Distribution and evolution of multiple-step phosphorelay in prokaryotes: lateral domain recruitment involved in the formation of hybrid-type histidine kinases

Weiwen Zhang and Liang Shi

Microbiology Department, Pacific Northwest National Laboratory, 902 Battelle Blvd, PO Box 999, Mail Stop P7-50, Richland, WA 99352, USA

Correspondence
Weiwen Zhang
Weiwen.Zhang{at}pnl.gov
Liang Shi
Liang.Shi{at}pnl.gov


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although most two-component signal transduction systems use a simple phosphotransfer pathway from one histidine kinase (HK) to one response regulator (RR), a multiple-step phosphorelay involving a phosphotransfer scheme of His–Asp–His–Asp was also discovered. Central to this multiple-step-type signal transduction pathway are a hybrid-type HK, containing both an HK domain and an RR receiver domain in a single protein, and a histidine-containing phosphotransfer (HPT) that can exist either as a domain in hybrid-type HKs or as a separate protein. Although multiple-step phosphorelay systems are predominant in eukaryotes, it has been previously suggested that they are less common in prokaryotes. In this study, it was found that putative hybrid-type HKs were present in 56 of 156 complete prokaryotic genomes, indicating that multiple-step phosphorelay systems are more common in prokaryotes than previously appreciated. Large expansions of hybrid-type HKs were observed in 26 prokaryotic species, including photosynthetic cyanobacteria such as Nostoc sp. PCC 7120, and several pathogenic bacteria such as Coxiella burnetii. Phylogenetic analysis indicated that there was no common ancestor for hybrid-type HKs, and their origin and expansion was achieved by lateral recruitment of a receiver domain into an HK molecule and then duplication as one unit. Lateral recruitment of additional sensory domains such as PAS was also evident. HPT domains or proteins were identified in 32 of the genomes with hybrid-type HKs; however, no significant gene expansion was observed for HPTs even in a genome with a large number of hybrid-type HKs. In addition, fewer HPTs than hybrid-type HKs were identified in all prokaryotic genomes.


Abbreviations: GAF, cyclic nucleotide-binding domain of GAF; HAMP, HAMP domain for histidine kinases, adenylyl cyclases, methyl-binding proteins and phosphatases; HK, histitidine kinase; HPT, histidine-containing phosphotransfer domain (or protein); PAS, PAS domain; RR, response regulator; TCSTS, two-component signal transduction system


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Micro-organisms must modulate their gene expression repertoire in order to adapt to changing environments. One of the predominant signal transduction mechanisms employed by microbes is the phosphotransfer pathway commonly referred to as ‘two-component’ signal transduction systems (TCSTSs), which typically consist of a sensor histidine kinase (HK) and a response regulator (RR), and have been found across all three domains of life, the Bacteria, Archaea and Eukarya (Hoch, 2000). The sensor HK, generally an integral membrane protein, consists of a signal recognition domain, with unique specificity, coupled to an autokinase domain. In most cases, binding of extracellular signalling molecules to the signal recognition domain causes activation of the autokinase domain, resulting in phosphorylation of a conserved histidine (His) residue on the His-containing subdomain of the autokinase. The His-containing subdomain is associated with the receiver domain of the cognate RR, to which another phosphotransfer to an aspartate (Asp) on the RR occurs. Regulator domains normally inhibit the output domain of RRs and phosphorylation relieves this inhibition, freeing the output domain to carry out its function, which is usually transcription activation (Stock et al., 1989; Parkinson & Kofoid, 1992).

In addition to the prototypical TCSTSs described above, a more complex version of this phosphotransfer scheme was also discovered in both prokaryotic and eukaryotic cells (Appleby et al., 1996). This system involves multiple phosphotransfer steps with often more than two proteins. Three types of multiple-step phosphorelay systems have been revealed. The first pathway is exemplified by the system that governs the initiation of sporulation in Bacillus subtilis. This phosphorelay cascade begins with the autophosphorylation of one of the three sensor kinases, KinA, KinB or KinC. The phosphoryl group is then transferred to a receiver domain in the regulator Spo0F. The Spo0F then serves as a phosphodonor for Spo0B, which is phosphorylated on a His residue. Finally, the phosphoryl group completes its course by transfer to an Asp in Spo0A (Burbulys et al., 1991). The second and third type of multiple-step phosphorelay involve a hybrid-type HK in which both the HK domain and the RR receiver domain are present within a single protein. The second type has an intermediate His-containing phosphotransfer (HPT) domain in the same molecule, whereas in the third type, the HPT domain is contained on a separate protein. In the hybrid-type HK systems the phosphoryl group is first transferred from a His to an Asp residue within the hybrid HKs, then through the HPT domain or protein and is subsequently transferred to a cytoplasmic RR (Fig. 1). It has also been suggested that protein phosphatases, such as the sixA gene in Escherichia coli, may be implicated in the His–Asp phosphorelay through regulating the phosphorylation state of the HPT domain (Ogino et al., 1998). There are three well-studied cases where these hybrid HK mechanisms are utilized. The first is the BvgS–BvgA system controlling the transcriptional regulation of virulence factors in Bordetella pertussis, in which the BvgS protein contains the HK domain, the receiver domain and the HPT domain (Uhl & Miller, 1996) (Fig. 1a). The second is the Sln1p–Ypd1p–Ssk1p system governing osmoregulation in the yeast Saccharomyces cerevisiae. In this system the HPT domain is contained on Ypd1p, a separate protein of 167 residues (Posas et al., 1996) (Fig. 1b). The third is the RcsC–YojN–RcsB signalling pathway, implicated in capsular synthesis and swarming behaviour in E. coli (Takeda et al., 2001; Clarke et al., 2002). In this system, the HPT domain is present at the C terminus of the protein YojN, which shows a similarity to RcsC, particularly in the HK domain, although the crucial autophosphorylation His site is missing (Takeda et al., 2001; Clarke et al., 2002) (Fig. 1c). Early results from the analysis of HK domain architecture from a limited number of prokaryotic and eukaryotic genomes showed that most eukaryotic HKs are of the hybrid type, while only a small proportion of prokaryotic HKs contain both the kinase and receiver domains in a single HK molecule. It has thus been suggested that TCSTSs in prokaryotes generally use a simple two-component phosphotransfer scheme, whereas phosphorelays and hybrid HKs dominate two-component signalling in eukaryotes (West & Stock, 2001; Oka et al., 2002; Catlett et al., 2003). However, in recent years more hybrid-type HKs have been identified from various bacterial genomes (Xu et al., 2003; Rabus et al., 2004; W. Zhang and others, unpublished data), suggesting that the role of multiple-step phosphorelay systems in prokaryotes might have been underestimated.



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Fig. 1. Schemes of multiple-step phosphorelay signal transduction systems containing a hybrid-type HK and an HPT protein. (a) The scheme with hybrid-type HK containing all autokinase, receiver and HPT domains in one protein, as in the case of the BvgS–BvgA system of Bortedella pertussis; (b) the scheme with a separate HPT protein as in the case of Sln1p–Ypd1p–Ssk1p system of the yeast Sac. cerevisiae; (c) the scheme with an HPT domain located in another protein as in the case of RcsC–YojN–RcsB of E. coli.

 
TCSTSs were first identified decades ago and several model systems, including sporulation in Bac. subtilis, chemotaxis and osmoregulation in E. coli, have been extensively studied (Forst et al., 1989; Bourret et al., 1989; Leonardo & Forst, 1996; Stephenson & Hoch, 2002). However, until recently, few studies have been conducted on their origin and evolution. In an extensive study conducted recently, the TCSTSs from 14 complete and six partial genomes were phylogenetically analysed by distance methods (Koretke et al., 2000). The results suggested that the TCSTSs are of bacterial origin, and may share a common ancestor with heat-shock protein Hsp90, DNA-mismatch repair protein MutL and type II topoisomerases (Dutta et al., 1999). Phylogenetic analysis also indicated that the hybrid-type HKs in eukaryotes were acquired from bacteria through lateral gene transfer (Koretke et al., 2000). However, little information is available on how hybrid-type HKs evolved in bacteria other than that they cluster together in phylogenetic trees of HKs (Koretke et al., 2000). With the progress of bacterial genome sequencing programmes, more than 150 microbial genomes from almost all major phylogenetic lineages have been fully sequenced. The availability of complete genome sequences from numerous microbial species allows us to perform a detailed evaluation of the presence of multiple-step phosphorelay systems in prokaryotes and their evolutionary relationships, which was not previously possible. In this paper, we describe the results of a detailed survey and phylogenetic analysis of the hybrid-type HK and HPT proteins involved in multiple-step phosphorelay systems from 156 complete genomes. Our survey results show that more than one-third of bacterial genomes possessed hybrid-type HKs. In addition, the hybrid-type HKs were also identified from several archaeal genomes (Koretke et al., 2000; Kim & Forst, 2001). The results indicated that the role of multiple-step phosphorelay systems in prokaryotes may have been underestimated. Phylogenetic analysis indicated that there was no common ancestor for hybrid-type HKs, and that their origin and expansion were achieved by lateral recruitment of a receiver domain into an HK molecule and then duplication as one unit. In addition, survey and phylogenetic analysis of HPT proteins from prokaryotic sources were performed to infer their evolutionary course.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of hybrid-type HKs.
Sequences of all of the putative HKs from 156 complete microbial genomes (as of 22 September 2004) were extracted from the Comprehensive Microbial Resource (CMR) database of the Institute for Genomic Research (TIGR) (http://www.tigr.org), and then subjected to domain identification using the molecular architecture research tools provided by SMART (http://smart.embl-heidelberg.de/) with an E value of <0·01 (Letunic et al., 2002). The Che protein kinases involved in chemotaxis were excluded from this study because they have different domain constitution and organization where their His-containing subdomain has lost the catalytic His and is used exclusively for dimerization, while a HPT domain that may have originally served a regulatory function is now used for phosphorelay to CheY and CheB (Dutta et al., 1999; Koretke et al., 2000). A hybrid-type HK was determined by the presence of complete kinase and receiver domains in a single protein according to the definition by Catlett et al. (2003). The protein sequences of kinase domains (including both autokinase and His-containing subdomains), and the receiver domains from RR, HPT and various sensory domains were extracted separately. To confirm tentatively identified domains, the protein sequences of these domains were used to search for conserved domains from the Conserved Domain Databases, Pfam and COG using BLAST with a cut-off E value of <0·01 (Marchler-Bauer et al., 2003). In addition, visual inspection was used to eliminate domains that lacked the minimum complement of conserved sequence features considered necessary for each type of domain (Stock et al., 1989; Kato et al., 1997; Taylor & Zhulin, 1999).

Identification of HPT.
All known HPT proteins from microbial sources, along with HPT domains from proteins identified as hybrid-type HKs by SMART in this study, were used as query sequences in two separate searches for HPT gene homologues. The first search was performed against the 156 complete microbial genomes contained in the OMINOME Pep database of TIGR using BLASTP (http://tigrblast.tigr.org/cmr-blast/) and the second was against the NCBI sequence database using BLASTP (http://www.ncbi.nih.gov/blast). Both searches used an E value threshold of <0·01.

Sequence alignment and phylogenetic analysis.
Sequence alignments were performed using the default parameters of the CLUSTALW program originally developed by Higgins & Sharp (1988), available from the LaserGene software package (DNAStar) and PAUP* 4.0 beta version (Blumenberg, 1988) with an alignment gap penalty of 10·00 and a gap length penalty of 0·1. Confidence levels were determined by analysing 100 bootstrap replicates. For phylogenetic classification of kinase and receiver domains, functional domains of all known HKs and RRs in E. coli and Bac. subtilis were extracted and used as indicators for each phylogenetic group according to the method described previously (Koretke et al., 2000).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Distribution of hybrid-type HKs in prokaryotic genomes
From 156 complete microbial genomes listed in the CMR of TIGR (representing 18 archaea and 138 bacteria as of 22 September 2004), a total of 505 protein sequences encoding putative hybrid-type HKs were identified from a total of 2041 putative HKs present in the genomes surveyed. Hybrid-type HKs were more widely spread in prokaryotes than previously expected, with representatives found in four archaeal and 52 bacterial genomes (Table 1). Genes encoding hybrid-type HKs have previously been identified in Bacteria and Eukarya (Koretke et al., 2000; Kim & Forst, 2001; West & Stock, 2001). Our survey revealed that putative hybrid-type HKs were also present in four of 18 archaeal genomes, with Met. acetivorans containing five, and Halobacterium sp. NRC-1 containing three hybrid-type HKs, Met. mazei Goe1 containing two, and Arc. fulgidus DSM 4304 containing one. Among the Bacteria surveyed, the largest number of hybrid-type HKs were found in the Nostoc sp. PCC 7120 genome (49 of 122 putative HKs); followed by Bact. thetaiotaomicron (41 hybrid-type HKs of 85 putative HKs). By proportion, Chl. tepidum was found to contain the largest percentage of hybrid-type HKs (six of eight HKs; 75 %), followed by the Cox. burnetii genome (four of seven HKs; 57 %) (Table 1).


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Table 1. List of prokaryotic genomes with hybrid-type HKs

 
Among the prokaryotic genomes containing hybrid-type HKs, more than half possessed fewer than five hybrid-type HKs. However, 26 bacterial species were identified that contained unexpectedly large numbers of hybrid-type HKs (more than 10 in each genome or more than 25 % of all HKs in the genome) (Table 1). This group of species with enriched hybrid-type HKs consists of Bact. thetaiotaomicron and Bra. japonicum involved in bacterial–eukaryotic interactions (Xu et al., 2003, 2004; Hagiwara et al., 2004), photosynthetic cyanobacteria, such as Nostoc sp. PCC 7120 (Kaneko et al., 2001) and Synechocystis sp. PCC 6803 (Kaneko et al., 1996), and several bacterial species that possess versatile metabolic capabilities, such as Geo. sulfurreducens, which is capable of both anaerobic and aerobic respiration, one-carbon and complex carbon metabolism, motility and chemotactic behaviour (Methe et al., 2003), and Des. vulgaris, with an extraordinary ability to reduce (and bioremediate) multiple pollutants, including uranium and chromium (Heidelberg et al., 2004).

Phylogenetic analysis of kinase and receiver domains of hybrid-type HKs
The finding that hybrid-type HKs were unevenly distributed across microbial species leads to several immediate questions. First, did all hybrid-type HKs share the same ancestor or were they formed as the result of lateral events independently occurring in each species under specific selective pressure? Second, in 26 bacterial genomes with large numbers of hybrid-type HKs, what evolutionary mechanism was involved in their expansion? Is the same mechanism shared by all species? To address these questions, independent phylogenetic analyses were performed using sequences of functional kinase domains from HKs, and using the functional receiver domains from RRs, respectively. To help define the phylogenetic subfamilies to which these domains belong, the functional domains of all known TCSTSs from E. coli and Bac. subtilis were also extracted and included in the phylogenetic analysis. Phylogenetic trees of kinase and receiver domains were generated for each species from the aligned sequences and the confidence of the tree topology was evaluated.

In an earlier study (Koretke et al., 2000), the phylogeny constructed from a limited number of genomes showed that hybrid-type HKs were clustered together in one clade. In addition, they all shared the same root in the phylogenetic tree, implying that the hybrid-type HKs may have been generated before the divergence of microbial species, and that the kinase and receiver domains then evolved as a single unit into the present-day hybrid-type HKs. The group was thus conveniently named as ‘Hybrid’ phylogenetic group (Pao & Saier, 1997; Koretke et al., 2000).

In this study, we included all the kinase and receiver domains from the hybrid-type HKs we identified, and were therefore able to perform phylogenetic analyses in more detail. The domains were assigned to several known phylogenetic subfamilies (Cit, Nar, Ntr, Pho and Hybrid groups) according to their clustering characteristics. For those domains not showing a clear clustering pattern with any of the above subfamilies, they were classified as the ‘Other’ category. The results showed that although most of the kinase and receiver domains belonged to the ‘Hybrid’ phylogenetic subfamily, some of them were clustered into the Ntr phylogenetic subfamily (containing systems regulating nitrogen assimilation, acetoacetate metabolism and hydrogenase activity in E. coli) (Stoker et al., 1989), and the Pho phylogenetic subfamily (containing systems involved in phosphate regulation, virulence, osmoregulation and anaerobic nitrite reduction in E. coli) (Stock et al., 1989), suggesting that the members of hybrid-type HKs could be phylogenetically different.

Further analysis showed that the kinase and receiver domains from the same hybrid-type HKs were not necessarily located in the corresponding phylogenetic subfamily. For example, in Nostoc sp. PCC 7120, 49 kinase domains were clustered into Cit (1, number of kinase domains), Ntr (11), Pho (4), ‘Hybrid’ (29) and ‘Other’ (4) subfamilies, while its 59 receiver domains were clustered into Ntr (21), Pho (1), ‘Hybrid’ (32) and ‘Other’ (5) subfamilies, respectively (Table 2). In Bact. thetaiotaomicron all 41 kinase domains were clustered into the ‘Hybrid’ clade, but only 8 of the receiver domains located to their cognate phylogenetic clade, while the other 33 actually belonged to the Pho phylogenetic subfamily (Table 2). Examination of our data suggests that species from the same genus may have similar, but not identical, patterns of their HK and receiver domains in term of the subfamilies to which they belong. This is exemplified in the cases of Xan. axonopodis and Xan. campestris, or of Ps. aeruginosa, Ps. putida and Ps. syringae. Differences become even more obvious when comparing species across higher classification groups to determine to which subfamilies their domains belong. For example, two cyanobacteria have very different phylogenetic origination patterns for their kinase and receiver domains: in Nostoc sp. PCC 7120 the HK and receiver domains are mainly from the ‘Hybrid’ and Ntr phylogenetic groups, while those in Synechocystis sp. PCC 6803 are mainly from the ‘Hybrid’ and Pho phylogenetic groups (Table 2). These results suggest that hybrid-type HKs might not have originated from a common ancestor and that domain recruitment events occurred as lateral events during evolution. In addition, we found that some of the hybrid-type HKs contain more than one receiver domain (Table 2).


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Table 2. Phylogenetic origin of kinase and receiver domains of hybrid HKs from bacteria

 
Several types of bacteria with more complex metabolic activities and/or complex cell–cell interactions possessed a larger number of hybrid-type HKs. To explore the evolutionary mechanism operating in these bacteria, detailed phylogenetic analysis of kinase and receiver domains from each species was performed. The results from Bra. japonicum are presented as an example (Fig. 2). There are 28 hybrid-type HKs in Bra. japonicum and domain phylogeny analysis showed that they can be divided into five categories: Ntr (kinase domain)–Hybrid (receiver domain), Hybrid–Hybrid, Hybrid–Cit, Hybrid–Ntr and an Unknown–Unknown domain combination. The Ntr–Hybrid, Hybrid–Hybrid and Hybrid–Cit types were found to be the major categories and contained 14, six and six hybrid-type HKs, respectively (Fig. 2). Most hybrid-type HKs in the same category shared very high sequence similarity for both their kinase and receiver domains. This implies that each category might be a result of a single domain recruitment event at an early evolutionary stage, and that the genes for the hybrid proteins were then duplicated and mutated to provide new functional specializations (Fig. 2). Using the same approach, phylogenetic analysis of the genomes with large numbers of hybrid-type HKs, including Nostoc sp. PCC 7120, Cau. crescentus CB15 and V. vulnificus CMCP6, was performed and the results showed that a similar mechanism was operating in the expansion of hybrid-type HKs in these species as well.



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Fig. 2. Phylogenetic analysis of the kinase and receiver domains from Bra. japonicum. Different phylogenetic groups are indicated by colours and labels beside the clades. The definition of each phylogenetic subfamily was according to Koretke et al. (2000). The domain sequences isolated from E. coli are those with protein ID starting with ‘Ec’, followed by protein ID. The domain sequences isolated from Bac. subtilis are those with protein ID starting with ‘Bs’, followed by protein ID. The domain sequences isolated from Bra. japonicum are those with protein ID starting with ‘BLL’ or ‘BLR’, followed by protein ID. The kinase and receiver domains located in the same hybrid-type HKs in Bra. japonicum are indicated by lines linking the phylogenetic trees. The scale bar represents 0·1 expected amino acid replacements per site.

 
Evolution of HPT proteins/domains
With both kinase and receiver domains in a single protein, one obvious question is whether hybrid-type HKs can act directly on target proteins in a manner similar to the chemotaxis paradigm (Koretke et al., 2000). However, the analysis of protein sequences of hybrid-type HKs showed that, except in Bact. thetaiotaomicron, where 32 hybrid-type HKs were found to contain a putative DNA-binding domain in addition to the kinase and receiver domains and might be able to accomplish all the signal transduction processing within one protein (Xu et al., 2004), no DNA-binding domain was detected in any hybrid-type HKs from other bacterial species, suggesting that there must be intermediate proteins that can transfer signal to their cognate RRs. Although the possibility cannot be excluded that the hybrid-type HKs can also work other types of intermediate proteins, so far almost all hybrid-type HKs from both prokaryotic and eukaryotic sources worked with an intermediate HPT protein/domain, which mediates phosphotransfer reaction from the receiver domain in hybrid-type HKs to their cognate RRs (Kato et al., 1997; Chang & Stewart, 1998; West & Stock, 2001; Catlett et al., 2003) (Fig. 1). A total of 67 HPT domains were identified from hybrid-type HKs by SMART using an E value of <0·1. These sequences, along with previously identified bacterial HPT proteins, were used as queries to search multiple protein databases using an E value threshold of <0·01. The approach identified 26 separate HPT proteins from 156 prokaryotic genomes (Table 1). The results showed that among the 56 genomes with hybrid-type HKs, only 32 possessed HPT(s). Surprisingly, the genomes without any identifiable HPTs included those with a large number of hybrid-type HKs, such as Bact. thetaiotaomicron and Cau. crescentus. In addition, the number of HPTs found in each individual genome, ranging from one to eight, is smaller than that of hybrid-type HKs, and no evidence of significant duplication of HPTs was found even in the genomes where hybrid-type HKs were significantly duplicated (Table 1).

Sequence alignments of HPT domains from hybrid-type HKs or from separate HPT proteins were performed using the CLUSTALW program (Higgins & Sharp, 1988). The result showed that overall sequence similarity between various HPTs was low. The relatively conserved region was a region of approximately 30 aa in length starting from the forty-first amino acid in the N termini (the number shown in the case of TLR0349 from The. elongatus) (Fig. 3), consistent with an early study with a limited number of HPT sequences (Rodrigue et al., 2000). Histidine H44 is the only residue conserved through all HPTs, although several other residues, lysine K47, glycine G48, glycine G54 and glutamic acid E66 were also conserved in most HPTs (the number shown in the case of TLR0349) (Fig. 3). Phylogenetic analyses were conducted with the HPTs identified (Fig. 4). Although the HPT domains/proteins showed less than 20 % sequence identity, several recognizable clusters can be identified. However, no obvious correlation between the distribution of HPTs in each cluster and their taxonomic relationship was found, suggesting that the sequence diversification resulted mainly from specialization of function rather than bacterial speciation. All HPTs shared a single root in the phylogenetic tree, suggesting that there is a common ancestor for HPTs. This result is consistent with a previous observation that all HPTs share a common structural motif and active site (Kato et al., 1997; Xu & West, 1999).



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Fig. 3. Sequence alignments of HPT domains of prokaryotic sources. (a) Alignment of sequences of HPT domains located within hybrid-type HK protein. (b) Alignment of sequences of HPT domains located in separate HPT proteins. The sequences are indicated by their protein ID and species name. The conserved residues are shaded.

 


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Fig. 4. Phylogenetic analysis of HPT domains/proteins. The domain sequences are indicated by protein ID, followed by species name. The HPTs present as a separate protein are indicated by an S in parentheses following their species names. Several obvious clusters were indicated by dashes beside the tree. The HPT proteins of eukaryotic sources are marked in bold.

 
The HPT proteins from eukaryotes were grouped as a single subgroup within one cluster of bacterial HPTs, rather than clustering as a sister group of bacterial HPT (Fig. 4). This indicates that HPTs are likely to have originated in bacteria and then later radiated into the eukaryotic lineage through horizontal gene transfer event(s) before the divergence of eukaryotic species. This finding is consistent with the previously proposed hypothesis that eukaryotic TCSTSs evolved through horizontal transfer of bacterial hybrid-type HKs (Pao & Saier, 1997; Koretke et al., 2000).

Phylogenetic analysis of additional sensory domains in hybrid-type HKs
The analysis showed that a fraction of prokaryotic hybrid-type HKs contained sensory domains (Table 1). Three types of sensory domains were most frequently found in these hybrid-type HKs: PAS domains that bind flat heterocyclic molecules such as haem and flavin and are involved in sensing energy-related environmental factors such as oxygen, redox potential or light (Taylor & Zhulin, 1999; Taylor et al., 1999); GAF domains involved in binding cyclic nucleotides (Aravind & Ponting 1997); and the HAMP domain that is often found in various HKs, adenylyl cyclases, methyl-binding proteins and phosphatases (Galperin et al., 2001). A total of 245 PAS domain sequences (mean of 65–100 aa in length) were identified from hybrid-type HKs in prokaryotes using the SMART program. These sequences were then used in the construction of a phylogenetic tree. To help in the classification and definition of each phylogenetic cluster, a few dozen PAS domains with known function, obtained from other bacterial sources, were also used in the phylogenetic tree construction as described previously (Taylor & Zhulin, 1999; Zhang & Shi, 2004). It is obvious from the phylogenetic analysis that, although individual exceptions are present and overall bootstrap support was not high, PAS domains extracted from hybrid-type HKs tend to be clustered based on their putative physiological function rather than taxonomic relationship (data not shown). This finding suggested that PAS domains with different functional specialties were recruited into hybrid-type HKs as lateral events.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although the multiple-step phosphorelay and hybrid HKs were found in both prokaryotes and eukaryotes, they were previously thought to be less common in prokaryotes than in eukaryotes (Uhl & Miller, 1996; Posas et al., 1996; Robinson et al., 2000; West & Stock, 2001; Oka et al., 2002; Catlett et al., 2003). In this paper, a detailed survey and phylogenetic analysis were performed using the sequences from 156 complete prokaryotic genomes. The results showed that more than one-third of the bacterial and archaeal genomes possessed hybrid-type HKs, demonstrating that multiple-step phosphorelay systems are substantially more common in prokaryotes than previously thought. Although the functions of these HKs are still unknown, the existence of large numbers of hybrid-type HKs certainly indicates that these enzymes play a significant role in multiple-step phosphorelay systems in prokaryotes.

Hybrid-type HKs were found selectively enriched in very diverse bacterial species, from photosynthetic cyanobacteria to various pathogenic bacteria (Table 1), indicating that multiple-step phosphorelay may have special signalling properties such that the evolution and expansion of this unique family of signalling molecules occurred in response to unique challenges that these bacteria faced. Compared with simple scheme of TCSTSs, multiple-step phosphorelay has been suggested to have three major advantages: (i) presence of kinase and receiver domains in one protein may constrain signal amplification, modularity or cross-talk between components of TCSTSs (Bijlsma & Groisman, 2003); (ii) because of the involvement of HPT, the mechanism provides greater versatility in signalling strategies and a greater number of potential sites for regulation (Grossman, 1995; Appleby et al., 1996); and (iii) the multiple phosphorylation sites of the phosphorelay could provide more junction points for communicating with other signalling pathways (Appleby et al., 1996).

Unlike HKs in other phylogenetic subfamilies, such as Pho and Ntr (Koretke et al., 2000), several observations emerging from this study suggest that there was no single ancestor for hybrid-type HKs. First, the survey of hybrid-type HKs across prokaryotic genomes showed that their distribution did not follow any taxonomic relationship; species with very close relationship could be quite different in terms of the total numbers and percentage of hybrid-type HKs. For example, Synechocystis sp. PCC 6803 contains 11 hybrid-type HKs, while Synechococcus sp. WH8102 has none. Second, independent phylogenetic analysis of kinase and receiver domains from hybrid-type HKs showed that hybrid-type HK may have kinase and receiver domains with different phylogenetic origins. Further support was also provided by the observation that hybrid-type HKs from the same species often have multiple combinations of individual kinase and receiver domains (based on their phylogenetic origins), indicating that lateral recruitment events were involved in the evolution of these proteins. The results demonstrated that domain recruitment followed by gene duplication may be responsible for the expanding of hybrid-type HKs in bacteria.

No correlation was found between the number of hybrid-type HKs and HPTs in prokaryotic genomes, which was consistent with a previous study in fungal genomes (Catlett et al., 2003). Even more interesting, 41 % of the prokaryotic genomes with hybrid-type HKs do not have any identifiable HPT sequences. This observation raises questions regarding the mechanism by which prokaryotic multiple-step phosphorelay systems function and how the specificity of signal transduction is being controlled. One plausible explanation might be that the prokaryotic systems are indeed functioning like the eukaryotic systems, but that most of the bacterial HPT proteins were not identified in this study because of low sequence similarity to known HPT proteins (Rodrigue et al., 2000). Another hypothesis is suggested by a mechanism that has been proposed for Arabidopsis HPTs involving chaperone-like proteins that associate with TCSTSs at the membrane/cytoplasm interface and/or guide the phosphorylated HPT into the nucleus or other subcellular compartments (Pawson & Scott 1997; Grefen & Harter, 2004). However, it is unclear whether there is any similar mechanism involved in guiding the specialization of phosphorylation in prokaryotes. Finally, although almost all known hybrid-type HKs appear to function in multiple-step phosphorelays, in which the phosphate is transferred from the receiver domain of the hybrid HK to a second His residue in an HPT domain and then to RR (Chang & Stewart, 1998; West & Stock, 2001; Catlett et al., 2003), it is still possible that phosphorelay may not be the only use of this architectural design and it is therefore possible that not all of the prokaryotic hybrid-type HKs are involved in multiple-step phosphorelay. An example of this is seen in Agr. tumefaciens, where the attached receiver domain of VirA, a transmembrane hybrid HK, functions as an autoinhibitory domain. In its unphosphorylated state, this receiver domain interacts with the transmitter module and prevents the transmitter from autophosphorylating and serving as a phosphodonor to its cognate response regulator VirG (Chang et al., 1996; Appleby et al., 1996).

In conclusion, this study presents a survey of the distribution and evolutionary analysis of the components involved in multiple-step phosphorelay in prokaryotes, and constitutes a basis for further exploration of their physiological functions.


   ACKNOWLEDGEMENTS
 
We would like to thank Drs David E. Culley and Brian H. Lower of Pacific Northwest National Laboratory for their critical reading of this manuscript. The research described in this paper was conducted under the LDRD Program at the Pacific Northwest National Laboratory, a multi-program national laboratory operated by Battelle for the US Department of Energy under Contract DE-AC06-76RLO1830.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Appleby, J. L., Parkinson, J. S. & Bourret, R. B. (1996). Signal transduction via the multi-step phosphorelay: not necessarily a road less traveled. Cell 86, 845–848.[CrossRef][Medline]

Aravind, L. & Ponting, C. P. (1997). The GAF domain: an evolutionary link between diverse phototransducing proteins. Trends Biochem Sci 22, 458–459.[CrossRef][Medline]

Bijlsma, J. J. E. & Groisman, E. A. (2003). Making informed decisions: regulatory interactions between two-component systems. Trends Microbiol 11, 359–366.[CrossRef][Medline]

Blumenberg, M. (1988). Concerted gene duplications in the two keratin gene families. J Mol Evol 27, 203–211.[Medline]

Bourret, R. B., Hess, J. F., Borkovich, K. A., Pakula, A. A. & Simon, M. I. (1989). Protein phosphorylation in chemotaxis and two-component regulatory systems of bacteria. J Biol Chem 264, 7085–7088.[Abstract/Free Full Text]

Burbulys, D., Trach, K. A. & Hoch, J. A. (1991). Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell 64, 545–552.[CrossRef][Medline]

Catlett, N. L., Yoder, O. C. & Turgeon, B. G. (2003). Whole-genome analysis of two-component signal transduction genes in fungal pathogens. Eukaryot Cell 2, 1151–1161.[Abstract/Free Full Text]

Chang, C. & Stewart, R. C. (1998). The two-component system. Regulation of diverse signaling pathways in prokaryotes and eukaryotes. Plant Physiol 117, 723–731.[Free Full Text]

Chang, C. H., Zhu, J. & Winans, S. C. (1996). Pleiotropic phenotypes caused by genetic ablation of the receiver module of the Agrobacterium tumefaciens VirA protein. J Bacteriol 178, 4710–4716.[Abstract/Free Full Text]

Clarke, D. J., Joyce, S. A., Toutain, C. M., Jacq, A. & Holland, I. B. (2002). Genetic analysis of the RcsC sensor kinase from Escherichia coli K-12. J Bacteriol 184, 1204–1208.[Abstract/Free Full Text]

Dutta, R., Qin, L. & Inouye, M. (1999). Histidine kinases: diversity of domain organization. Mol Microbiol 34, 633–640.[CrossRef][Medline]

Forst, S., Delgado, J. & Inouye, M. (1989). Phosphorylation of OmpR by the osmosensor EnvZ modulates expression of the ompF and ompC genes in Escherichia coli. Proc Natl Acad Sci U S A 86, 6052–6056.[Abstract/Free Full Text]

Galperin, M. Y., Nikolskaya, A. N. & Koonin, E. V. (2001). Novel domains of the prokaryotic two-component signal transduction systems. FEMS Microbiol Lett 203, 11–21.[CrossRef][Medline]

Grefen, C. & Harter, K. (2004). Plant two-component systems: principles, functions, complexity and cross talk. Planta 219, 733–742.[Medline]

Grossman, A. D. (1995). Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. Annu Rev Genet 29, 477–508.[CrossRef][Medline]

Hagiwara, D., Yamashino, T. & Mizuno, T. (2004). Genome-wide comparison of the His-to-Asp phosphorelay signaling components of three symbiotic genera of Rhizobia. DNA Res 11, 57–65.[Medline]

Heidelberg, J. F., Seshadri, R., Haveman, S. A. & 32 other authors (2004). The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. Nat Biotechnol 22, 554–559.[CrossRef][Medline]

Higgins, D. G. & Sharp, P. M. (1988). CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73, 237–244.[CrossRef][Medline]

Hoch, J. A. (2000). Two-component and phosphorelay signal transduction. Curr Opin Microbiol 3, 165–170.[CrossRef][Medline]

Kaneko, T., Sato, S., Kotani, H. & 21 other authors (1996). Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions (supplement). DNA Res 3, 185–209.[Medline]

Kaneko, T., Nakamura, Y., Wolk, C. P. & 19 other authors (2001). Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res 8, 205–213.[Medline]

Kato, M., Mizuno, T., Shimizu, T. & Hakoshima, T. (1997). Insights into multistep phosphorelay from the crystal structure of the C-terminal HPt domain of ArcB. Cell 88, 717–723.[CrossRef][Medline]

Kim, D. & Forst, S. (2001). Genomic analysis of the histidine kinase family in bacteria and archaea. Microbiol 147, 1197–1212.[Abstract/Free Full Text]

Koretke, K. K., Lupas, A. N., Warren, P. V., Rosenberg, M. & Brown, J. R. (2000). Evolution of two-component signal transduction. Mol Biol Evol 17, 1956–1970.[Abstract/Free Full Text]

Leonardo, M. R. & Forst, S. (1996). Re-examination of the role of the periplasmic domain of EnvZ in sensing of osmolarity signals in Escherichia coli. Mol Microbiol 22, 405–413.[CrossRef][Medline]

Letunic, I., Goodstadt, L., Dickens, N. J. & 7 other authors (2002). Recent improvements to the SMART domain-based sequence annotation resource. Nucleic Acids Res 30, 242–244.[Abstract/Free Full Text]

Marchler-Bauer, A., Anderson, J. B., DeWeese-Scott, C. & 24 other authors (2003). CDD: a curated Entrez database of conserved domain alignments. Nucleic Acids Res 31, 383–387.[Abstract/Free Full Text]

Methe, B. A., Nelson, K. E., Eisen, J. A. & 31 other authors (2003). Genome of Geobacter sulfurreducens: metal reduction in subsurface environments. Science 302, 1967–1969.[Abstract/Free Full Text]

Ogino, T., Matsubara, M., Kato, N., Nakamura, Y. & Mizuno, T. (1998). An Escherichia coli protein that exhibits phosphohistidine phosphatase activity towards the HPt domain of the ArcB sensor involved in the multistep His-Asp phosphorelay. Mol Microbiol 27, 573–585.[CrossRef][Medline]

Oka, A., Sakai, H. & Iwakoshi, S. (2002). His-Asp phosphorelay signal transduction in higher plants: receptors and response regulators for cytokinin signaling in Arabidopsis thaliana. Genes Genet Syst 77, 383–391.[CrossRef][Medline]

Pao, G. M. & Saier, M. H., Jr (1997). Nonplastid eukaryotic response regulators have a monophyletic origin and evolved from their bacterial precursors in parallel with their cognate sensor kinases. J Mol Evol 44, 605–613.[Medline]

Parkinson, J. S. & Kofoid, E. C. (1992). Communication modules in bacterial signaling proteins. Annu Rev Genet 26, 71–112.[CrossRef][Medline]

Pawson, T. & Scott, J. D. (1997). Signaling through scaffold, anchoring, and adaptor proteins. Science 278, 2075–2080.[Abstract/Free Full Text]

Posas, F., Wurgler-Murphy, S. M., Maeda, T., Witten, E. A., Thai, T. C. & Saito, H. (1996). Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 "two-component" osmosensor. Cell 86, 865–875.[CrossRef][Medline]

Rabus, R., Ruepp, A., Frickey, T. & 15 other authors (2004). The genome of Desulfotalea psychrophila, a sulfate-reducing bacterium from permanently cold Arctic sediments. Environ Microbiol 6, 887–902.[CrossRef][Medline]

Robinson, V. L., Buckler, D. R. & Stock, A. M. (2000). A tale of two components: a novel kinase and a regulatory switch. Nat Struct Biol 7, 626–633.[CrossRef][Medline]

Rodrigue, A., Quentin, Y., Lazdunski, A., Méjean, V. & Foglino, M. (2000). Cell signalling by oligosaccharides. Two-component systems in Pseudomonas aeruginosa: why so many? Trends Microbiol 8, 498–504.[CrossRef][Medline]

Stephenson, K. & Hoch, J. A. (2002). Evolution of signalling in the sporulation phosphorelay. Mol Microbiol 46, 297–304.[CrossRef][Medline]

Stock, J. B., Ninfa, A. J. & Stock, A. M. (1989). Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev 53, 450–490.[Medline]

Stoker, K., Reijnders, W. N., Oltmann, L. F. & Stouthamer, A. H. (1989). Initial cloning and sequencing of hydHG, an operon homologous to ntrBC and regulating the labile hydrogenase activity in Escherichia coli K-12. J Bacteriol 171, 4448–4456.[Medline]

Takeda, S., Fujisawa, Y., Matsubara, M., Aiba, H. & Mizuno, T. (2001). A novel feature of the multistep phosphorelay in Escherichia coli: a revised model of the RcsC->YojN->RcsB signalling pathway implicated in capsular synthesis and swarming behaviour. Mol Microbiol 40, 440–450.[CrossRef][Medline]

Taylor, B. L. & Zhulin, I. B. (1999). PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol Mol Biol Rev 63, 479–506.[Abstract/Free Full Text]

Taylor, B. L., Zhulin, I. B. & Johnson, M. S. (1999). Aerotaxis and other energy-sensing behavior in bacteria. Annu Rev Microbiol 53, 103–128.[CrossRef][Medline]

Uhl, M. A. & Miller, J. F. (1996). Integration of multiple domains in a two-component sensor protein: the Bordetella pertussis BvgAS phosphorelay. EMBO J 15, 1028–1036.[Abstract]

West, A. H. & Stock, A. M. (2001). Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem Sci 26, 369–376.[CrossRef][Medline]

Xu, Q. & West, A. H. (1999). Conservation of structure and function among histidine-containing phosphotransfer (HPt) domains as revealed by the crystal structure of YPD1. J Mol Biol 292, 1039–1050.[CrossRef][Medline]

Xu, J., Bjursell, M. K., Himrod, J., Deng, S., Carmichael, L. K., Chiang, H. C., Hooper, L. V. & Gordon, J. I. (2003). A genomic view of the human–Bacteroides thetaiotaomicron symbiosis. Science 299, 2074–2076.[Abstract/Free Full Text]

Xu, J., Chiang, H. C., Bjursell, M. K. & Gordon, J. I. (2004). Message from a human gut symbiont: sensitivity is a prerequisite for sharing. Trends Microbiol 12, 21–28.[CrossRef][Medline]

Zhang, W. & Shi, L. (2004). Evolution of the PPM-family protein phosphatases in Streptomyces: duplication of catalytic domain and lateral recruitment of additional sensory domains. Microbiology 150, 4189–4197.[CrossRef][Medline]

Received 22 February 2005; revised 5 April 2005; accepted 18 April 2005.



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