Genome-wide comparison of the His-to-Asp phosphorelay signaling components of three symbiotic genera of Rhizobia

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1 DNA Research 11, (2004) Short Communication Genome-wide comparison of the His-to-Asp phosphorelay signaling components of three symbiotic genera of Rhizobia Daisuke Hagiwara, Takafumi Yamashino, and Takeshi Mizuno Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Aichi , Japan (Received 7 November 2003; revised 28 November 2003) Abstract Histidine-to-aspartate (His-Asp) phosphorelay (or two-component) systems are very common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli. The His-Asp phosphorelay components include sensor histidine kinase (HK), phosphotransfer intermediate (HPt), and response regulator (RR). With special reference to three bacterial species (Mesorhizobium loti, Bradyrhizobium japonicum, Sinorhizobium meliloti), each of which belongs to a different genera of Rhizobia, here we attempted to compile all of the His-Asp phosphorelay components in order to reveal a comparative genome-wide overview as to the His-Asp phosphorelay. It was revealed that M. loti has 47 HKs, 1 HPts, and 58 RRs; B. japonicum has 80 HKs, 3 HPts, and 91 RRs; whereas S. meliloti has 40 HKs, 1 HPt, and 58 RRs. These His-Asp phosphorelay components were extensively compiled and characterized. The resulting overview as to the His-Asp phosphorelay of Rhizobia will provide us with a basis for understanding of the fundamental mechanisms underlying interactions between plants and microorganisms (including symbiosis), as well as nitrogen fixation. Key words: phosphorelay signal transduction; histidine-kinase; response regulator; Mesorhizobium loit; Bradyrhizobium japonicum; Sinorhizobim meliloti Histidine-to-aspartate (His-Asp) phosphorelay (or twocomponent) systems are very common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli. 1 To date, a number of instances of such His-Asp phosphorelay systems have been uncovered not only in many prokaryotic species but also in certain eukaryotic species, including higher plants. 2 A classical His-Asp phosphorelay system consists of two types of common signal transducers, a sensor kinase (HK) that exhibits a histidine (His)- kinase activity and a response regulator (RR) containing a phospho-accepting aspartate (Asp) in its receiver domain. 3 A crucial event underlying this signal transduction mechanism is a His-Asp phosphorelay from an HK to its cognate RR. In some sophisticated cases, however, a histidine-containing phosphotransfer (HPt) factor plays an essential role as a phosphotransfer intermediate of phosphorelay. 4 In this case, a phosphoryl group moves from an HK to a receiver, then to an HPt, and finally to another receiver in a given phosphorelay pathway. 5 Such His-Asp-His-Asp signaling is often referred to as a Communicated by Satoshi Tabata To whom correspondence should be addressed. Tel , Fax , tmizuno@agr. nagoya-u.ac.jp multistep phosphorelay. When the entire nucleotide sequence of the model bacterium Escherichia coli (Gramnegative bacterium) has been determined, it was revealed that this prokaryotic microorganism has 29 HKs and 32 RRs. 6 Similarly, Bacillus subtilis (Gram-positive bacterium) has 33 HKs, 7 and Synechocystis PCC 6803 (unicellular cyanobacterium) has 42 HKs, 8 whereas Pseudomonas aeruginosa has as many as 63 HKs. 9 It should thus be emphasized again that the His-Asp phosphorelay is one of the most common and evolutionarily conserved molecular tactics for the intracellular signal transduction in response to a variety of external stimuli in bacteria. It is thus of interest to compile such His-Asp phosphorelay components for Rhizobia, which contains a genera of α-proteobacteria, such as Rhizobium, Sinorhizobium, Mesorhizobium, and Bradyrhizobium. The reason for this is twofold. They are soil and rhizosphere bacteria of agronomic importance because they form nitrogen-fixing symbioses with leguminous plants, and therefore they must have evolved common strategies of signal transduction because nodule formation and the subsequent nitrogen-fixation involve a series of interactions between these symbiotic bacteria and host plants. Note also that these species are relatively divergent from a phy-

2 58 His-to-Asp phosphorelay components in Rhizobia [Vol. 11, logenetic viewpoint, and each of them displays distinctive host specificities. Taking advantage of the available genome sequences of three species (Mesorhizobium loti, Bradyrhizobium japonicum, Sinorhizobium meliloti), each belonging to a different genera of Rhizobia, here we attempted to compile all of the His-Asp phosphorelay components in order to provide a comprehensive genomewide overview as to the His-Asp phosphorelay. Rationale of analyses In the current databases (RhizoBase, the entire nucleotide sequences of the genomes of M. loti, B. japonicum, and S. meliloti are available, each of which includes nicely characterized genome organizations and a set of useful search tools. In these databases, the His-Asp phosphorelay components are denoted as twocomponent sensor kinase, two-component response regulator probable sensor/regulator hybrid proteins, and response sensor protein. However, they were classified into the gene category list of regulatory functions, collectively together with many other gene products with regulatory functions that are not apparently relevant to the His-Asp phosphorelay components. At first glance, therefore, we noticed that these classifications and annotations do not necessarily provide us with a unified picture as to the His-Asp phosphorelay systems in these interesting symbiotic microorganisms. Furthermore, some of the computer-aided annotations in the databases appear to be misleading. We thus decided to invistigate the comparative and comprehensive pictures of the His-Asp phosphorelay components of M. loti, B. japonicum, and S. meliloti, more intensively and precisely. A typical histidine kinase (HK) domain of about 240 amino acids contains certain conserved short stretches of amino acids, consisting of two distinctive functional domains. They are referred to as HisKA [histidine kinase A (phosphoacceptor) domain] and HATPase c [histidine kinase-like ATPase domain], respectively. For these, see the Simple Modular Architecture Research Tool (SMART) ( motifs.pl). The former domain contains a phospho-accepting invariant histidine residue, and the latter serves as a nucleotide-binding catalytic domain. A typical receiver domain of about 120 amino acids, commonly found in RRs, also contains several invariant amino acid residues in a characteristic context of amino acid sequences. The receiver domains are referred to as REC (or response reg) [response regulator receiver domain] in the SMART classification. A typical HPt domain is most difficult to recognize, because the amino acid sequences are quite variable. However, they contain an invariant histidine residue, which is crucial for the phosphotransfer between two given receiver domains. Such an HPt domain is referred to as HPT in the SMART classification, which allows us to identify the HPt domian in a given protein. In addition to the SMART program, we adopted the Pfam program (Protein families database of alignments and HMMS), which also allows us to search for predicted open reading frames (ORFs) that contain either one of the HisKA, HATPase c, REC, or HPT ( Adopting these programs, we inspected every possible ORF to gain a comprehensive picture as to the His-Asp phosphorelay components of M. loti, B. japonicum, and S. meliloti. The resulting candidate ORFs were further inspected one by one with the BLAST program (Basic Local Alignments Search TOOL) to confirm whether they indeed contain each one of the characteristic domains ( The results are summarized in Fig. 1 (M. loti), Fig. 2 (B. japonicum), and Fig. 3 (S. meliloti), respectively. Overview of His-Asp phosphorelay components In Figs. 1, 2, and 3, the uncovered ORFs encoding any one of the His-Asp phosphorelay components were tentatively classified as follows (see the inset panel in Fig. 1). Histidine kinases (HKs) were classified into three types. An authentic HK contains a single Hiskinase domain consisting of HisKA and HATPase c. M. loti ORF mlr0397 is such an example (designated as mlr0397-h in Fig. 1). A hybrid HK contains both a His-kinase domain and a receiver domain (REC), represented by M. loti ORF mll0767. In Figs. 1, 2, and 3, these hybrid HKs were denoted by ORF-ID-hybrid(h)H, and they are highlighted by blue letters. In some cases, two REC domains were found in either the N-terminal or C-terminal end of such hybrid HKs (e.g., mll2385- hh, mll2385, mll3725, mlr3786, mlr6689, blr2287, and SMb20356). One instance (bll2599) contains three receiver domains in a single molecule. The last classification is CheA-type HKs that have a structural design very similar to E. coli CheA sensor involved in chemotaxis. M. loti ORF mll9511 is such an example (designated as mll9511-chea). These CheA-type HKs are easily recognized as judged by their unique structural design, each of which contains an HPt domain at its N-terminal end, followed by a HATPase c domain. In principle, HKs could be classified into more complicated subgroups based on their phylogenetic relationships. 1 To make this overview concise and clear, however, we did not classify these HKs any further. Only one HPt factor was found in M. loti (designated as mll6695- HPt). Response regulators (RRs) were classified into six subgroups (NtrC-family, NarL-family, OmpR-family, CheY-family, unique CheB-family, and unclassified ones). In general, a common REC domain in a given RR is followed by a C-terminal domain, which often acts as a DNA-binding transcriptional regulator. In E. coli, NtrC, NarL, and OmpR are such typical examples of RRs. 13 However, they are distinctive from each other, as judged by their C- terminal amino acid sequences. Indeed, most of the RRs identified in this study are classified into the NtrC-family [e.g., mlr0398-r(c)], NarL-family [e.g., mll0990-r(l)], or

3 No. 1] D. Hagiwara et al. 59 Figure 1. A list of His-Asp phosphorelay components of M. loti. ORFs (designated as mll-series and mlr-series), each encoding one of the His-Asp phosphorelay components, are listed along with the schematically illustrated chromosome and pmlb. The practical designations for these components are summarized in the inset panel (details are given in the text). If a pair of HK/RR was located closely on the genomes, they are placed together. If the orthologous HKs and/or RRs were found both in B. japonicum and S. meliloti, they are highlighted in red (see also Fig. 5). Hybrid HKs and HPt are indicated by blue and green colors, respectively. The symbiosis island is indicated by shading with green color. Note also that we inspected the nucleotide sequence of the symbiosis island of the close relative M. loti (R7A), and the results are shown as an appendix of this figure.

4 60 His-to-Asp phosphorelay components in Rhizobia [Vol. 11, Figure 2. The list of His-Asp phosphorelay components in B. japonicum. ORFs (designated as bll-series and blr-series), each encoding either one of His-Asp phosphorelay components, are listed along with the schematically illustrated chromosome. The practical designations for these components are those summarized in the inset panel of Fig. 1. If the orthologous HKs and/or RRs were found both in M. loti and S. meliloti, they are highlighted in red. The symbiosis islands (referred to as A and B) are indicated by green shading. Other details are the same as those given in Fig. 1. reasonably assumed that mll9510-r(b) is implicated, together with mll9511-chea, in the chemotaxis response in M. loti. Based on these practical classifications and designations, here one can easily see: (i) how many HKs and RRs are present in M. loti (Fig. 1), B. japonicum (Fig. 2), and S. meliloti (Fig. 3); (ii) where they are located on the chromosomes; (iii) to which family a given RR belongs; and (iv) whether or not they are closely lo- OmpR-family [e.g., mlr1334-r(r)]. However, like E. coli CheY, some RRs contain only the REC domain without any C-terminal extension [e.g., mll0861-r(y)]. Remaining RRs could not be classified into these typical types of RRs, thus referred to as, for instance, mll0859-r(x). Note that RRs homologous to E. coli CheB are unique in that they have a C-terminal methyltransferase domain, preceded by an REC domain. M. loti has such a CheB homolog on pmlb [designated as mll9510-r(b)]. It is

5 No. 1] D. Hagiwara et al. 61 Figure 3. The list of His-Asp phosphorelay components in S. meliloti. ORFs (designated as SMc-series, Sma-series, and SMb-series), each encoding one of the His-Asp phosphorelay components, are listed along with the schematically illustrated chromosome, psyma, and psymb. Other details are the same as those given in Fig. 1.

6 62 His-to-Asp phosphorelay components in Rhizobia [Vol. 11, Figure 4. The collective list of His-Asp phosphorelay components among the three species belonging to Rhizobia. Numbers of histidine kinases (HKs) and HPt factors (HPt), and response regulators (RRs), found in each species, are summarized (each left hand side panel). Sixteen sets of orthologous HKs were found among these species (panel at right hand side). Note that a few HKs were found redundantly in the same species (e.g., mlr5841/mlr7238) (for details, see Fig. 5). Note that there are many HKs unique to a given species (e.g., 20 unique HKs in M. loti, 54 unique HKs in B. japonicum, and 16 unique HKs in S. meliloti). cated on the chromosomes (e.g., an inferred functional pair of HK/RR). His-Asp phosphorelay components in M. loti M. loti has 47 HKs, including 11 hybrid HKs. These HKs are all located on the chromosome, except for mll9511 on pmlb (Figs. 1 and 4). This species has 58 RRs (5 members in NtrC-family, 12 in NarL-family, 23 in OmpR-family, 12 in CheY-family, and one CheB homolog) (Fig. 1). There is no relevant gene on pmla, whereas pmlb contain 5 RRs. As mentioned earlier, M. loti has only one gene, which most likely encodes an HPt factor (mll6695-hpt). On the genome, there are 30 pairs of HK/RR (e.g., mll0983- R(l)/mll0984-H), each pair of which is most likely implicated together in a certain signaling system. Among these, there is a cluster of genes in pmlb, which together are most likely responsible for chemotaxis, as mentioned above. M. loti has a symbiosis island of about 500 kb at the coordinate of about 5 Mb on the chromosome, in which a pair of HK/RR, two orphan RRs, and one orphan HK were found (see the greenish region in Fig. 1). The nucleotide sequences of such symbiosis islands have also been determined for some close relatives. 14 The entire nucleotide sequence of the symbiosis island of M. loti (R7A) is available (see RhizoBase, mentioned above). In this region of M. loti (R7A) too, a set of HK and RR genes are present, all of which are very homologous to those found in M. loti (MAFF303099) (see appendix in Fig. 1). His-Asp phosphorelay components in B. japonicum B. japonicum has 80 HKs including 30 hybrid HKs (Figs. 2 and 4). Three CheA-like HK homologs were found, which might be implicated in chemotaxis. This species has 91 RRs (6 members in NtrC-family, 27 in NarL-family, 22 in OmpR-family, 29 in CheY-family including two CheB homologs) (Fig. 2). Notably, there is a unique RR in B. japonicum, in that this RR (blr2864) encodes a response regulator, followed by a putative adenylate cyclase domain. In this respect, it would also be noteworthy that a hybrid HK (blr2288-hh) encodes a sensor containing a putative adenylate cyclase domain at its C-terminus. Note also that this HK appears to be defective in that it contains a HisAK domain, but not a HATPase c domain. B. japonicum has three genes, each of which most likely encodes an HPt factor. For instance, bll5886-hpt might be a component of the His-Asp phosphorelay system, in concert with a pair of bll5888- R(l)/bll5889-hH. There are 46 pairs of HK/RR, both of which are co-localized next to each other (or closely) on the chromosome. B. japonicum also has the symbiosis islands (referred to as A and B) on the chromosome (see the greenish regions in Fig. 2). Interestingly, HKs and RRs found in this region are not analogous to those found in the symbiosis island of M. loti (see also the greenish region in Fig. 1). If a certain His-Asp phosphorelay system(s) is crucial commonly for these symbiotic species, the corresponding genes might be located outside of the symbiosis islands. In any event, the content of the phosphorelay-associated genes (174 ORFs) in the whole B. japonicum genes (8317 ORFs) turned out to be strikingly high (approximately 2%). His-Asp phosphorelay components in S. meliloti This species has 40 HKs, including 8 hybrid HKs (Figs. 3 and 4). Two CheA-like HK homologs were found, one of which is on the chromosome, and the other is in psyma.

7 No. 1] D. Hagiwara et al. 63 Figure 5. A summarized list of orthologous sets of HKs commonly found among three species belonging to Rhizobia. Sixteen sets of orthologous HKs are listed with serial numbers. The numbers in parentheses indicated the amino acid residues. The location of these orthologous HKs on the genome are indicated with the same serial numbers as in Figs. 1, 2, and 3. Characteristics relevant to each orthologous set of HKs are also noted at the right hand side (with regard to each reference for the remarks, and each abbreviation for the bacterial species, see the text). S. meliloti has 58 RRs (5 members in NtrC-family, 12 in NarL-family, 17 in OmpR-family, 15 in CheY-family including two CheB homologs) (Fig. 3). Notably, these HK and RR genes are located evenly throughout the chromosome and two large plasmids (psyma and psymb) (Fig. 4). Again, many pairs of HK/RR were found (31 pairs). In S. meliloti, psyma is thought to be mainly involved in symbiosis. However, no characteristic feature as to the His-Asp phosphorelay components in psyma was revealed, as compared with those in the symbiosis islands in M. loti and B. japonicum. Comparative analyses of HKs amongst three genera of Rhizobia Among the His-Asp phosphorelay components, HKs are most characteristic because each HK serves as a sensor for each specific environmental stimulus. To gain more specific insight into the His-Asp phosphorelay networks in these symbiotic microorganisms, it is thus of interest to compare the amino acid sequences of HKs with each other, particularly, with regard to those signal-input domains outside of the common HK domains. We inspected all HKs of these three species, one by one, in order to access whether a given HK of M. loti is orthologous to any one of B. japonicum HKs, or whether they are just paralogous. Because considerable similarity was observed throughout the entire amino acid sequences including not only the HK domains but also the signalinput domains, they were classified as orthologs. Such intensive analyses revealed a surprising result, as shown in Fig. 4 (see panel at right hand side). Only 16 orthologous sets of HKs were revealed amongst three species. These orthologous sets of HKs that are highlighted by red color, and they were coordinately numbered in Figs. 1, 2 and 3. It was revealed that the inferred orthologous sets of HKs were located on each chromosome in a divergent manner with regard to both the positions and orders on the chromosomes, suggesting that the genome organizations of these related genera has been highly diversified. In addition, some orthlogous HKs were found only in two out of three species. In other words, many HKs are unique to a given species (e.g., among 40 HKs of S. meliloti, 16 of them were found only in this particular species). In any event, these orthologous HKs are most likely implicated in certain His-Asp phosphorelays, which must be common in these species. Common HKs among three genera The orthologous sets of HKs among these genera of Rhizobia are summarized in more detail in Fig. 5, which provided us with several insights. For instance, an or-

8 64 His-to-Asp phosphorelay components in Rhizobia [Vol. 11, thologous set of mlr0397-h/blr4487-h/smc0104 appears to act as sensors in a signaling pathway common to these species. This view was further strengthened by the fact that each of them makes a pair with a certain RR partner, mlr0397-h/mlr0398-r(c)/blr4487- H/blr4488-R(c)/SMc01042-H/SMc01043(c), and these RRs are also orthologous to each other. This view is generally true for the other orthologous sets listed here. Thus, these His-Asp phosphorelay systems had been evolutionarily conserved among three divergent genera of Rhizobia (belonging to α-proteobacteria). As listed in Fig. 5, it is further noteworthy that three HKs are conserved even in E. coli (belonging to γ-proteobacteria). M. loti mlr0397-h is orthologous to E. coli NtrB (or NRII) that is involved in nitrogen regulation, 15 M. loti mll3127-h is orthologous to E. coli KdpD that is involved in potassium transport regulation, 16 whereas M. loti mll9511-chea is analogous to E. coli CheA that is crucial for chemotaxis. 17 As mentioned earlier, one of the most characteristic features of Rhizobia is the nitrogen-fixing symbiosis. Four orthologous sets of HKs are clearly relevant to nitrogen fixation regulation: NtrB (nitrogen regulation), 15 NtrY (nitrogen regulation), 18 RegS (nitrogen fixation regulation), 19 and FixL (oxygen sensor), 20 which have already been characterized to some extent in Rhizobia. In addition, C4- dicarboxyate transport sensor DctB also appears to be conserved in three species. 21 For some other sets of HKs, highly homologous HKs have been identified in other bacterial species: (tyrosine kinase DivL of Caulobactor crescentus), 22 (ph sensor ChvG of Agrobacterium tumefaciens), 23 (putative transporter-like proline sensor of Aeromonas hydrophila), and (C1-metabolite sensor FlhS of Paracoccs denitrificans), 24 as shown in Fig. 5. With other sets of HKs, no information is available so far. However, most of them are conserved also in the distantly related α-proteobacterial genera (Agrobactrium tumefaciens) (as also indicated in Fig. 5). 25 Notably, HKs highly homologous to mll2385-hh/bll2599-hh are found in some eukaryotic species, DnkJ of Dictyosterium discoideum 26 and Nik-1 of Neurospora crassa. 27 In addition, blr2288-hh/smb20358-hh are unique in that they contain a putative adenylate cyclase domain at their C- terminus. However, note that these HKs appear to be defective in that each contains a HisAK domain but not a HATPase c domain. In any case, future analyses of these common orthologous sets of HKs might be promising for understanding crucial mechanisms underlying signal transduction in response to certain environmental stimuli, which is characteristic for these genera of Rhizobia. In short, the compiled genome-wide overview as to the His-Asp phosphorelay components of Rhizobia will provide us with a crucial basis for understanding the fundamental mechanisms underlying the interaction between plants and microorganisms (including symbiosis), as well as nitrogen fixation. This study was supported by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan (a priority area of , and COE), and also the Ministry of Agriculture, Forestry and Fisheries of Japan. References 1. Hoch, J. A. and Silhavy, T. J. 1995, Two-component signal transduction, ASM Press, Washington, D. 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