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Original Paper	Pdf file on Synergy omments
Acta Biochim Biophys Sin 2007, 39: 317-325
doi:10.1111/j.1745-7270.2007.00282.x

Comparative Analysis of Two-component Signal Transduction System in Two Streptomycete Genomes

Wu WEI^1,3#, Weihua WANG^2#, Zhiwei CAO^3#, Hong YU³, Xiaojing WANG¹, Jing ZHAO³, Hao TAN³, Hao XU³, Weihong JIANG²*, and Yixue LI^1,3*

1 Bioinformation Center, Key Lab of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China;

2 Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200032, China;

3 Shanghai Center for Bioinformation Technology, Shanghai 200235, China

Received: November 29, 2006������

Accepted: February 27, 2007

This study was supported by the grants from the National Natural Science Foundation of China (30470029, 30500107), Key Program of Basic Research of Shanghai (No. 06PJ14072), and the Major State Basic Research Development Program of China (2004CB720103, 2006CB910705, and 2007CB707803)

^# These authors contributed equally to this work

*Corresponding authors:

Yixue LI: Tel, 86-21-64363311; Fax, 86-21-64838882; E-mail, [email protected]

Weihong JIANG: Tel, 86-21-54924172; Fax, 86-21-54924015; E-mail, [email protected]

Abstract������� Species of the genus Streptomyces are major bacteria responsible for producing most natural antibiotics. Streptomyces coelicolor A3(2) and Streptomyces avermitilis were sequenced in 2002 and 2003, respectively. Two-component signal transduction systems (TCSs), consisting of a histidine sensor kinase (SK) and a cognate response regulator (RR), form the most common mechanism of transmembrane signal transduction in prokaryotes. TCSs in S. coelicolor A3(2) have been analyzed in detail. Here, we identify and classify the SK and RR of S. avermitilis and compare the TCSs with those of S. coelicolor A3(2) by computational approaches. Phylogenetic analysis of the cognate SK-RR pairs of the two species indicated that the cognate SK-RR pairs fall into four classes according to the distribution of their orthologs in other organisms. In addition to the cognate SK-RR pairs, some potential partners of non-cognate SK-RR were found, including those of unpaired SK and orphan RR and the cross-talk between different components in either strain. Our study provides new clues for further exploration of the molecular mechanism for regulation of industrially important streptomycetes.

Key words������� Streptomyces; two-component system; cross-talk; phylogenetic analysis

Two-component signal transduction systems (TCSs), consisting of a histidine (His) sensor kinase (SK) and a cognate response regulator (RR), serve as a basic stimulus-response coupling mechanism to allow organisms to sense and respond to changes in many different environmental conditions in prokaryotes [1]. They are widespread not only in almost all prokaryotes and many archaea, but also in some eukaryotes, such as fungi and plants, in which they play an important role in light and hormone signaling.

Most of the SKs are membrane-associated His kinases. Extracellular stimuli are sensed by the periplasmic domain of the SK, and serve to modulate the activities of the SK. The SK catalyzes ATP-dependent autophosphorylation of a specific His residue located in its dimerization domain. The phosphoryl group subsequently transfers from the phosphohistidine of the SK to a specific aspartate (Asp) residue within the conserved regulatory domain of the RR. Phosphorylation of the regulatory domain activates a downstream output domain that elicits the specific cellular response [2].

Streptomyces is a genus of Gram-positive bacteria. Unlike normal bacteria, streptomycetes have a complex development life cycle such as mycelial growth and spore formation. To adapt the particularly complex and variable environment, streptomycetes possess a broad range of metabolic processes and biotransformations [3,4]. The most interesting property of streptomycetes is their ability to produce most natural antibiotics used in human and veterinary medicine and agriculture. Therefore it is quite essential to understand the biological process of remarkable morphological differentiation and antibiotic production in streptomycetes [5]. Over 20 different pleiotropic genes influence antibiotic production in Streptomyces coelicolor A3(2), three of which are TCSs, indicating an important role for protein phosphorylation and phosphory�lation cascades in the regulation of antibiotic production [6].

Two complete genomic sequences of the genus Streptomyces are now available. S. coelicolor A3(2), the best-known representative of streptomycetes, was sequenced in 2002 and has an 8.7 Mbp linear chromosome containing 7825 protein-encoding sequences [3,4]. The second streptomycete genome, S. avermitilis, was published in 2003 [4]. The linear chromosome of this genome is just over 9 Mbp, which is larger than that of S. coelicolor A3(2) but contains� fewer open reading frames (7574 instead of 7825). Comparative analysis of the two streptomycete genomes revealed that there is a common highly conserved 6.5 Mbp region with respect to gene order and content [4]. SKs and RRs of S. coelicolor A3(2) have been analyzed in detail [7]. However, to date, a detailed analysis of TCSs of S. avermitilis has not been reported.

In this study, we attempt to conduct a comparative analysis that will constitute a basis for further exploration of the signal transduction systems of streptomycetes.

Materials and Methods

Identification and classification of SKs and RRs of S. avermitilis

The genome sequences of S. avermitilis (http://avermitilis.ls.kitasato-u.ac.jp/) were searched against the Interpro database [8] using InterproScan on a local workstation. The SKs were identified by visual inspection of all the proteins that contain the ATPase domain [7,10]. According to the alignments of the 16 amino acids around the conserved histidine for each of the five groups of SKs in Bacillus subtilis [11], we made five hidden Markov models (HMMs) using the hmmbuild program of the HMMER package Version 2.3.2 [12] (http://hmmer.janelia.org/). The identified SKs were assigned to five groups by searching the five HMMs using the hmmpfam program of the HMMER suite (E<10-5). The transmembrane (TM) domains of each SK were acquired using TopPred II (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html) [13]. The two best matches for each SK were assumed to be the true TM domains, and all the residues between the end of the first and the start of the second TM domain were taken as the sensor domain. Similar to SKs, RRs were identified by checking all the proteins that contain the CheY-like domain. Alignments of the C-terminal output domains of S. avermitilis RRs with those of Escherichia coli RRs revealed the five different groups of RRs [11]. All the functional domains of SKs and RRs were obtained from the InterproScan results.

Comparison of TCSs in S. coelicolor A3(2) and S. avermitilis

All the orthologs of the TCSs were retrived through KEGG API (http://www.genome.jp/kegg/soap/; SOAP interface to KEGG) from KEGG SSDB using best-best relations (Smith-Waterman score>100). The orthologs of SKs in the two streptomycete genomes with less than 50% amino acid sequence identity in their sensor domains were ignored.

Sequence alignment and phylogenetic analysis

In addition to trans-phosphorylation between cognate SK-RR pairs, cross-talk in trans-phosphorylation between non-adjacent SK-RR pairs was also reported [14]. To explore those potential cross-talks in S. coelicolor A3(2) and S. avermitilis, we inferred functional coupling between SKs and RRs based on conservation of SK-RR pairs between genomes, which is employed as a classical computational method to predicting functionally coupled genes [15]. If orthologs of non-cognate SK and RR in S. coelicolor A3(2) and S. avermitilis are adjacent in some other organisms, we assumed that the potential cross-talk in signal transduction might take place between them or they are a potential pair (Fig. 1). We took those non-cognate SK and RR pairs with more than 10 support ortholog pairs in other organisms as the potential pairs or the potential cross-talks of TCSs in either streptomycete genomes. The multiple alignments were obtained by aligning with the program CLUSTAL W [16] and phylogenetic trees were constructed using PHYLIP based on the neighbor-joining algorithm [17] and the bootstrap value was 1000.

Results

Identification of TCSs of S. avermitilis and comparison with S. coelicolor A3(2)

Sixty-seven SKs were obtained after the visual inspection of S. avermitilis proteins that contain both the ATPase domain and the conserved His domain. Similarly, 68 RRs were found by checking the CheY-like domain and the RR output domain. Of these 67 SKs, 53 are adjacent to RRs (one pair is assumed), and 14 are unpaired. Besides the 53 paired RRs, there are 15 orphan RRs as well (Table 1).

The sensor domains of SKs vary greatly to sense diverse environment stimuli, and the ATP domains, conserved in all SKs, do little to differentiate them . The region around the conserved His, the site of autophosphorylation, proved to be more informative for classification of SKs. Alignment with the region around the His that becomes phosphorylated revealed that the SKs of S. avermitilis fell into the five main groups (I, II, IIIa, IIIb and IV), as defined in B. subtilis [11]. Similar to S. coelicolor A3(2), most of the SKs of the S. avermitilis fell into group II and IIIa (32 and 30, respectively), with only one in group I, one in group IIIb and five in group IV. The RRs were classified to NarL, OmpR, DBD, LytTR and wHtH groups by the relatedness of their output domains (Table 1). The largest number of RRs fell into the NarL group, which are all contain the bacterial regulatory protein LuxR domain (InterPro IPR000792). It was found that the second largest number of RRs containing the C-terminal transcriptional regulatory domain (Trans_reg_C; IPR001867) was related to the OmpR group. LuxR domain and the C-terminal transcriptional regulatory domain are subfamilies of winged helix repressor DNA-binding domain (IPR011991), which contains the winged helix-turn-helix DNA-binding motif. It is notable that SAV6219 contains the ANTAR domain (IPR005561), which is an RNA-binding domain found in bacterial transcription antitermination regulatory proteins [18]. This domain has been detected in various RRs of TCSs and also some one-component sensory regulators from a variety of bacteria. Most activated RRs interact with DNA to activate or repress the transcription of a series of genes, however, ANTAR-containing RR might interact with RNA to resist the termination of special gene transcription.

It is interesting that, as in B. subtilis and S. coelicolor A3(2), all of the SKs in group II are linked with RRs classified as NarL family, whereas all of the group IIIa SKs are paired with RRs belonging to the OmpR family without exception. Two SKs of group IV are paired with RRs containing a typical winged helix-turn-helix domain, while� the SKs of group I and IIIb are unpaired. The conclusion for the conserved relationship is that the catalytic domain of the SKs and both domains of the RRs might have evolved as a unit from a common ancestor [11]. Consistent with this conclusion, the gene orders of SK and RR in the transcription units are preserved within different classes (Table 1).

Most SKs contain a variety of extracellular, intracellular and/or transmembrane functional domains to respond to specific environmental stimuli. As a result, there is little sequence similarity in the N-terminal domains of the SKs. The membrane topology predictions of the SKs indicate that over half of the SKs have three or more TM domains. Three soluble cytoplasmic proteins were predicted for those SKs that contain no TM domain. They might be activated by another SK in the transduction pathway, as for E. coli CheA [19]. The region between the end of the first and the start of the second TM domain was taken as the sensor domain (Table 1). The size of the sensor domains ranges from 3 to 275 amino acids. Eight SKs containing sensor domains of less than 20 amino acids were predicted to belong to a new subfamily of SKs, which is almost entirely buried in the cytoplasmic membrane and frequently linked to ABC transporters. SKs of this new subfamily were speculated to sense changes in membrane structure or topology [20].

PAS and GAF domains, as cytosolic sensing modules, have been found in a large number of SKs. Three SKs that contain PAS domain (IPR000014) were found in the S. avermitilis genome. PAS domain proteins function to detect some signals, such as oxygen, redox potential and light, by binding flavins, haems, chromophores or some other cofactors [21]. The GAF domain (IPR003018) is found in six SKs of S. avermitilis. GAF domains appear to act as binding sites for small ligands that induce the autophosphorylation of the SK and subsequent signal transduction to activate specific gene transcription [22]. One SK (SAV6889) was detected that contains the nitrate and nitrite-sensing� domain (IPR010910), which responds to changes in nitrate and nitrite concentrations.

Phylogenetic analysis of the TCSs in S. avermitilis and S. coelicolor A3(2) genomes

All the potential pairs of the TCSs in two streptomycete genomes were analyzed and the numbers of all supported pairs of each potential pair were counted. The organism distribution of each cognate SK-RR pair and the support pairs consisting of their orthologs showed that the paired TCSs fell into four classes: present in most bacteria; present in most actinobacteria; specific to streptomycetes; and specific to either of the two streptomycete strains.

KdpD-KdpE of S. coelicolor A3(2), have orthologs in S. avermitilis and 45 other bacteria. KdpD and KdpE, which regulate the expression of the high affinity K+ transport system most notably under K+ limiting conditions [23], have been extensively studied in E. coli and appear to be ubiquitous in most bacteria. Independent phylogenetic analyses were carried out using amino acid sequences of the regions assigned for classification of KdpD, KdpE and their orthologs (Fig. 2). The similarity of the two phylogenetic trees could imply the inherent functional connection between the catalytic domain of the SKs and both the regulatory and output domains of the RRs.

In the S. coelicolor A3(2) genome, 12 pairs of TCSs are present in most actinobacteria, and 42 TCS pairs are specific to streptomycetes. CutRS, which might play roles in the production of actinorhodin [24], and ChiRS, which is related to chitinase production [25], might be specific to streptomycete strains. The TCS pairs specific in streptomycetes exceed more than those present in most bacteria or in the actinobacteria group, suggesting that this genus might be well equipped to adapt to a wide range of environmental stimuli and stresses, and to regulate complex multicellular development, a broad range of metabolic processes and biotransformation.

Phylogenetic analysis using all orthologs of available organisms revealed that 26 pairs of TCSs are specific to S. coelicolor A3(2). As mentioned above, the S. coelicolor A3(2) genome contains 31 paired TCSs that are specific compared to the S. avermitilis genome. Of these paired TCSs, five have orthologs in some other organisms but not in S. avermitilis. While in the 17 paired TCSs specific to S. avermitilis, only one pair has orthologs in other bacteria.

Discussion

The basic TCS elements can be combined to produce a His-Asp-His-Asp phosphorelay. Central to this phos�phorelay pathway is a hybrid-type SK that contains both an SK core and an RR receiver domain in a single protein [26]. Two hybrid-type SKs and their orthologs (SAV1085 with SCO7327, SAV2512 with SCO5748) were identified in S. avermitilis and S. coelicolor A3(2). The third hybrid-type SK in S. avermitilis, SAV 5564, has no ortholog in S. coelicolor A3(2). The complexity of phosphorelay systems permits the integration of multiple check points and regulatory steps into the pathway [27].

In addition to the cognate TCS pairs we have discussed in detail, some potential pairs that are not adjacent in the genome were found by the ortholog analysis. The SK SAV1990 and RR SAV1988 are not back-to-back in the S. avermitilis genome. However, our analysis suggested that they are cognate, as their orthologs (SCO6253 and SCO6254) are adjacent and they accord with the rule that all SKs falling into group II are linked with an RR classified to the NarL family (Table 1). Orthologs of S. avermitilis TCSs, SAV7118 (SK) and SAV7115 (RR) are adjacent in 47 organisms (Table 2), and orthologs of SAV3017 (SK) and SAV6219 (RR) are adjacent in 10 organisms. These four TCSs had been supposed to be unpaired SKs or orphan RRs in S. avermitilis because they did not have an adjacent pair in the genome, but the cases in other organisms indicated that they might be two paired TCSs.

Microarray analysis for the TCS mutants of E. coli has represented that TCSs functionally interact with each other, at least for certain combinations, to expand the signal transduction network so as to allow some genes to respond to a wide range of environmental stimuli [28]. Trans-phosphorylation in vitro was detected between non-cognate SK-RR pairs in E. coli at a rate of approximately 3% [14], raising the possibility that the cross-talk in signal transduction takes place between non-cognate SKs and RRs. In this study, some non-cognate SK-RR pairs were found in both the S. avermitilis and S. coelicolor A3(2) genomes that have cognate ortholog SK-RR pairs in other organisms (Table 2), suggesting that cross-talk might take place between them. TCSs, such as SCO7534, SCO3741, SCO1136 and SCO1801 in S. coelicolor A3(2), and SAV2430, SAV2971, SAV4416 and SAV4047 in S. avermitilis might be inclined to take part in the cross-talk. The streptomycete strain-specific TCSs have insufficient support pairs, so that the cross-talk referencing to these TCSs might be omitted. While our results accord with the corollary that if cross-talk between SK-RR pairs is of regulatory significance, it is likely to occur only within a group [11].

The identification and classification of the TCSs in the S. avermitilis genome provide the foundation to understand the signal transduction system of the strain. Approximately 80% of the commercially available antibiotics are produced by the streptomycetes, therefore comparison analysis of the TCSs of the two streptomycete strains can improve our understanding of the two-component systems of this organism and also provide insight into the molecular mechanisms of regulation in industrially important strains of streptomycetes.

Acknowledgements

We are grateful to Drs. Govind CHANDRA and Matthew I. HUTCHINGS from the John Innes Centre (Nowich, UK), for their useful discussion and valuable advice.

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