Molecular Evolution of the H-NS Protein - Journal of Bacteriology

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Jul 27, 2006 - Mailing address: Department of Microbiol- ... molecular-mass proteins (8.6 and 8 kDa, respectively) that .... Basic local alignment search tool.
JOURNAL OF BACTERIOLOGY, Jan. 2007, p. 265–268 0021-9193/07/$08.00⫹0 doi:10.1128/JB.01124-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Vol. 189, No. 1

NOTES Molecular Evolution of the H-NS Protein: Interaction with Hha-Like Proteins Is Restricted to Enterobacteriaceae䌤 Cristina Madrid,1 Jesu ´s Garcı´a,2 Miquel Pons,2 and Antonio Jua´rez1* Department of Microbiology, University of Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain,1 and Laboratory of Biomolecular NMR, Institut de Recerca Biome`dica, Parc Cientific de Barcelona, Josep Samitier 1-5, 08028 Barcelona, Spain2 Received 27 July 2006/Accepted 25 September 2006

We show here that chromosomal hha-like genes are restricted to the Enterobacteriaceae. The H-NS Nterminal 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. oid 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 do-

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 nucle-

TABLE 1. Bacterial species included in the study Accession no. Abbreviation

Species name Complete genome

EC YP YPT YE WG SG BA-APS BP EW PL STM ST SPT SC SF SB SD SS HI HS PM AP MS PP VC VV VF VP VS XA XC XO XF AH PV SO PC

Escherichia coli K-12 Yersinia pestis CO92 Yersinia pseudotuberculosis IP32953 Yersinia enterocolitica Wigglesworthia glossinidia Sodalis glossinidius strain “morsitans” Buchnera aphidicola APS Blochmania pennsylvanicus BPEN Erwinia carotovora subsp. atroseptica SCRI1043 Photorhabdus luminescens subsp. laumondii TTO1 Salmonella enterica serovar Typhimurium LT2 Salmonella enterica serovar Typhi Ty2 Salmonella enterica serovar Paratyphi A strain ATCC 9150 Salmonella enterica serovar Cholerasuis strain SC-B67 Shigella flexneri 2a 2457T Shigella boydii Sb227 Shigella dysenteriae Sd197 Shigella sonnei Ss046 Haemophilus influenzae 86-028NP Haemophilus somnus 129PT Pasteurella multocida subsp. multocida strain Pm70 Actinobacillus pleuropneumoniae serovar 1 strain 4074 Manheimia succiniproducens MBEL55E Photobacterium profundum SS9 Vibrio cholerae O1 biovar eltor strain N16961 Vibrio vulnificus CMCP6 Vibrio fisheri ES114 Vibrio parahaemolyticus RIMD 2210633 Vibrio splendidus 12B01 Xanthomonas axonopodis pv. citri strain 306 Xanthomonas campestris pv. campestris strain 8004 Xanthomonas oryzae pv. oryzae KACC10331 Xylella fastidiosa Temecula 1 Aeromonas hydrophila Proteus vulgaris Shewanella oneidensis Psychrobacter cryohalolentis K5

* Corresponding author. Mailing address: Department of Microbiology, University of Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain. Phone: 34934034624. Fax: 34934034629. E-mail: [email protected]. 䌤 Published ahead of print on 13 October 2006. 265

NC㛬000913 NC㛬003143 NC㛬006155 NC㛬004344 NC㛬007712 NC㛬002528 NC㛬007292 NC㛬004547 NC㛬005126 NC㛬003197 NC㛬004631 NC㛬006511 NC㛬006905 NC㛬004741 NC007613 NC㛬007606 NC㛬007384 NC㛬007146 NZ㛬AABO01000002 NC㛬002663 NZ㛬AACK01000021 NC㛬006300 NC㛬006370 NC㛬002505 NC㛬004459 NC㛬006840 NC㛬004603 NZ㛬AAMR01000029 NC㛬003919 NC㛬007086 NC㛬006834 NC㛬004556 NC㛬004347 NZ㛬AAJC01000010

H-NS-like protein

Hha-like protein

NP㛬415753 NP㛬405719 YP㛬070618 CAC36307 NP㛬871370 YP㛬455050 NP㛬240096 YP㛬050423 YP㛬050423 NP㛬929734 NP㛬460710 NP㛬805439 YP㛬150400 YP㛬216733 NP㛬836928 YP㛬408259 YP㛬402927 YP㛬310845 YP㛬248940 ZP㛬00122343 NP㛬245809 ZP㛬00124899 YP㛬088514 YP㛬129295 NP㛬230775 NP㛬761728 YP㛬205014 NP㛬797512 ZP㛬00991642 NP㛬643875 YP㛬244659 YP㛬199447 NP㛬779825 CAD29802 P18818 NP㛬718702 ZP㛬00654501

NP㛬414993 NP㛬406613 YP㛬069519 CAA41091 NP㛬871527 YP㛬454361 YP㛬049271 NP㛬931058 NP㛬459468 NP㛬806117 YP㛬151447 YP㛬215502 NP㛬836131 YP㛬406900 YP㛬402156 YP㛬309458

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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.

main and a C-terminal DNA-binding domain that are separated by a linker domain. H-NS binds DNA in a non-sequencespecific 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 lowmolecular-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

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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.

modulators of bacterial gene expression (3, 17). Members of this family are widespread among different genera of gramnegative 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 ␣-, ␤-, 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. The structural details of Hha–H-NS interaction are avail-

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able. 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). 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 ␣-, ␤-, 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 Tecnologı´a (BIO2004-02747 to A.J., BIO2004-05436 and GEN200320642-C09-04 to M.P., and Ramo ´n 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. REFERENCES 1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410.

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