Genomic analysis of the histidine kinase family in bacteria and archaea

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1 Microbiology (2001), 147, Printed in Great Britain Genomic analysis of the histidine kinase family in bacteria and archaea Dong-jin Kim and Steven Forst Author for correspondence: Steven Forst. Tel: Fax: sforst uwm.edu Department of Biological Sciences, PO Box 413, University of Wisconsin, WI 53201, Milwaukee, USA Two-component signal transduction systems, consisting of histidine kinase (HK) sensors and DNA-binding response regulators, allow bacteria and archaea to respond to diverse environmental stimuli. HKs possess a conserved domain (H-box region) which contains the site of phosphorylation and an ATP-binding kinase domain. In this study, a genomic approach was taken to analyse the HK family in bacteria and archaea. Based on phylogenetic analysis, differences in the sequence and organization of the H-box and kinase domains, and the predicted secondary structure of the H-box region, five major HK types were identified. Of the 336 HKs analysed, 92% could be assigned to one of the five major HK types. The Type I HKs were found predominantly in bacteria while Type II HKs were not prevalent in bacteria but constituted the major type (13 of 15 HKs) in the archaeon Archaeoglobus fulgidus. Type III HKs were generally more prevalent in Gram-positive bacteria and were the major HK type (14 of 15 HKs) in the archaeon Methanobacterium thermoautotrophicum. Type IV HKs represented a minor type found in bacteria. The fifth HK type was composed of the chemosensor HKs, CheA. Several bacterial genomes contained all five HK types. In contrast, archaeal genomes either contained a specific HK type or lacked HKs altogether. These findings suggest that the different HK types originated in bacteria and that specific HK types were acquired in archaea by horizontal gene transfer. Keywords: classification scheme, phylogenetic analysis, secondary structure analysis, horizontal gene transfer INTRODUCTION In bacteria and archaea, two-component signal transduction systems (Kofoid & Parkinson, 1988; Parkinson & Kofoid, 1992; Forst & Roberts, 1994; Hoch & Silhavy, 1995), also referred to as His Asp phosphorelay systems (Egger et al., 1997), mediate adaptive responses to changes in environmental conditions. A typical twocomponent signal transduction system consists of a membrane-bound sensor histidine kinase (HK) and a cognate regulatory protein referred to as a response regulator (RR). HKs usually function as dimeric proteins that undergo transautophosphorylation on a conserved histidine residue in response to specific stimuli (Dutta et al., 1999). The phosphoryl group is subsequently transferred to an Asp residue in the receiver domain on the... Abbreviations: HK, histidine kinase; RR, response regulator. RR. Modulation of the phosphorylated state of the RR controls either expression of target genes or cellular behaviour, such as swimming motility (Hoch & Silhavy, 1995). Environmental stimuli received by highly divergent sensory input domains provides specificity for the signal transduction pathway and controls the level of the phosphorylated state of the RR. Bacteria can possess more than 30 different two-component signal transduction systems (Mizuno, 1997). Bacteria that possess a large number of HKs generally are able to adapt to a broad spectrum of environmental stimuli. While HKs have also been identified in fungi (Ota & Varshavsky, 1993; Loomis et al., 1998), amoeba (Schuster et al., 1996; Chang et al., 1998), Neurospora (Alex et al., 1996) and Arabidopsis (Chang et al., 1993; Suzuki et al., 1998), the HK content in eukaryotic genomes is much lower than that found in bacterial genomes. HKs consist of an ATP-binding kinase domain and the H-box domain which includes the histidine site of SGM 1197

2 D. -j. K I M a n d S. F O R S T HK subtype HK H-box X-region + (a) Type IA IB IC Type II Type III Type IV CheA (b) Kinase type HK N G1 F G2 G3 Orthodox Unorthodox CheA... Fig. 1. For legend see facing page. 1198

3 Histidine kinase family 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 His Asp His Asp 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. METHODS Analysis of bacterial and archaeal HK families. The HK family of each genome was assembled using the gene tables of the completed genomes listed in TIGR (http: Additionally, BLAST analysis using the transmitter domains (H-box plus kinase domain) of EnvZ, CheA, NarX, YehU and DcuS as the search sequences was performed. During this analysis ORFs were retrieved which lacked either H-box or kinase consensus motifs. Also, proteins such as HipA of E. coli and SpoIIAB of B. subtilis, which contained kinase domains but lacked an identifiable H-box domain were retrieved. Only those proteins that contained both an identifiable H-box and complete kinase domain were included in the HK gene family. 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: 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. RESULTS Characterization of the HK family of E. coli K-12 HKs were retrieved using the BLAST server within each genome listed in the TIGR microbial database. Various transmitters, which included both the H-box region and kinase domain, were used as search probes. Retrieved ORFs containing both an H-box region and a kinase domain were included in the HK gene family. We initially analysed the genomes containing large HK families. The HK family of the completed genomic sequence of E. coli K-12 (Blattner et al., 1997) was analysed first since it has been extensively studied and Fig. 1. Alignment of the H-box region of the HKs of E. coli. H-box consensus sequences are boxed. Bold characters depict invariable residues and characteristic conserved residues of a given subtype are represented by white letters. The predicted helix loop helix structures are underlined. The helix loop helix structure of EnvZ is shown by thick lines above the EnvZ sequence. denotes the position of the conserved positive amino acid residue at the end of helix 2. Proline residues in the putative loop region are shaded. (b) Alignment of the kinase domain of the HKs of E. coli. The conserved motifs are enclosed in boxes and shaded. Bold characters depict invariable residues and characteristic conserved residues of a given subtype are represented by white letters. The number of amino acid residues between motifs are represented by either a single dot (less than 10 residues), by double dots (10 25 residues) or double open circles (more than 25 residues). Dashes represent deleted residues. 1199

4 D.-j. KIM and S. FORST BasS YgiY PhoR YbcZ BaeS RstB EnvZ CpxA IA KdpD YedV CreC YfhK BarA RcsC IB TorS ArcB Type I IC EvgS AtoS HydH NtrB PhoQ Type II Che A Type III DcuS DpiB CheA NarX NarQ UhpB Type IV YehU 0 1 changes B Fig. 2. Phylogenetic analysis of the HKs of E. coli. Dendrogram of the 29 HKs of E. coli analysed using the UPGMA algorithm. The alignments shown in Fig. 1(a) and (b) were used in the analysis. the function of 18 of its HKs are presently known (Egger et al., 1997). 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. Phylogenetic analysis separated the HK gene family into five major branches (Fig. 2). The analysis reveals that Type I and II HKs are related to each other while Type III and IV HKs occupy separate branches. Type I and II HKs both possess orthodox kinase domains, which contain the N, G1, F and G2 consensus motifs. Type III and IV HKs possess so-called unorthodox kinase domains in which N1 of the N-box motif is either a glycine (Type III) or a proline (Type IV) residue, the F- box is absent and the G2 motif is truncated (Fig. 1b). The conserved glycine residue identified on the C- terminal side of the G2 motif in almost all kinase domains is referred to as the G3 site. The Type I group contained the largest number of members (72%). Within this group, three separate subtypes could be distinguished. The Type IA group contained 12 HKs, the Type IB group contained the hybrid HKs and the Type IC group contained three HKs, including the nitrogen 1200

5 Histidine kinase family Table 1. Characteristic features of HK types HK type H-box consensus* Kinase domain Mean H to N distance (aa) Type I H EhR P Orthodox 116 Type II H E N Orthodox 96 Type III R EhHD h Unorthodox 110 Type IV P H Fh N Unorthodox 92 CheA H ShKG Orthodox 325 * The bold H represents the invariable histidine residue in the H-box. Conserved hydrophobic residues (I, L, V, M) are designated by h. regulator, NtrB. While PhoQ contained a Type I H-box motif and an orthodox kinase domain, it did not branch within the Type IA, IB and IC subgroups. Each of the five HK types contained a characteristic H- box motif (Fig. 1a). The H-box of Type I HKs contained the consensus motif HEhRTPh. Secondary structure analysis predicted a helix loop helix structure (underlined in Fig. 1) in the H-box region. This structure was shown to exist in the NMR solution structure of EnvZ (Tomomori et al., 1999). Proline residues were present in the loop region of numerous HKs and positively charged residues were found at the end of the X-region. The H-box motif of the Type II HKs possessed a conserved asparagine residue at position 5 (the invariant H residue is defined as position 1) and lacked the positively charged residue and the proline at positions 4 and 6, respectively. These HKs lacked a predicted helix loop helix structure. The H-box motif of Type III HKs was characterized by an R-E-L sequence on the N- terminal side of the histidine site of phosphorylation and lacked the conserved positively charged and proline residues. A helix loop helix structure was predicted in these molecules. The H-box motif of Type IV HKs contained a proline residue on the N-terminal side and the conserved sequence FLFNAL on the C-terminal side of the histidine site of phosphorylation. Finally, the P1 domain of CheA contained the consensus motif HSIKG and exists at the N terminus of the protein. 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). HK family of Pseudomonas aeruginosa To determine whether the five major HK types found in E. coli were present in other bacteria, a phylogenetic analysis of HK gene family of Psd. aeruginosa (Stover et al., 2000) using the UMPGA method was performed (Fig. 3). Psd. aeruginosa possesses 63 HKs. The five HK types found in E. coli were also identified in Psd. aeruginosa. The majority of the family members (86%) were Type I HKs, containing typical orthodox kinase domains and H-box motifs. One cluster within the Type IA group (PA1396, 1976, 1992, 3271 and 4936) contained orthodox kinase domains while the H-box motifs contained a non-polar residue at position 4 and a glutamine residue at position 5. This clade of HKs formed a distinct branch within the Type IA group. In addition, a cluster of HKs in the Type IC group possessed the consensus H-box motif HDLNQPL in which the asparagine residue replaced the typical positively charged residue at position 4 and the glutamine residue at position 5 was highly conserved. Psd. aeruginosa lacked Type II HKs and possessed four CheAs. Two HKs, PA3078 and PA4380, could not be assigned to the defined type, so were categorized as unclassified. Helix loop helix structures were predicted in the H-box region of the Type I and III HKs and the H to N distances for each of the HK types were similar to those found in the different HK types of E. coli. Finally, the majority of the HKs were found in operons with cognate RRs. HK families in bacterial genomes The HK gene families of bacterial genomes listed as completed in the TIGR database were characterized using the cluster analysis approach. We began the analysis with free-living micro-organisms containing relatively large genomes. 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 1201

6 D.-j. KIM and S. FORST IA Type I IC Type II Che A Type III Type IV IB BaeS CpxA PA0930 PA2687 PA3206 RstB PA1158 PA1798 EnvZ PA5199 PA3191 PA4102 KdpD PA1636 PA5484 BasS YgiY PA4777 PA0757 PA2480 PhoR PA5361 YedV PA4886 YbcZ PA2810 PA1438 PA2524 CreC PA0464 YfhK PA1396 PA1976 PA1992 PA3271 PA4036 BarA PA0928 PA4112 TorS PA1611 PA3462 PA2824 PA4982 PA2583 PA3044 PA3946 RcsC ArcB EvgS PA4856 PA3974 PA3078 PA4380 AtoS HydH PA4725 PA4398 PA2571 PA2882 PA1336 PA5165 PA4293 PA5512 PA4197 PA4546 PA1243 PA2177 NtrB PA5124 PA1098 PA4494 PA4117 PhoQ PA1180 PA2656 DcuS DpiB CheA PA0178 PA1458 PA0413 PA3704 NarX PA3878 NarQ UhpB PA1979 PA0600 YehU B2380 PA5262 IA IB IC 0 1 changes... Fig. 3. Phylogenetic analysis of the HKs of Psd. aeruginosa. Dendrogram showing the analysis of a combined dataset of E. coli and Psd. aeruginosa HKs. HKs of Psd. aeruginosa are represented by bold type. 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 helix loop helix 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. 1202

7 Histidine kinase family Table 2. Distribution of HK types in bacteria HK type Escherichia coli Vibrio cholerae Pseudomonas aeruginosa Xylella fastidiosa Bacillus subtilis Synechocystis sp. Deinococcus radiodurans Thermotoga maritima Aquifex aeolicus Type I A Type I B Type I C Type II Type III Type IV CheA Unclassified Total HKs 29* 44* 63* 12* Total proteins Percentage HKs * Includes PhoQ-like HK. 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. Xylella fastidiosa (Simson, 2000), like E. coli and V. cholerae, is a member of the γ-subclass of the Proteobacteria. X. fastidiosa contained predominantly Type I HKs (Table 2) and lacked Type II and III HKs. The HKs in this bacterium existed in operons with cognate RRs. The HK family of the Gram-positive bacterium B. subtilis (Kunst, 1997) contained all five HK types. The Type I group, containing six Type IA, five Type IC HKs and no Type IB HKs, was not as large as that found in the Gram-negative bacteria analysed above, while 25% of the HKs belonged to the Type III group. B. subtilis contained a relatively large number of HKs which did not cluster within the five defined HK types. Three unclassified HKs (YtsB, YvcQ, YxdK) clustered together into one clade while three other unclassified HKs (YbdK, YrkQ, YccG) formed a separate clade (see Table 5). All of the HKs of B. subtilis existed in operons with cognate RR with the exception of the Type IC group which were orphans (Fabret et al., 1999). A markedly different HK distribution was found in the cyanobacterium, Synechocystis sp. (Kaneko et al., 1996). This bacterium possessed a large Type IB group but lacked Type IC HKs. Several of the Type IB HKs did not contain D1 or HPt modules. Whether these Type IB molecules represent HKs to which additional phosphorelay modules had not been added or are hybrid types which lost the phosphorelay modules, remains to be determined. Interestingly, the two CheA proteins identified in Synechocystis sp. contained additional phosphorelay modules (Mizuno et al., 1996). Synechocystis sp. contains two type III HKs, slr0331 and slr1212. While slr0331 was a typical Type III HK, slr1212 contained an atypical H-box sequence (HHRhKNNLQ) connected to a typical unorthodox kinase domain. Type II and IV HKs were not present in Synechocystis sp. Finally, only 13 of the HKs were organized in operons with cognate RRs (Mizuno et al., 1996). 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 1203

8 D.-j. KIM and S. FORST Table 3. Distribution of HK types in pathogenic bacteria HK type Haemophilus influenzae Neisseria meningitidis MC58 Rickettsia prowazekii Helicobacter pylori Campylobacter jejuni Chlamydophila pneumoniae CWL029 Chlamydia trachomatis serovar D Borrelia Treponema burgdorferi pallidum Mycobacterium tuberculosis Type I A Type I B Type I C Type II Type III Type IV CheA Unclassified Total HKs Total proteins Percentage HKs (a) HK subtype Type I HK H-box + Type II CheA (b) Kinase type Orthodox HK N G1 F G2 G3 CheA... Fig. 4. Alignment of the kinase and H-box regions of Arc. fulgidus. (a) Sequence alignment of the H-box regions. Shadings and symbols are as in Fig. 1. Only AF1483 and CheA possessed predicted helix loop helix structures. (b) Sequence alignment of the kinase domains. 1204

9 Histidine kinase family IA Type I IB Type II Type III Type IV IC 0 1 changes BaeS BasS YgiY PhoR YbcZ CpxA RstB EnvZ KdpD YedV CreC YfhK AtoS HydH NtrB BarA RcsC TorS ArcB EvgS AF1483 mth444 PhoQ DcuS DpiB AF1721 AF1639 AF1515 AF0021 AF0208 AF AF0450 AF0770 AF1467 AF2109 AF1184 AF1452 CheA AF1040 NarX NarQ UhpB mth292 mth356 mth619 mth174 mth468 mth446 mth560 mth823 mth985 mth123 mth1124 mth459 mth901 mth902 YehU B Fig. 5. Phylogenetic analysis of the HKs of Arc. fulgidus and Mbc. thermoautotrophicum. Dendrogram showing the analysis of a combined dataset of HKs of E. coli, Arc. fulgidus and Mbc. thermoautotrophicum. HKs of Arc. fulgidus and Mbc. thermoautotrophicum are represented by bold type. 1205

10 D.-j. KIM and S. FORST Table 4. Distribution of HK types in archaea HK type Methanobacterium thermoautotrophicum Archaeoglobus fulgidus Pyrococcus horikoshii Methanococcus jannaschii Aeropyrum pernix Type I A Type I B 1 1 Type I C Type II 13 Type III 14 Type IV CheA 1 1 Total HKs Total proteins Percentage HKs ). 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. Helicobacter pylori and Campylobacter jejuni, both of which belong to the δ-subclass of the Proteobacteria, contained several unclassified HKs. This finding suggests that this group of bacteria may possess a unique HK type that is not yet identified in the current HK dataset. 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. Earlier analysis of the Methanobacterium thermoautotrophicum genome identified 15 HKs (Smith et al., 1997). The H-box region of 14 of these HKs contained the motif HHRVKNNLQ which was identical to the H-box sequence found in Mycobacterium and Synechocystis. HKs containing this sequence belong to the Type IIIB group. Cluster analysis also showed that the HKs of Mbc. thermoautotrophicum branched within the Type III group forming a clade that was distinct from the E. coli group (Fig. 5). The mean H to N distance of the Mbc. thermoautotrophicum HKs was 110 residues. Mbc. thermoautotrophicum possessed one orthodox HK subtype and did not contain semi-orthodox, minor, CheA, tripartite or hybrid HKs. Finally, most of the HKs were not organized in operons with cognate RRs. 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. The genome sequence of the archaeon Halobacterium sp. was recently completed (Ng et al., 2000). Of the 14 reported HKs, we retrieved 12 HKs, 8 of which formed a clade within the Type I group but were distinct from the IA, IB and IC subtypes. These HKs appear to represent a subtype that is so far unique to Halobacterium. Three Type II HKs and one CheA were also identified. Finally, as found in Archaeoglobus, there were more HKs (14) than RRs (6) in Halobacterium. DISCUSSION Phylogenetic analysis of HKs in numerous bacterial and archaeal genomes led to the identification of five major HK types. Of the 336 HKs analysed in this study 92% could be assigned to one of the five HK types. Type I and II HKs possessed orthodox kinase domains while Type 1206

11 Histidine kinase family Table 5. Classification of HK family of completed genomes Genome Type I Type II Type III Type IV CheA Unclassified IA IB IC PhoQ* Escherichia coli BaeS PhoR ArcB AtoS PhoQ DcuS NarQ B2380 CheA K-12 BasS RstB BarA HydH DpiB NarX YehU CpxA YbcZ EvgS NtrB UhpB CreC YehU RcsC EnvZ YfhK TorS KdpD YgiY Vibrio cholerae VC0303 VC0622 VC1084 VC1639 VC0791 VC1276 VC0694 VC1397 VCA0257 serotype O1 VC0720 VC1349 VC1085 VC1088 VCA0675 VCA0851 VC2063 VCA0565 VC1319 VC1445 VC1156 VC1605 VCA0683 VCA1095 VC2693 VC1653 VC1315 VCA0238 VC2713 VC1831 VC1521 VCA0531 VC2369 VC1925 VCA0522 VCA1104 VC2453 VC2136 VCA0709 VC2748 VCA0736 VCA0141 VCA0211 VCA0705 VCA0719 Haemophilus HI1378 HI0220 HI0267 influenzae KW20 HI1707 Pseudomonas PA0464 PA0928 PA1098 PA1180 PA0600 PA5262 PA0178 PA3078 aeruginosa PAO1 PA2810 PA4293 PA0757 PA1611 PA1243 PA2656 PA1979 PA0413 PA3191 PA4398 PA0930 PA2583 PA1336 PA3878 PA1458 PA3206 PA4494 PA1158 PA2824 PA2177 PA3704 PA3271 PA4546 PA1396 PA4036 PA3044 PA2571 PA4725 PA1438 PA4102 PA3462 PA2882 PA5124 PA1636 PA4380 PA3946 PA4117 PA5165 PA1798 PA4777 PA3974 PA4197 PA5512 PA1976 PA4112 PA4886 PA1992 PA4856 PA5199 PA2480 PA4982 PA5361 PA2524 PA5484 PA2687 Xylella fastidiosa XF0323 XF0853 XF1455 XF0390 XF1625 XF1952 XF2577 9a5c XF0973 XF1849 XF2535 XF2546 XF

12 D.-j. KIM and S. FORST Table 5 (cont.) Genome Type I Type II Type III Type IV CheA Unclassified IA IB IC PhoQ* Neisseria meningitidis NMB0594 NMB0114 NMB1249 NMB1606 MC58 NMB1792 Neisseria meningitidis NMA0670 NMA0160 NMA1418 NMA1803 serogroup A Z2491 NMA0797 Rickettsia prowazekii RP426 RP229 RP614 Madrid E RP465 Helicobacter pylori HP0244 HP0392 HP HP1364 Campylobacter jejuni Cj0793 Cj0284c Cj1262 NCTC Cj1226c Cj0889c Cj1222c Cj1492c Chlamydophila AtoS pneumoniae CWL029 Chlamydophila CP0164 pneumoniae AR39 Chlamydia NtrB trachomatis serovar D Chlamydia TC0752 trachomatis MoPn Borrelia burgdorferi BB0420 BB0764 BB0567 B31 BB0669 Treponema pallidum TP0520 TP0363 Nichols Mycobacterium Rv0490 Rv0845 tuberculosis H37Rv Rv1028c Rv0758 Rv2027c Rv1032c Rv0982 Rv3132c Rv3245c Rv0601c Rv3220c Rv3764c Rv0902c Bacillus subtilis 168 PhoR KinA CitS ComP YocF LytS CheA YtsB YbdK ResE KinB YdbF DegS YvfT YesM YvcQ YrkQ YclK KinC YufL YdfH YvqE YwpD YxdK YccG YkoH YkrQ YcbA YfiJ YxjM YvrG YkvD YhcY SpaK YvqB YycG Synechocystis sp. PCC 6803 sll0337 sll0474 slr0210 slr0311 sll0043 sll

13 Histidine kinase family Table 5 (cont.) Genome Type I Type II Type III Type IV CheA Unclassified IA IB IC PhoQ* sll0790 sll0798 slr0533 slr0640 slr1147 sll0698 sll0750 slr1400 slr0473 slr2099 sll1228 slr0484 sll1353 slr1393 sll1672 slr1759 sll1871 slr1969 sll1888 slr2098 sll1905 slr2104 slr1212 sll1296 sll1590 slr1805 sll1555 sll1003 sll1124 sll1475 slr0222 slr1324 DR0860 DR0577 DR0892 Deinococcus radiodurans R1 DR0744 DR2419 DR1174 DRB0028 DR1227 DRA0050 DR1175 DR1556 DRA0205 DR1606 DRA0009 DRB0090 DR2244 DRB0029 DR2328 DRB0082 DR2416 Thermotoga maritima TM0400 TM1359 TM0702 TM0127 MSB8 TM0853 TM0187 TM1258 TM1654 Aquifex aeolicus hksp1 VF5 Methanobacterium thermoautotrophicum delta H mth444 Type III B mth123 mth560 mth174 mth619 mth292 mth823 mth356 mth901 mth446 mth902 hksp2 hksp4 1209

14 D.-j. KIM and S. FORST Table 5 (cont.) Genome Type I Type II Type III Type IV CheA Unclassified IA IB IC PhoQ* Archaeoglobus fulgidus DSM4304 Pyrococcus horikoshii OT3 mth459 mth985 mth468 mth1124 AF1483 Type II B AF1040 AF0021 AF1452 AF0208 AF1467 AF0450 AF1515 AF0770 AF1639 AF0893 AF1721 AF1184 AF CheA * PhoQ-type HKs were found only in the γ-subclass of the Proteobacteria. HK not listed in the TIGR gene table. Subtypes (III B) that exist within Type III HK family. Subtypes (II B) that exist within Type II HK family. III and IV HKs possessed unorthodox kinase domains. The predicted secondary structure of the different H- box and X regions and the H to N distances in the different HKs were characteristic for the various HK types. The fifth HK type was composed of CheA molecules in which the H-box (P1) domain is located at the N terminus of the protein. Based of these findings, several conclusions can be made concerning the HK family of bacteria and archaea. (i) All bacteria sequenced to date, with the exception of mycoplasmas, contain HKs. The HK content was found to increase as the size of the genome increased. In free-living bacteria possessing larger genomes the HK content was relatively high while in pathogenic bacteria possessing smaller genomes, the HK content was relatively low. On the other hand, the HK content in archaea was highly variable with some archaeal genomes completely lacking HKs. (ii) Type I HKs were predominant in bacteria with the content of the different Type I subtypes varying greatly. For example, V. cholerae contained 12 Type IC HKs while Synechocystis lacked these HKs. Similarly, Synechocystis sp. contained 19 Type IB HKs while none was present in B. subtilis. In contrast, Type I HKs were not prevalent in archaea. (iii) Unlike bacterial HKs, archaeal HKs were generally not organized in operons with RRs. Some archaeal genomes possessed significantly more HKs than RRs. (iv) The Gram-positive bacteria analysed in this study contained a relatively high content of Type III HKs. Similarly, Grebe & Stock (1999) found that four of the eight HKs of the Grampositive bacterium Streptomyces coelicolor belonged to the Type III (HPK7) group. (v) Hybrid HKs were found in bacteria but have not yet been identified in archaea. Interestingly, all known eukaryotic HKs belong to the hybrid (Type IB) HK group (Grebe & Stock, 1999). (vi) Finally, the HKs of Aqu. aeolicus and many of the HKs in Helicobacter, Campylobacter and Halobacterium remain unclassified. As more bacterial genomes are sequenced, new HK types may be established that will encompass these as yet unclassified HKs. 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 1 4) in the Grebe & Stock (1999) study. For example, the NtrBrelated HKs were placed in the HPK 4 group while phylogenetic analysis (Fig. 2) placed these HKs within 1210

15 Histidine kinase family 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. 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