with each other, bind to the same chromosomal regions, and function coordinately [21], [22]. This strain cannot tolerate the loss of both MvaT and MvaU because it results in the induction of Pf4 phage, which superinfects and kills cells or inhibits their growth [23]. "
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    ABSTRACT: Enterobacterial H-NS-like proteins and Pseudomonas MvaT-like proteins share low homology at the amino acid sequence level, but both can function as xenogeneic silencers and are included in the H-NS family of proteins. H-NS family members have dimerization/oligomerization and DNA-binding domains connected by a flexible linker and form large nucleoprotein complexes using both domains. Pmr, an MvaT-like protein encoded on the IncP-7 carbazole-degradative plasmid pCAR1, is a key regulator of an interaction between pCAR1 and its host Pseudomonas putida KT2440. KT2440 has two transcribed genes that encode the MvaT-like proteins TurA and TurB. Our previous transcriptome analyses suggested that the functions of Pmr, TurA and TurB are non-equivalent, although the detailed underlying mechanisms remain unclear. In this study, we focused on the protein-protein interactions of Pmr, and assessed the homo-oligomerization capacity of various substituted and truncated Pmr derivatives by protein-protein cross-linking analysis. Six of the seven residues identified as important for homo-oligomerization in Pmr were located near the N-terminus, and the putative flexible linker or the region near that was not involved in homo-oligomerization, suggesting that Pmr homo-oligomerization is different from that of enterobacterial H-NS and that the functional mechanism differs between H-NS-like and MvaT-like proteins. In addition, we assessed homo- and hetero-oligomerization of Pmr by surface plasmon resonance analysis and found that the coupling ratio of TurB-Pmr oligomers is smaller than that of Pmr-Pmr or TurA-Pmr oligomers. These results raised the possibility that composition of the hetero-oligomers of Pmr, TurA, and TurB could explain why the different gene sets were affected by either pmr, turA, or turB disruption in our previous studies.
    PLoS ONE 08/2014; 9(8):e105656. DOI:10.1371/journal.pone.0105656 · 3.23 Impact Factor
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    • "To investigate protein-protein interactions in E. coli, PCR amplified fragments encoding VipA or mutants thereof, VipB, full-length or truncated ClpV (first 178 residues), were ligated into plasmids pBRGPω (directs the synthesis of a Gal11P-ω fusion protein and can be used to create fusions to the N-terminus of the ω subunit of E. coli RNAP) and pACTR-AP-Zif (directs the synthesis of the zinc finger DNA-binding domain of the murine Zif268 protein and can be used to create fusions to the N-terminus of Zif268) [32]. Plasmids were introduced into the reporter strain KDZif1ΔZ by electroporation. "
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    ABSTRACT: Background Many Gram-negative bacteria rely on a type VI secretion system (T6SS) to infect eukaryotic cells or to compete against other microbes. Common to these systems is the presence of two conserved proteins, in Vibrio cholerae denoted VipA and VipB, which have been shown to interact in many clinically relevant pathogens. In this study, mutagenesis of a defined region within the VipA protein was used to identify residues important for VipB binding in V. cholerae O1 strain A1552. Results A dramatically diminished interaction was shown to correlate with a decrease in VipB stability and a loss of hemolysin co-regulated protein (Hcp) secretion and rendered the bacterium unable to compete with Escherichia coli in a competition assay. Conclusions This confirms the biological relevance of the VipA-VipB interaction, which is essential for the T6SS activity of many important human pathogens.
    BMC Microbiology 05/2013; 13(1):96. DOI:10.1186/1471-2180-13-96 · 2.98 Impact Factor
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    • "As the reporter strain for the bacterial two-hybrid experiments, the E. coli strain KDZif1ΔZ was used. It harbors an F9 episome containing the lac promoter-derivative placZif1-61 driving expression of a linked lacZ reporter gene [51]. Cells were grown with aeration at 37°C in LB supplemented with 0.4 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside), permeabilized with SDS-CHCl3 and assayed for β-galactosidase (β-gal) activity as described previously [52]. "
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    ABSTRACT: Background A prerequisite for the virulence of the facultative intracellular bacterium Francisella tularensis is effective intramacrophage proliferation, which is preceded by phagosomal escape into the cytosol, and ultimately leads to host cell death. Many components essential for the intracellular life cycle are encoded by a gene cluster, the Francisella pathogenicity island (FPI), constituting a type VI secretion system. Results We characterized the FPI mutant ΔpdpC of the live vaccine strain (LVS) of F. tularensis and found that it exhibited lack of intracellular replication, incomplete phagosomal escape, and marked attenuation in the mouse model, however, unlike a phagosomally contained FPI mutant, it triggered secretion of IL-1β, albeit lower than LVS, and markedly induced LDH release. Conclusions The phenotype of the ΔpdpC mutant appears to be unique compared to previously described F. tularensis FPI mutants.
    BMC Microbiology 01/2013; 13(1):20. DOI:10.1186/1471-2180-13-20 · 2.98 Impact Factor
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