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Journal of Bacteriology, January 2007, p. 265-268, Vol. 189, No. 1
0021-9193/07/$08.00+0     doi:10.1128/JB.01124-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Molecular Evolution of the H-NS Protein: Interaction with Hha-Like Proteins Is Restricted to Enterobacteriaceae

Cristina Madrid,¹ Jesús García,² Miquel Pons,² and Antonio Juárez^1*

Department of Microbiology, University of Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain,¹ Laboratory of Biomolecular NMR, Institut de Recerca Biomèdica, Parc Cientific de Barcelona, Josep Samitier 1-5, 08028 Barcelona, Spain²

Received 27 July 2006/ Accepted 25 September 2006

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We show here that chromosomal hha-like genes are restricted to the Enterobacteriaceae. The H-NS N-terminal domain of members of this family includes an unaltered seven-amino-acid sequence located between helixes 1 and 2, termed the Hha signature, that contains key residues for H-NS-Hha interaction.

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The nucleoid-associated protein H-NS is widespread in gram-negative bacteria (5, 24, 25). Best characterized in Escherichia coli and related genera, this protein plays a dual role, both as an architectural protein that contributes to the nucleoid structure and as a global modulator of gene expression (8, 20, 21). As a regulatory protein, H-NS has been shown to modulate gene expression in response to different environmental factors. H-NS consists of an N-terminal dimerization domain and a C-terminal DNA-binding domain that are separated by a linker domain. H-NS binds DNA in a non-sequence-specific manner but with a preference for intrinsically curved AT-rich regions. The H-NS protein is not only capable of interacting with DNA but also with itself and other proteins. The generation of homodimers, tetramers, and oligomers appears to be a key process in allowing H-NS to modulate gene expression (8, 9, 22, 23). H-NS is also capable of heteromeric interactions. E. coli and other members of the Enterobacteriaceae such as Salmonella enterica serovar Typhimurium or Shigella dysenteriae express a paralogous protein termed StpA. This latter protein shares 58% sequence identity to the H-NS protein and is able to form heteromers with H-NS (26). Interaction of H-NS with members of the Hha-YmoA family of proteins represents a well-characterized example of a different heteromeric interaction of H-NS. Hha and YmoA are low-molecular-mass proteins (8.6 and 8 kDa, respectively) that show 82% sequence identity. These proteins have been shown to participate in the modulation of the expression of virulence factors, such as the E. coli alpha-hemolysin or the Y. enterocolitica Yop proteins, invasin, and YadA adhesin (7, 10, 19). They have been considered as representatives of a new family of modulators of bacterial gene expression (3, 17). Members of this family are widespread among different genera of gram-negative bacteria and are also present in various large conjugative plasmids that belong to different incompatibility groups (12). Hha-H-NS complexes modulate the expression of, among other genes, the E. coli toxin alpha-hemolysin (18), and YmoA-H-NS complexes modulate the expression of the invasin in Y. enterocolitica (11, 15).

Previous studies based on biochemical and in silico analysis have shown that members of the H-NS family of proteins are widespread among -, ß-, and -proteobacteria (24). Considering the reported interaction between H-NS and Hha proteins, we decided to test whether the ubiquity of the hha-like gene is similar to that of hns-like genes. To do this, we searched for the presence of genes codifying H-NS and Hha-like proteins in a significant number of bacterial genomes (Table 1) . We used protein-protein BLAST (Blastp) and protein query versus translated database (tBlastn) (1). The sequences corresponding to the N-terminal domain of the different H-NS proteins were used to develop a phylogenetic tree, using the neighbor-joining method in the molecular evolutionary genetic analysis (MEGA 3.1) and marked in it the presence of hha-like genes (Fig. 1). Interestingly, genomes encoding for both hha and hns genes correspond exclusively to members of the family Enterobacteriaceae, including some of the endocellular obligate symbionts that belong to the family (Wigglesworthia glossinidia, Photorhabdus luminiscens, and Sodalis glossinidius). These endosymbionts have retained copies of both the hha and the hns genes. Different phylogenetic studies have related them to the Enterobacteriaceae (4, 6), and the presence of the hha gene further establishes a link with the family. Some of the endosymbiont genera that encode hns (Buchnera aphidicola strain APS and Blochmania pennsylvanicus strain BPEN) do not encode hha, whereas others do. The likely interpretation is that the adaptation as obligate endosymbionts to a far less variable environment rendered the H-NS-mediated modulatory functions not relevant for the cell physiology, and the drastic genome reduction that some of them experienced included the hha gene and, in some instances, the hns gene as well. In fact, from the two species of Blochmania whose genomes have been completely sequenced, one of them (B. pennsylvanicus strain BPEN) contains the hns gene and the other does not (B. floridanus). In a similar case, of the three genomes of strains of Buchnera aphidicola completely sequenced, only one (strain APS) contains an hns gene.

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TABLE 1. Bacterial species included in the study

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FIG. 1. Phylogenetic relationship of the amino acid sequences of the N-terminal end of H-NS protein from different bacterial species. The presence of either hns-like or hha-like genes is indicated.

The structural details of Hha-H-NS interaction are available. With respect to the Hha protein, amino acid residues interacting with H-NS are scattered along the whole molecule (14). In contrast, H-NS amino acid residues interacting with Hha are located mainly within helixes H1 and H2 of the H-NS N-terminal domain (13). We used the sequence corresponding to the N-terminal end of the H-NS protein to perform a sequence alignment, using CLUSTAL W (16). In spite of the variability, a seven-amino-acid sequence (LNNIRTL) located within helixes H1 and H2 is absolutely conserved among the H-NS proteins encoded by those microorganisms that encode a chromosomal hha-like gene (Fig. 2). That seven-amino-acid stretch is included within the H-NS domain that interacts with Hha and corresponds to the residues most affected after interaction with Hha (13). We propose for that sequence the term "Hha signature." Other aminoacidic residues located in the N-terminal domain of the H-NS proteins of the species that carries a copy of a hha-like gene are also conserved, but they are located outside the H1-H2 region (13). With the genomic data available, the intact Hha signature of the H-NS protein is exclusively found in chromosomally encoded H-NS proteins from members of the family Enterobacteriaceae. The H-NS protein from Proteus vulgaris contains the Hha signature. Therefore, it is conceivable that its complete genome sequence will reveal the presence of an hha-like gene. This hypothesis is supported by the fact that the P. mirabilis HI4320 genome sequencing project, carried out at the Wellcome Trust Sanger Institute, has revealed the presence of an hns gene encoding an H-NS protein with an intact Hha signature, as well as the presence of an hha gene (ftp://ftp.sanger.ac.uk/pub/pathogens/pm).

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FIG. 2. Sequence alignment of the N-terminal end of H-NS protein from bacterial species listed in Table 1. The seven amino acid residues corresponding to the Hha signature have a gray background. + or –, presence of absence of an Hha-like protein. *, Genomes not completely sequennced.

Formerly, H-NS proteins were thought to be restricted to the enterobacteria and related genera such as Haemophilus (2). Further studies identified H-NS-like proteins in other groups, and it is now well established that that H-NS-like proteins are present in -, ß-, and -proteobacteria. We show here that, in fact, the H-NS proteins from the Enterobacteriaceae exhibit at least one evolutionary trait that differentiates them from other H-NS proteins: they have evolved to be able to interact with Hha-like proteins. Whether all or only some of the amino acid residues of the Hha signature are essential for H-NS-Hha interaction deserves future investigation.

Phylogenetic studies based on genomic data have led to a much more complete understanding of the relationships between different bacterial groups. We show here that analysis of the molecular evolutionary characteristics of global modulators can help to establish regulatory links between different bacterial groups and can lead to a better understanding of their physiological properties.

 
This study was supported by grants from the Ministerio de Ciencia y Tecnología (BIO2004-02747 to A.J., BIO2004-05436 and GEN2003-20642-C09-04 to M.P., and Ramón y Cajal contract to J.G.) and from the Generalitat de Catalunya (2005SGR00635 to A.J.).

We thank Francisco J. Silva for critical reading of the manuscript.

 
* Corresponding author. Mailing address: Department of Microbiology, University of Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain. Phone: 34934034624. Fax: 34934034629. E-mail: ajuarez{at}ub.edu.

Published ahead of print on 13 October 2006.

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Journal of Bacteriology, January 2007, p. 265-268, Vol. 189, No. 1
0021-9193/07/$08.00+0     doi:10.1128/JB.01124-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

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• Vivero, A., Banos, R. C., Mariscotti, J. F., Oliveros, J. C., Garcia-del Portillo, F., Juarez, A., Madrid, C. (2008). Modulation of Horizontally Acquired Genes by the Hha-YdgT Proteins in Salmonella enterica Serovar Typhimurium. J. Bacteriol. 190: 1152-1156 [Abstract] [Full Text]  

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