Department of Biological Sciences, PO Box 413, University of Wisconsin, WI 53201, Milwaukee, USA1
Author for correspondence: Steven Forst. Tel: +1 414 229 6373. Fax: +1 414 229 3926. e-mail: sforst{at}uwm.edu
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ABSTRACT |
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Keywords: classification scheme, phylogenetic analysis, secondary structure analysis, horizontal gene transfer
Abbreviations: HK, histidine kinase; RR, response regulator
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INTRODUCTION |
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HKs consist of an ATP-binding kinase domain and the H-box domain which includes the histidine site of phosphorylation. The kinase domain consists of three conserved consensus motifs called the N-, G1- and G2-boxes, and a fourth, more variable sequence, the F-box (Kofoid & Parkinson, 1988 ; Stock et al., 1988
, 1995
). In most HKs, the kinase domain is directly connected to the C-terminal side of the H-box domain. In contrast, in the chemosensor CheA, the H-box (P1 domain) resides at the N terminus of the protein and is separated from the kinase domain by the intervening P2 and P3 modules (Garzon & Parkinson, 1996
; Robinson & Stock, 1999
).
The structures of the H-box domain of the osmosensor, EnvZ (Tomomori et al., 1999 ) and the P1 domain of CheA (Zhou & Dalquihst, 1997
) have been determined. The H-box region of EnvZ consists of a four-helix bundle structure formed by the dimeric association of two identical subunits while the P1 domain is a monomeric four-helix bundle structure. While the structure of H-box domains differ, the structure of the kinase domains of EnvZ of Escherichia coli (Tanaka et al., 1998
) and CheA of Thermatoga maritima (Bilwes et al., 1999
) were shown to be homologous to each other and to the ATP-binding domains of DNA gyrase B and Hsp 90. The phosphotransfer reaction can be reconstituted using liberated H-box and kinase domains (Garzon & Parkinson, 1996
; Park et al., 1998
), indicating that the individual domains can be obtained as functionally intact modules.
Besides the typical two-component organization, multistep HisAspHisAsp phosphorelay systems can be composed of individual phosphotransfer proteins. This modular organization has been extensively investigated in the multi-step pathway controlling sporulation in Bacillus subtilis (Appleby et al., 1996 ; Fabret et al., 1999
; Hoch, 1995
; Perraud et al., 1999
). Multistep phosphotransfer reactions can also occur within a single HK. These so-called hybrid HKs contain additional phosphotransfer modules referred to as the D1 receiver and the HPt phosphotransfer domains that are attached to the C-terminal side of the kinase domain (Appleby et al., 1996
).
The number of HKs recognized has expanded enormously with the advent of microbial genomic sequencing projects. The HK superfamily has been classified by numerous criteria. Recently, Grebe & Stock (1999) separated the HK family into 11 different subtypes based on cluster analysis of 348 HKs. In the present study, the HK families in the completed genomes of 22 bacteria and 4 archaea was analysed. This genomic analysis divided the HK family into five major types. The HK type distribution differed markedly between bacteria and archaea.
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METHODS |
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Alignment of H-box and kinase domains.
The transmitter domains were initially aligned using the multi-sequence alignment program MSA version 2.1. Refinement of the alignments was aided by BLAST search analysis and visual inspection.
Phylogenetic analysis.
A distance dendrogram of each HK family was constructed using the unweighted pair-group method with arithmetic means (UPGMA) algorithm. Using this method, five different HK types were identified in E. coli. For each genome analysed, a dataset, which included the HKs from E. coli, was created and subsequently analysed using the UPMGA method. The assignment of HK types and subtypes within each genome analysed was accomplished using this approach.
Secondary structure analysis.
The PredictProtein server (http://www.embl-heidelberg.de/predictprotein/predictprotein.html) was used to predict the secondary structure of the H-box region in each HK retrieved. A predicted secondary structure was assigned to sequences that possessed a liability value of greater than 7.
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RESULTS |
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Twenty-nine HKs were retrieved from the genome of E. coli. Fig. 1(a) shows the amino acid sequence alignment of the H-box and X regions and Fig. 1(b)
shows the alignment of the kinase domains. During the BLAST analysis, proteins which contained phosphorelay subdomains but lacked kinase domains were retrieved. For example, YojN contains an HPt domain but no identifiable kinase domain. These types of proteins were not included in the HK gene family.
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The distance between the conserved histidine residue of the H-box and the conserved asparagine residue of the kinase domain (H to N distance) was characteristic for the different HK subtypes. The mean H to N distance was approximately 116 residues and 96 residues for the Type I and II HKs, respectively (Table 1). The mean H to N distance was 110 residues and 92 residues for the Type III and IV HKs, respectively. The kinase domain of CheA was characterized by insertions between the N and G1 boxes and the G1 and F boxes. The N-box of CheA contained a histidine residue at the N1 position. The localization of the H-box at the N terminus of CheA created an H to N distance of 325 residues (Table 1
; Kofoid & Parkinson, 1988
). Finally, it has been shown that 26 of the 29 HKs of E. coli are organized in operons with cognate RRs (Mizuno, 1997
).
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A total of 44 HKs were identified in the genome of Vibrio cholerae (Heidelberg et al., 2000 ), which included three new proteins (VCA0705, VC0694, VCA0851) not previously listed in the TIGR gene table. V. cholerae possessed a large Type I group which included 7 Type IA, 9 Type IB and 12 Type IC molecules (Table 2
). We noted that a cluster of HKs within the Type IC group possessed the H-box motif HDLNNP in which the typical positively charged residue at position 4 was substituted by an asparagine residue. The Type I HKs possessed helixloophelix structures in the H-box region and a mean H to N distance of 110 residues. Type II, III, IV and CheA HKs were also present in V. cholerae. Additionally, four HKs did not cluster with a defined group and were therefore placed in an unclassified category. Twenty-eight of the HKs were found on the large chromosome of V. cholerae (2·96 Mb) while 16 were found on the small chromosome (1·07 Mb). The majority of the HKs existed in operons with cognate RRs.
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The Gram-positive bacterium Deinococcus radiodurans (White et al., 1999 ) contained 20 HKs, 13 of which belonged to the Type IA group. The HK content of Type III HKs (4 out of 20) was relatively high. The genome of this bacterium possesses two chromosomes (2·6 and 0·41 Mb), a megaplasmid (0·18 Mb) and a small plasmid (45 kb). Thirteen of the HKs were present on the large chromosome, three were located on the smaller chromosome and four were located on the megaplasmid. In the thermophilic bacterium Thermatoga maritima (Nelson et al., 1999
) the majority of the HKs (5 out of 8) belonged to the Type I group while two HKs remained unclassified. Seven of the eight HKs of T. maritima existed in operons with cognate RRs. Finally, the hyperthermophilic bacterium, Aquifex aeolicus, which is considered to be one of the earliest diverging eubacteria (Deckert et al., 1998
), possessed three HKs, none of which could be classified. This organism is motile and possesses polytrichous flagella but does not contain an identifiable CheA protein.
In summary, 92% of the HKs analysed were able to be assigned to one of the five major HK types. Several bacteria contained all five HK types. The majority of HKs (63%) belonged to the Type I group while the distribution of the various subtypes varied considerably. In the bacteria, most of the HKs were organized in operons with cognate RRs, with the notable exception of Synechocystis.
HK families of human pathogens
The size of the genomes of pathogenic bacteria is generally smaller than that of free-living bacteria (Table 3). The mean HK content of the pathogenic bacteria was 0·26% as compared with 0·65% for the free-living bacteria. The human pathogenic bacteria contained predominantly Type I HKs (Table 3
). Interestingly, the Gram-positive bacterium, Mycobacterium tuberculosis (Davies et al., 1998
) contained a relatively high content (4 of 13) of Type III HKs. One of the Type III HKs, Rv3220, contained a typical unorthodox kinase domain and the atypical H-box sequence HHRhKNNLQ which was similar to the H-box of slr1212 of Synechocystis sp. These proteins are referred to as Type IIIB HKs (see Table 5
). The majority of the HKs and RRs in these bacteria were organized in operons with cognate RRs.
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Analysis of the HK family of archaeal genomes
The amino acid sequence alignment of the H-box regions and kinase domains of the 15 HKs of Archaeoglobus fulgidus (Klenk et al., 1998 ) is shown in Fig. 4(a)
and (b)
, respectively. Thirteen of the HKs belonged to the Type II subtype while only one belonged to the Type I group. The H-box module of Type II HKs contained the characteristic asparagine residue at position 5. The H-box of the Type II HKs lacked a predictable secondary structure. Highly conserved glutamic acid and positively charged residues were identified downstream of the H-box (shaded in Fig. 4a
). The kinase domains (Fig. 4b
) possessed a conserved glycine residue in the F-box and the mean H to N distance was 96 residues. Cluster analysis revealed that the Type II group of Archaeoglobus formed a separate clade, designated the IIB subtype, within the Type II group of E. coli (Fig. 5
). The 13 HKs did not exist in operons with the nine RRs identified in Archaeoglobus.
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Methanococcus jannaschii (Bult et al., 1996 ) and Aeropyrum pernix (Kawarabayasi et al., 1999
) were previously found to lack HKs. A re-examination of these genomes confirmed that HKs were missing in these organisms. The genome of Pyrococcus horikoshii has been completed recently (Kawarabayasi et al., 1998
). The only HK found in this genome was CheA (Table 4
). The HK and RR organization in archaea was markedly different than that found in bacteria. Most of the HKs in Arc. fulgidus and Mbc. thermoautotrophicum were not organized in operons with a cognate RR. Only AF0450 of Arc. fulgidus and MTH0902 and MTH0444 of Mbc. thermoautotrophicum (Smith et al., 1997
) were located in operons with a cognate RR.
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DISCUSSION |
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In this study, a genomics approach was taken to analyse the HKs of bacterial and archaeal genomes. A different approach was taken by Grebe & Stock (1999) in which cluster analysis of 348 HKs led to a classification scheme consisting of 11 HPK (histidine protein kinase) types. A primary difference in the respective classification schemes is found in the Type I group which was separated into four different HPK types (HPK 14) in the Grebe & Stock (1999)
study. For example, the NtrB-related HKs were placed in the HPK 4 group while phylogenetic analysis (Fig. 2
) placed these HKs within the Type I (Type IC) group. In addition, Type II HKs were separated into a bacterial group (HPK 5) and an Arc. fulgidus group (HPK 6) by Grebe & Stock (1999)
. Similarly, the Type III HKs were separated into a bacterial group (HPK 7) and an Mbc. thermoautotrophicum group (HPK 11). HKs that did not cluster within a defined HK group remained unclassified in our study while Grebe & Stock (1999)
either did not include these HKs or gathered them into a separate subgroup. Thus, we identified 36 HKs in B. subtilis with YvcQ, YxdK and YtsB remaining unclassified (Table 5
), while Grebe & Stock (1999)
identified 31 HKs and placed YvcQ, YxdK and YtsB in their own subgroup (HPK3i).
We show that bacteria possessing larger genomes contained several different HK types while archaeal genomes either lacked HKs or possessed a HK family consisting of a specific type. Arc. fulgidus and Mbc. thermoautotrophicum possessed one Type I HK and a large family of either Type II or III HKs, respectively. These findings raise the question of why Type II and III HKs, rather than Type I HKs, have expanded in different archaea. Furthermore, it appears that the different HK types arose in bacteria and were acquired by archaea via lateral gene transfer (Grebe & Stock, 1999 ). Presumably, Arc. fulgidus acquired a Type II HK gene from one bacterial source while Mbc. thermoautotrophicum acquired a Type III HK from a different bacterium. It is of interest to consider whether different HK types possess distinct functions that allow micro-organisms to exploit specific ecological niches. Biochemical studies have almost exclusively focused on the Type I HKs. A comparison of the biochemical and structural properties of the various HK types may reveal differences that could further our understanding of the role that HKs play in allowing micro-organisms to adapt to specific environmental conditions.
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ACKNOWLEDGEMENTS |
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Received 13 March 2000;
revised 8 January 2001;
accepted 25 January 2001.