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Evidence for direct protein-protein interaction between members of the enterobacterial Hha/YmoA and H-NS families of proteins.

ABSTRACT: Escherichia coli nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from Yersinia enterocolitica. This interaction has been found to be involved in the regulation of the expression of the toxin alpha-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the interaction between the proteins. The precise molecular mass of the H-NS protein retained by Hha, obtained by mass spectrometry analysis, does not show any posttranslational modification other than removal of the N-terminal Met residue. We constructed an H-NS-His recombinant protein and found that, as expected, it interacts with Hha. We used a Ni(2+)-nitrilotriacetic acid agarose method for affinity chromatography copurification of proteins to identify the H-NS protein of Y. enterocolitica. We constructed a six-His-YmoA recombinant protein derived from YmoA, the homologue of Hha in Y. enterocolitica, and found that it interacts with Y. enterocolitica H-NS. We also cloned and sequenced the hns gene of this microorganism. In the course of these experiments we found that His-YmoA can also retain H-NS from E. coli. We also found that the hns gene of Y. enterocolitica can complement an hns mutation of E. coli. Finally, we describe for the first time systematic characterization of missense mutant alleles of hha and truncated Hha' proteins, and we report a striking and previously unnoticed similarity of the Hha family of proteins to the oligomerization domain of the H-NS proteins.

SUBMITTER: Nieto JM

PROVIDER: S-EPMC139531 | biostudies-literature | 2002 Feb

REPOSITORIES: biostudies-literature

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Evidence for direct protein-protein interaction between members of the enterobacterial Hha/YmoA and H-NS families of proteins.

Nieto J M JM Madrid C C Miquelay E E Parra J L JL Rodríguez S S Juárez A A

Journal of bacteriology 20020201 3

Escherichia coli nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from Yersinia enterocolitica. This interaction has been found to be involved in the regulation of the expression of the toxin alpha-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the in ...[more]

PMID: 11790731

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Project description:Nucleoid-associated proteins (NAPs) silence xenogenes by blocking RNA polymerase binding to promoters and hindering transcript elongation. In Escherichia coli, H-NS and its homolog SptA interact with YmoA proteins Hha and YdgT to assemble nucleoprotein filaments that facilitate transcription termination by Rho, which acts in synergy with NusG. Countersilencing during initiation is facilitated by proteins that exclude NAPs from promoter regions, but auxiliary factors that alleviate silencing during elongation are not known. A specialized NusG paralog, RfaH, activates lipopolysaccharide core biosynthesis operons, enabling survival in the presence of detergents and antibiotics. RfaH strongly inhibits Rho-dependent termination by reducing RNA polymerase pausing, promoting translation, and competing with NusG. We hypothesize that RfaH also acts as a countersilencer of NAP/YmoA filaments. We show that deletions of hns and hha+ydgT suppress the growth defects of ΔrfaH by alleviating Rho-mediated polarity within the waa operon. The absence of YmoA proteins exacerbates cellular defects caused by reduced Rho levels or Rho inhibition by bicyclomycin but has negligible effects at a strong model Rho-dependent terminator. Our findings that the distribution of Hha and RfaH homologs is strongly correlated supports a model in which they comprise a silencing/countersilencing pair that controls expression of chromosomal and plasmid-encoded xenogenes. IMPORTANCE Horizontally acquired DNA drives bacterial evolution, but its unregulated expression may harm the recipient. Xenogeneic silencers recognize foreign genes and inhibit their transcription. However, some xenogenes, such as those encoding lipo- and exopolysaccharides, confer resistance to antibiotics, bile salts, and detergents, necessitating the existence of countersilencing fitness mechanisms. Here, we present evidence that Escherichia coli antiterminator RfaH alleviates silencing of the chromosomal waa operon and propose that plasmid-encoded RfaH homologs promote dissemination of antibiotic resistance genes through conjugation.

Project description:BACKGROUND:Hha facilitates H-NS-mediated silencing of foreign genes in bacteria. RESULTS:Two Hha monomers bind opposing faces of the H-NS N-terminal dimerization domain. CONCLUSION:Hha binds the dimerization domain of H-NS and may contact DNA via positively charged surface residues. SIGNIFICANCE:The structure of Hha and H-NS in complex provides a mechanistic model of how Hha may affect gene regulation. The bacterial nucleoid-associated proteins Hha and H-NS jointly repress horizontally acquired genes in Salmonella, including essential virulence loci encoded within Salmonella pathogenicity islands. Hha is known to interact with the N-terminal dimerization domain of H-NS; however, the manner in which this interaction enhances transcriptional silencing is not understood. To further understand this process, we solved the x-ray crystal structure of Hha in complex with the N-terminal dimerization domain of H-NS (H-NS(1-46)) to 3.2 Å resolution. Two monomers of Hha bind to symmetrical sites on either side of the H-NS(1-46) dimer. Disruption of the Hha/H-NS interaction by the H-NS site-specific mutation I11A results in increased expression of the Hha/H-NS co-regulated gene hilA without affecting the expression levels of proV, a target gene repressed by H-NS in an Hha-independent fashion. Examination of the structure revealed a cluster of conserved basic amino acids that protrude from the surface of Hha on the opposite side of the Hha/H-NS(1-46) interface. Hha mutants with a diminished positively charged surface maintain the ability to interact with H-NS but can no longer regulate hilA. Increased expression of the hilA locus did not correspond to significant depletion of H-NS at the promoter region in chromatin immunoprecipitation assays. However, in vitro, we find Hha improves H-NS binding to target DNA fragments. Taken together, our results show for the first time how Hha and H-NS interact to direct transcriptional repression and reveal that a positively charged surface of Hha enhances the silencing activity of H-NS nucleoprotein filaments.

Dataset Information

Evidence for direct protein-protein interaction between members of the enterobacterial Hha/YmoA and H-NS families of proteins.

Publications

Evidence for direct protein-protein interaction between members of the enterobacterial Hha/YmoA and H-NS families of proteins.

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