High-order oligomerization is required for the function of the H-NS family member MvaT in Pseudomonas aeruginosa

Division of Infectious Diseases, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Molecular Microbiology (Impact Factor: 4.42). 11/2010; 78(4):916-31. DOI: 10.1111/j.1365-2958.2010.07378.x
Source: PubMed


H-NS is an abundant DNA-binding protein that has been implicated in the silencing of foreign DNA in several different bacteria. The ability of H-NS dimers to form higher-order oligomers is thought to aid the polymerization of the protein across AT-rich stretches of DNA and facilitate gene silencing. Although the oligomerization of H-NS from enteric bacteria has been the subject of intense investigation, little is known regarding the oligomerization of H-NS family members from bacteria outside of the enterobacteriaceae, many of which share little sequence similarity with their enteric counterparts. Here we show that MvaT, a member of the H-NS family of proteins from Pseudomonas aeruginosa, can form both dimers and higher-order oligomers, and we identify a region within MvaT that mediates higher-order oligomer formation. Using genetic assays we identify mutants of MvaT that are defective for higher-order oligomer formation. We present evidence that these mutants are functionally impaired and exhibit DNA-binding defects because of their inability to form higher-order oligomers. Our findings support a model in which the ability of MvaT to bind efficiently to the DNA depends upon protein-protein interactions between MvaT dimers and suggest that the ability to form higher-order oligomers is a conserved and essential feature of H-NS family members.

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    • "In fact, H-NS or MvaT variants, which lack the ability to form higher-order oligomers, were defective for gene silencing [30], [34]. Both domains, which are responsible for higher-order oligomer formation and dimer formation, have been shown to exist in the N-terminal region of H-NS and MvaT [30], [32], [34]–[36]. In the present study, we found that Pmr also has a dimerization/oligomerization domain in its N-terminal region, similar to H-NS and MvaT (Figure 2). "
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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.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "Moreover a paralog of the P. aeruginosa gene mvaT was identified. MvaT belongs to the H-NS family of small DNA-binding proteins that are global regulators of gene expression [44]. Five homologues have been identified in P. putida and two homologues mvaT and mvaU have been identified in the P. aeruginosa core genome [45]. "
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    ABSTRACT: Adaptation of Pseudomonas aeruginosa to different living conditions is accompanied by microevolution resulting in genomic diversity between strains of the same clonal lineage. In order to detect the impact of colonized habitats on P. aeruginosa microevolution we determined the genomic diversity between the highly virulent cystic fibrosis (CF) isolate CHA and two temporally and geographically unrelated clonal variants. The outcome was compared with the intraclonal genome diversity between three more closely related isolates of another clonal complex. The three clone CHA isolates differed in their core genome in several dozen strain specific nucleotide exchanges and small deletions from each other. Loss of function mutations and non-conservative amino acid replacements affected several habitat- and lifestyle-associated traits, for example, the key regulator GacS of the switch between acute and chronic disease phenotypes was disrupted in strain CHA. Intraclonal genome diversity manifested in an individual composition of the respective accessory genome whereby the highest number of accessory DNA elements was observed for isolate PT22 from a polluted aquatic habitat. Little intraclonal diversity was observed between three spatiotemporally related outbreak isolates of clone TB. Although phenotypically different, only a few individual SNPs and deletions were detected in the clone TB isolates. Their accessory genome mainly differed in prophage-like DNA elements taken up by one of the strains. The higher geographical and temporal distance of the clone CHA isolates was associated with an increased intraclonal genome diversity compared to the more closely related clone TB isolates derived from a common source demonstrating the impact of habitat adaptation on the microevolution of P. aeruginosa. However, even short-term habitat differentiation can cause major phenotypic diversification driven by single genomic variation events and uptake of phage DNA.
    Full-text · Article · Jun 2013 · BMC Genomics
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    • "Others have shown that the binding of H-NS upstream of a regulatory region interferes with either promoter clearance or progression of RNAP,46 and H-NS has been shown to constrain local DNA topology to regulate the promoter activity of supercoiling-sensitive genes.47–49 Mutations impairing the ability of H-NS and its homologue of Pseudomonas aeruginosa termed MvaT to form higher-order oligomers have been shown to reduce their abilities to repress transcription of the E. coli proU and P. aeruginosa cupA fimbria genes.18,50,51 The formation of a DNA–protein filament through cooperative polymerization of MvaT or H-NS along DNA and of protein bridges to constrain DNA loops through the interaction of the DNA–protein filaments were suggested to be important for transcriptional repression.7,52 "
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    ABSTRACT: The Hha and YdgT proteins are suggested to modulate the expression of horizontally acquired genes by interacting with H-NS and StpA, which play central roles in the transcriptional silencing of such genes. However, it is also possible that Hha/YdgT repress gene expression independently of H-NS/StpA, as we have not fully understood the molecular mechanism through which Hha/YdgT modulate H-NS/StpA activity. To gain further insight into the basic functions of Hha/YdgT, we analysed the impact of hha/ydgT double inactivation on the transcriptome profile of Escherichia coli K-12, and compared the effects with that of hns/stpA double inactivation. In addition, we examined the effects of hha/ydgT inactivation on the chromosomal binding of H-NS, and conversely the effects of hns/stpA inactivation on the chromosomal binding of Hha. Our results demonstrated that the chromosomal binding of Hha requires H-NS/StpA, and is necessary for the repression of a subset of genes in the H-NS/StpA regulon. Furthermore, the distribution of H-NS binding around Hha/YdgT-dependent and -independent genes suggests that Hha/YdgT proteins modulate formation of the H-NS/StpA-DNA complex.
    Full-text · Article · Mar 2013 · DNA Research
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