Sensing and responding to diverse extracellular signals? Analysis of the sensor kinases and response regulators of Streptomyces coelicolor A3(2)

Matthew I. Hutchings, Paul A. Hoskisson, Govind Chandra and Mark J. Buttner

Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK

Correspondence
Matthew I. Hutchings
matt.hutchings{at}bbsrc.ac.uk


   ABSTRACT
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ABSTRACT
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Streptomyces coelicolor is a Gram-positive soil bacterium that undergoes a complex developmental life cycle. The genome sequence of this organism was recently completed and has revealed the presence of over 60 sigma factors and a multitude of other transcriptional regulators, with a significant number of these being putative two-component signal transduction proteins. The authors have used the criteria established by Hoch and co-workers (Fabret et al., 1999, J Bacteriol 181, 1975–1983) to identify sensor kinase and response regulator genes encoded within the S. coelicolor genome. This analysis has revealed the presence of 84 sensor kinase genes, 67 of which lie adjacent to genes encoding response regulators. This strongly suggests that these paired genes encode two-component systems. In addition there are 13 orphan response regulators encoded in the genome, several of which have already been characterized and are implicated in development and antibiotic production, and 17 unpaired and as yet uncharacterized sensor kinases. This article attempts to infer useful information from sequence analysis and reviews what is currently known about the two-component systems, unpaired sensor kinases and orphan response regulators of S. coelicolor from both published reports and the authors' own unpublished data.


The online version of this review (at http://mic.sgmjournals.org) contains supplementary figures showing results from the alignment of residues surrounding conserved histidine residue in SKs, and from TopPred 2 analysis predicting the membrane topology of all 84 SK sequences.

Two-component signal transduction systems (TCSs), consisting of a sensor kinase (SK) and a cognate response regulator (RR), are found across all three domains of life, the Bacteria, Archaea and Eukarya. They are most widespread in the Bacteria (with the exception of the mycoplasmas) but SK genes have also been identified in the Archaea (Kim & Forst, 2001), in fungi and protozoa (Thomason & Kay, 2000), and in plants (Hwang et al., 2002). SK genes are conspicuously absent from the animal genomes so far sequenced and it has been proposed that these proteins are not present in the animal kingdom as a whole (Wolanin et al., 2002). This potentially makes them an attractive target for antimicrobials (Barrett et al., 1998), especially since some bacteria, including Bacillus subtilis and the opportunistic pathogen Staphylococcus aureus, contain essential TCSs (Fabret & Hoch, 1998; Martin et al., 1999). The extracytoplasmic sensor domain of each SK responds to specific types of environmental stimuli. The signal is transferred via autophosphorylation of a conserved His residue in the cytoplasmic H box to the aspartate residue of the cognate RR, which then activates transcription of target genes (Hakenbeck & Stock, 1996; Fig. 1). In bacteria, generally speaking the range of environmental stimuli to which an organism can respond is directly linked to the number of SKs encoded by that organism's genome. The number of SKs encoded by a bacterial genome is also proportional to the size of the genome, such that in bacteria which are obligate pathogens, and generally have smaller genomes, the percentage of SK and RR pairs in relation to the total number of genes is quite small, approximately 0·26 % compared to 0·65 % in free-living bacteria (Kim & Forst, 2001). Pseudomonas aeruginosa, however, has a large number of regulatory genes including 118 encoding RRs (Stover et al., 2000), and this may be a defining trait of opportunistic pathogens.



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Fig. 1. Schematic representation of the basic two-component signalling pathway.

 
Streptomyces coelicolor is a high-G+C, Gram-positive bacterium that exhibits a complex developmental life cycle. Spore germination and subsequent outgrowth leads to a network of vegetative hyphae. In response to any of a number of proposed signals, including nutritional stress and an extracellular signalling cascade, the substrate mycelium gives rise to aerial hyphae that eventually become septate to form mature spores. Since S. coelicolor is a soil-dwelling organism it needs to respond to highly variable conditions within its environment.

The complete genome sequence of S. coelicolor (Bentley et al., 2002) has allowed us to analyse the TCSs, unpaired SKs and orphan RRs of this organism. Although these systems are widespread throughout the bacteria, very little is known about the signals sensed by the SK or, in many cases, the targets for the RR of each system. By searching the S. coelicolor genome for proteins containing both the SK ATPase domain and the conserved histidine motif (Fabret et al., 1999) we found 84 SK genes. Searching for the RR effector domain together with the CheY-like domain for interaction with SKs revealed 80 RR genes. This differs slightly from the figures (85 SKs and 79 RRs) reported in the genome sequence paper (Bentley et al., 2002), probably because the hybrid SCO4009 is listed as an SK gene in the sequence annotation whereas our analysis suggests it is not a true SK, since it lacks the conserved histidine for autophosphorylation. Of these 84 SK genes, 67 are adjacent to RR genes, while 17 are unpaired.

As well as the 67 paired RR genes there are 13 orphan RR genes. The paired SK and RR genes account for approximately 0·86 % of the total ORFs in S. coelicolor, which is 25 % higher than the average for non-pathogenic, free-living bacteria (Kim & Forst, 2001). This, as pointed out by Bentley et al. (2002), suggests that this organism might be well equipped to deal with a wide range of environmental stimuli.

Initial identification and classification of SKs and RRs
Taking as a starting point the analysis of Hoch and co-workers (Fabret et al., 1999) we set about classifying the 84 SK genes and 80 RR genes of S. coelicolor. The SKs were identified by searching the genome sequence for proteins containing SCOP domain 55874 (ATPase domain; Murzin et al., 1995) using hidden Markov models (HMMs) for this domain obtained from the Superfamily database (Gough et al., 2001) and the hmmsearch program of the HMMER package version 2.2 (2001; S. Eddy, http://hmmer.wustl.edu). To search for the conserved histidine, the site of autophosphorylation, alignments of the 16 amino acid residues surrounding the conserved histidine for each of the groups I, II IIIa, IIIb and IV were taken from Fabret et al. (1999) and used to make HMMs using the hmmbuild program of the HMMER package. The five HMMs thus made were used to search each of the 84 SKs of S. coelicolor using the program hmmpfam of the HMMER suite (see supplementary figure S1 with the online version of this paper at http://mic.sgmjournals.org). The scores of the proteins with these HMMs were used to assign them to different groups. In each case it was verified that the His alignment did indeed identify the conserved histidine in the S. coelicolor sequence. As pointed out by Fabret et al. (1999), alignment of this region is more informative than alignment of the whole protein sequence since the sensor domains of SKs differ greatly and are likely to skew the analysis. Alignment of these sequences revealed that the SKs of S. coelicolor fell into the five main groups, I, II, IIIa, IIIb and IV, as defined in B. subtilis (Fabret et al., 1999), where groups IIIa and IIIb are very closely related. As in B. subtilis, the vast majority fell into groups II and IIIa, with only one falling into group I, two into group IIIb and five into group IV (Table 1). It was notable that, as in B. subtilis, all of the group II SKs were paired with (i.e. adjacent to) RRs belonging to the NarL family, while all but two of the paired group IIIa SKs were linked with RRs belonging to the OmpR family. The remaining two paired group IIIa SKs (SCO0871 and SCO5748) are paired with RRs that lack a DNA binding domain, and three group IIIa SKs are unpaired. Three of the group IV SKs (SCO1136, SCO5434 and SCO5828) are paired with RRs containing atypical winged helix–turn–helix domains, while the remaining two group IV SKs and the only two group IIIb SKs are unpaired (Table 1). Similar to SKs, the RRs were identified by searching the genome for proteins containing SCOP domain 52172 (CheY-like domain for interaction with SK). This search identified 80 RRs, 75 of which contain SCOP domain 46894 (the C-terminal effector domain of the bipartite RRs, for DNA binding), three contain a different effector domain (SCOP 46785, winged helix–turn–helix domain), as mentioned above, and another two (SCO0870 and SCO5749) contain no recognized DNA binding domain (Table 1). A single gene (SCO4009) is annotated as a bifunctional protein (SK and RR) but, while it contains domains common to both, it does not appear to fit in either group. SCO4009 contains the ATPase domain common to all SKs, the homodimerization domain found in many SKs and the CheY-like receiver domain common to all RRs. However, it lacks both the conserved His residue for autophosphorylation and the C-terminal effector domain of the bipartite response regulators, which is required for binding to DNA.


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Table 1. Classification and novel domains of the SKs

Abbreviations: NADP, NADP binding domain; DNA, DNA binding domain; PAS, PAS domain; cAMP, cyclic AMP binding domain; GAF, GAF domain; ABP, adenine nucleotide binding protein. The TM domains column shows the positions of the most likely transmembrane helices as predicted by TopPred 2 analysis. For the RRs: *Lacks CheY-like domain for SK interaction; DBD, no DNA binding domain; wHtH, winged helix–turn–helix. HR or RH refers to the gene order of the RR and SK genes.

 
Sensor domains
The N-terminal domains of SKs have very low sequence homology, probably due to the diverse range of signals sensed by these proteins. As such these domains were excluded from the analysis used to group the 84 SKs in the S. coelicolor genome. However, in order to learn more about the sensor domains, the sequence of each SK was analysed using TopPred II, a membrane topology prediction package (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html; Claros & von Heijne, 1994). Where available, the two best matches for each SK were assumed to be the true transmembrane (TM) domains (Table 1). Surprisingly, five of the SKs are predicted to be soluble cytoplasmic proteins, four had only a single TM domain and over half are predicted to have three or more TM domains (see supplementary figure S2 with the online version of this paper at http://mic.sgmjournals.org). Of the five soluble SKs, four are unpaired (Table 1), and none of these contains the HPt domain (characteristic of proteins involved in signalling cascades). However, it is possible that these soluble kinases may be activated by another SK as in the case of Escherichia coli CheA (Levit et al., 1996).

The sensor domain includes all the residues between the end of the first and the start of the second TM domain, and the size of the sensor domains ranged from 3 to 314 amino acids (Table 1). Ten SKs contain sensor domains of less than 20 amino acids, suggesting that they may belong to a new subfamily of intramembrane sensing SKs (Mascher et al., 2003). SKs belonging to this recently discovered subfamily are predicted to sense changes in membrane structure or topology. Mascher and colleagues first identified SKs of this type in B. subtilis and then searched all the microbial genome sequences using a defined set of criteria. In order to fit into this subfamily the proteins must be less than 400 residues in length, with an N-terminal domain less than 100 amino acids in length (including both TM domains) and a sensor domain of less than 20 residues. They identified only four intramembrane sensing SKs in S. coelicolor, although our analysis shows that in fact five out of the ten SKs described here fit these criteria. One of these (SCO3740) therefore escaped their initial analysis (Mascher et al., 2003). The remaining five either have N-terminal domains that are too long or the length of the whole protein exceeds 400 residues so that, despite exceeding the cut-off criteria, these proteins may still be intramembrane sensing SKs. The 20 residue cut-off point for these domains appears to be critical since there is strong evidence that S. coelicolor VanS, which has a sensor domain of only 27 residues, responds to vancomycin and related glycopeptides directly, presumably by binding of the drug to the sensor domain (Hong et al., 2004).

GAF and PAS domains: new partners for the SK domains
The S. coelicolor genome encodes seven GAF-containing SKs (Table 1). GAF domains have been identified in many bacterial proteins, including adenylate cyclases, phosphotransferases and SKs, and appear to act as binding sites for small ligands which modulate the catalytic activity of the target protein (Aravind & Ponting, 1997). It seems likely therefore that the signals for these GAF-containing SKs are small ligands that induce autophosphorylation of the SK and subsequent signal transduction to activate or repress the expression of target genes.

The closely related PAS domain is also found in six of the S. coelicolor SKs (Table 1). Both PAS and GAF domains are implicated in signalling. However, while GAF domains bind ligands such as nucleotides and small molecules, PAS domains appear to bind flavins, haems and chromophores and are responsive to oxygen, redox potential and light (Zhulin et al., 1997; Taylor & Zhulin, 1999). S. coelicolor synthesizes a yellow/orange pigment when exposed to light but produces colourless colonies when grown in the dark. This raises the intriguing possibility that S. coelicolor may modulate gene expression in response to light, which is probably sensed by one or more of these PAS-containing SKs and the signal transduced through their cognate RRs.

Functions and phenotypes of TCSs
The functions and targets of the 67 S. coelicolor TCSs are largely unknown, and those with assigned functions are usually homologous to TCSs from other organisms. However, phenotypic and/or microarray data are available for many of the TCSs of S. coelicolor and here we summarize what is known so far.

PhoPR.
The phoPR operon of B. subtilis encodes a TCS that directly activates expression of Pho regulon genes under low-phosphate conditions. The Pho regulon includes operons encoding a high-affinity phosphate transport system, enzymes involved in synthesis of the cell wall polymer teichuronic acid (which replaces teichoic acid in phosphate-starved cells), enzymes involved in teichoic acid biosynthesis, two genes encoding the major alkaline phosphatases of B. subtilis, PhoA and PhoB, and the phoPR operon itself (Shi & Hulett, 1999). As with most TCSs the exact ligand bound by the SK, PhoR, has yet to be identified, but the signal for PhoPR activation of the Pho regulon appears to be phosphate starvation and results in an increase in the availability of phosphate in the cell.

In the actinomycete Mycobacterium tuberculosis the phoPR genes were identified on the basis of similarity to the B. subtilis genes (Cole et al., 1998). In M. tuberculosis, a phoP mutant strain is attenuated in vivo in a mouse infection model, and it was proposed that these genes are more similar to the phoPQ genes that control virulence in Salmonella sp. than they are to the phoPR genes that control the Pho regulon of B. subtilis (Perez et al., 2001). Alternatively, deletion of this TCS may result in down-regulation of the Pho-regulon genes, resulting in attenuation. In S. coelicolor and the closely related Streptomyces lividans the phoPR genes were also identified on the basis of similarity to the B. subtilis genes, but these genes were found to lie close to genes involved in phosphate transport, strengthening the case for their being true phoPR homologues (Sola-Landa et al., 2003). The S. lividans genes were cloned and characterized, and phosphorylated PhoP was found to activate expression of phoA (alkaline phosphatase), apparently in response to low phosphate concentrations (Sola-Landa et al., 2003). Phosphorylated PhoP is also implicated in the regulation of a high-affinity phosphate transporter, since there is a drastic reduction in phosphate uptake in a phoPR mutant strain (Sola-Landa et al., 2003). The same authors reported that production of actinorhodin and undecylprodigiosin was greatly increased in the phoPR background, suggesting that PhoP may negatively regulate production of these secondary metabolites.

CseBC.
Cse denotes control of sigma E, where {sigma}E is an ECF (extra-cytoplasmic function) sigma factor. The cse genes are found in the sigE operon, which includes cseA, encoding a negative regulator of sigE expression (M. I. Hutchings, H. J. Hong, E. Leibowitz & M. J. Buttner, unpublished), cseB, encoding the RR, and cseC, encoding the SK. Expression of sigE is induced by a range of cell-wall-specific antibiotics, which inhibit late steps of peptidoglycan biosynthesis, and also by the cell wall hydrolytic enzyme lysozyme (Hong et al., 2002). The sigE and cseB phenotypes are identical, i.e. hypersensitivity to lysozyme and a requirement for high concentrations of magnesium for normal growth, because sigE expression is absolutely dependent on CseB. This suggests a model in which CseC, sensing cell wall damage, is autophosphorylated at conserved histidine residue 271 and in turn phosphorylates CseB at Asp-55, which then activates expression of the sigE operon (Paget et al., 1999). The sigE promoter is not transcribed by {sigma}E-containing RNA polymerase (E{sigma}E) holoenzyme (Paget et al., 1999) and the only targets so far identified for E{sigma}E are one of two promoters for hrdD, a sigma factor of unknown function, and the promoter of the cwg operon, which appears to encode enzymes involved in the synthesis of a cell wall glycan (Hong et al., 2002). It seems likely from the evidence gathered so far that at least some of the targets for E{sigma}E are involved in cell wall homeostasis.

VanRS.
The SK VanS and its cognate RR VanR control inducible vancomycin resistance in enterococci and in glycopeptide-producing strains such as Streptomyces toyacaensis (Pootoolal et al., 2002). Vancomycin is a cell-wall-specific antibiotic that binds to the D-ala-D-Ala terminus of the stem peptide of Lipid II to prevent transpeptidation and, hence, to prevent cell wall assembly, resulting in osmotic lysis. Vancomycin-resistant strains reprogramme their cell walls so that the stem peptide ends D-ala-D-Lac, a substrate for which vancomycin has 1000-fold lower specificity (Billot-Klein et al., 1997). The enzymes that catalyse the reprogramming of the cell wall peptidoglycan are VanH, a D-lactate dehydrogenase, VanA, a D-ala-D-Lac ligase, and VanX, a D-alanyl-D-alanine dipeptidase that cleaves stem peptides ending D-ala-D-Ala.

A vancomycin resistance gene cluster was recently identified in S. coelicolor, containing seven genes: vanRS, vanJ, vanK and vanHAX. The vanHAX operon encodes the enzymes that reprogramme peptidoglycan biosynthesis, vanJ encodes a protein of unknown function that is not required for vancomycin resistance and vanK encodes a homologue of the Fem family of non-ribosomal peptide synthetases (Rohrer & Berger-Bachi, 2003). Deletion of vanK resulted in a drug-sensitive phenotype, suggesting that S. coelicolor has a novel mechanism of vancomycin resistance, since this gene has not been found in the vancomycin resistance clusters of any other bacteria (Hong et al., 2004). The expression of the seven genes in this cluster, divided into four transcription units, is absolutely dependent on the VanRS TCS, and microarray analysis has revealed that these are the only genes in the VanR regulon (M. I. Hutchings & M. J. Buttner, unpublished). Interestingly, of the four transcripts encoding these van genes, all but the vanJ transcript are leaderless (Hong et al., 2004), a situation that appears to be more common in the streptomycetes than in other bacteria (Janssen, 1993).

KdpDE.
Annotated as a putative turgor pressure sensor (KdpD) and putative turgor pressure regulator (KdpE) based on primary sequence similarity to the E. coli turgor-sensing TCS proteins, the Kdp TCS has been most extensively studied in E. coli but appears to be ubiquitous amongst bacteria. The signal sensed by KdpD is still unclear, although autophosphorylation in a reconstituted in vitro system appears to require the presence of divalent Mg2+ or Ca2+ and monovalent Na+ or K+. It has, however, been widely proposed that KdpD senses turgor pressure directly and that this pressure results in conformational change, autophosphorylation and subsequent phosphotransfer (Jung & Altendorf, 2003). In E. coli, kdpD and kdpE are the last two genes in the kdpFABCDE operon, which, besides the TCS, encodes the potassium-transporting KdpFABC ATPase. Expression of the kdp operon is activated by the KdpDE TCS in response to osmotic stress and adjusts the potassium content of the cytoplasm to maintain turgor pressure (Wood, 1999).

The kdpDE genes of S. coelicolor appear to be the first two of a four-gene operon, with the other genes encoding a hypothetical and a putative membrane protein. This operon is convergent with another two-gene operon encoding putative potassium uptake proteins showing homology to E. coli TrkA. In the actinomycete M. tuberculosis the kdpDE genes are divergent from the kdpFABC operon and expression of the operon is highly induced by low potassium concentrations and depends on KdpDE. In M. tuberculosis, KdpD interacts with two lipoproteins, LprJ and LprF, which appear to act as accessory proteins positively regulating KdpD and enhancing the expression of the kdpFABC operon (Steyn et al., 2003). The proximity of the S. coelicolor operon designated kdpDE to putative potassium uptake genes strongly suggests that they do indeed encode homologues of the E. coli KdpDE TCS, that these genes are regulated by KdpDE, and that they encode a potassium transporter. Interestingly, the kdpFABC operon in S. coelicolor is in a separate location on the chromosome and it will be interesting to see if these genes are regulated by KdpDE.

AbsA.
The AbsA1-2 TCS was identified through the analysis of mutations that blocked production of actinorhodin, undecylprodigiosin, calcium-dependent antibiotic and the plasmid-encoded methylenomycin, the four antibiotics produced by S. coelicolor (Adamidis et al., 1990). The point mutations accounting for this phenotype were all located in the absA1 (SK) gene, whereas deletion of either absA1 or absA2 resulted in hyperproduction of actinorhodin and undecylprodigiosin, known as the Pha (precocious hyperproduction of antibiotics) phenotype (Brian et al., 1996). This implies that the original point mutants were gain-of-function mutations that led to constitutive phosphorylation of the RR. The similarity of the phenotypes for both the absA1 and absA2 deletion strains suggests that AbsA2 is not subject to phosphorylation by other SKs (i.e. cross-talk). Mutation of the conserved His-202 residue, the proposed site of autophosphorylation, resulted in a strain exhibiting the Pha phenotype, although the strain produced undecylprodigiosin before actinorhodin, rather than simultaneously as seen in the absA1 deletion strain, and neither antibiotic was overproduced to the same levels as seen in the absA1 background. No alternative site of phosphorylation could be identified on AbsA1 and the differences between the two strains have not yet been explained.

AfsQ.
Closely related to the cseBC TCS of S. coelicolor, the afsQ genes were identified by screening KpnI-digested fragments of S. coelicolor genomic DNA for their ability to stimulate actinorhodin production in S. lividans when introduced on a multicopy plasmid. The afsQ1 (RR), but not the afsQ2 (SK), gene stimulated actinorhodin production in S. lividans. Deletion of the afsQ genes in S. coelicolor had no effect either on the normal growth cycle or on antibiotic production (Ishizuka et al., 1992). Since the original work was published the availability of the genome sequence has revealed that not only are the AfsQ1 and 2 proteins homologous to CseC and B (respectively) but they are also divergently transcribed from a gene encoding an ECF sigma factor that is highly similar to {sigma}E. There is a third gene in the afsQ operon, encoding a lipoprotein that may be an accessory protein to the AfsQ TCS (discussed below). Like the vanRS and absA transcripts, the transcript encoding all three afsQ genes in both S. lividans and S. coelicolor is leaderless (Ishizuka et al., 1992; M. I. Hutchings & M. Buttner, unpublished).

CutRS.
CutRS was the first TCS to be identified in the streptomycetes. It was isolated in a screen for DNA fragments that could restore melanin production in a melC1-132 mutant strain of S. lividans (Tseng & Chen, 1991). Insertion mutagenesis of either cutR or cutS in S. lividans resulted in overproduction of actinorhodin both on solid and in liquid medium, and overexpression of cutRS in S. coelicolor repressed production of actinorhodin in the same way as the AbsA TCS (Chang et al., 1996). As the authors of this work pointed out, the AbsA and Cut systems may act in some kind of hierarchical manner, otherwise disruption of both loci would be required to relieve the repression of actinorhodin production. Cross-talk between the two systems can be ruled out for the same reason, since knocking out either of the RRs, AbsA1 or CutR, resulted in actinorhodin overproduction (Brian et al., 1996; Chang et al., 1996).

ChiRS.
The chiRS operon was first identified and characterized in Streptomyces thermoviolaceus (Tsujibo et al., 1999) and subsequently in S. coelicolor on the basis of homology (Bentley et al., 2002). In both organisms the chiRS genes are next to the chi40 gene, which encodes a chitinase, an enzyme that hydrolyses chitin into chitooligosaccharides before further enzymic breakdown results in the production of N-acetylglucosamine, a source of carbon and nitrogen. The model proposed suggests that ChiS is autophosphorylated at His-1199 in response to chitibiose (a dimer subunit of chitin) and subsequently transfers the phosphoryl group to Asp-54 of ChiR, which then binds to the chi40 promoter to activate the expression of Chi40 (Tsujibo et al., 1999).

Phenotypic and expression analysis
Recent microarray analysis of growth-phase-dependent gene expression and the regulation of antibiotic biosynthetic pathways in S. coelicolor revealed that the expression of two operons encoding group II TCSs is coordinated with the expression of the red gene cluster. These TCS genes were subsequently designated ecrA1 (SCO2518) and ecrA2 (SCO2517), ecrE1 (SCO6421) and ecrE2 (SCO6422) for expression coordinated with red. Expression of EcrA1-2 is also growth phase dependent (Huang et al., 2001) and an EcrA1-2-deficient strain produces less undecylprodigiosin than wild-type S. coelicolor (Li et al., 2004). In addition, the ecrA1 SK gene was identified in a screen for developmentally impaired mutants of S. coelicolor using the transposon Tn4560 (P. A. Hoskisson & M. Buttner, unpublished), strongly suggesting that this TCS is involved in development. One other TCS (SCO3654/SCO3653) was found to be developmentally impaired in this screen (P. A. Hoskisson, J. Towle & M. Buttner, unpublished), although in this case expression was not coordinated with red or growth phase dependent (Huang et al., 2001). Finally, expression of four more genes encoding TCSs [SCO0203/SCO0204 and SCO5748/5749 (osaAB)] was found to be growth phase dependent. Disruption of the osaB (RR) gene results in a strain that is sensitive to osmotic change, is conditionally bald (i.e. cannot raise aerial hyphae), and has uncoordinated antibiotic production, with actinorhodin and undecylprodigiosin massively over-expressed (Bishop et al., 2004). The osaA (SK) and osaB genes are separated by 500 bp, and are not co-transcribed (Bishop et al., 2004). However, the SK primary sequence shows strong similarity to putative osmosensing SKs from yeast (Tao et al., 2002).

Unpaired histidine kinases
The 17 unpaired SKs span all four groups and include the single group I (SCO1217) and the two group IIIb (SCO1596 and SCO7297) SKs. None of these unpaired SKs have been characterized, although the group IIIb SK SCO1596 was identified in a screen for developmentally impaired mutants (P. A. Hoskisson & M. Buttner, unpublished), suggesting that it may be involved in development. Another interesting unpaired SK is encoded by one of the S. coelicolor conservons (SCO6794 or cvnA7). The 13 conservons of S. coelicolor each consist of a cluster of four genes (cvnA–D), of which cvnD encodes a nucleotide-binding protein, cvnB and cvnC encode proteins of unknown function, and cvnA encodes a membrane protein with weak similarity to SKs (Bentley et al., 2002), although, judging by our analysis, only cvnA7 encodes a true SK. The presence of so many unpaired kinases in the genome suggests that they might specifically activate other RRs, responding to signals that differ from that of the RR's cognate kinase. This would allow subsets of genes to be activated in response to a wider range of environmental stimuli than might be sensed by a single SK.

Orphan response regulators
Typically, the N-termini of RRs have several conserved residues which form the phosphorylation pocket; these conserved residues include two adjacent aspartates near the N-terminus of the protein (DD), an aspartate in the middle of the N-terminal domain, usually close to position 54 (D54), a hydroxylated residue at position 82, normally serine or threonine (S/T82) and a lysine residue near the end of the N-terminal domain, close to position 105 (K105). Here the RRs are characterized as typical where they contain the conserved residues of the phosphorylation pocket (including at least one of the adjacent aspartates) or where they have been shown to be dependent on phosphorylation experimentally and the atypical RRs are those which lack two (both DD and another residue) or more of the conserved residues which make up the phosphorylation pocket. Of the 13 orphan RR genes in the S. coelicolor genome, seven have typical phosphorylation pockets, five have atypical phosphorylation pockets and a single RR (BldM) has a ‘pseudo’-phosphorylation pocket (Table 2). BldM contains all the conserved amino acids but has been demonstrated to be active in the absence of phosphorylation (Molle & Buttner, 2000). Only five of these orphan RRs have been studied experimentally and the findings are described below.


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Table 2. Orphan response regulators

Conserved residues refers to those in the N-terminal phosphorylation pocket; the presence or absence of these residues was used to classify these pockets as either typical or atypical (see text). Phenotypes and functions are given where available, along with references describing the experimental evidence.

 
Atypical orphan response regulators
BldM.
The BldM N-terminal phosphorylation domain is apparently typical; indeed the original point mutations isolated in the bldM locus lay in the putative phosphorylation pocket (Molle & Buttner, 2000). However, the conserved Asp-54 residue is not required for function. Replacement of this residue with unphosphorylatable residues such as asparagine (Asp-54N) or alanine (Asp-54A) did not affect its function, with wild-type levels of aerial hyphae and spores formed (Molle & Buttner, 2000). An Asp-54N substitution in CheY resulted in an active protein in vivo as a result of phosphorylation of the adjacent Ser-56 residue (Bourret et al., 1990; Appleby & Bourret, 1999). Phosphorylation at alternative residues has also been reported in FixJ of Rhizobium meliloti and NtrC of E. coli (Reyrat et al., 1994; Moore et al., 1993). However, no potentially phosphorylatable hydroxyamino acid residues are found adjacent to Asp-54 in BldM, suggesting that BldM has a pseudo-phosphorylation pocket.

WhiI.
The whiI mutant is unable to sporulate and the lack of grey spore pigment gives a white colony phenotype. The whiI gene encodes an orphan RR that is required for normal septum formation early in the development of aerial hyphae. The phosphorylation pocket, however, is atypical, lacking the lysine residue and one aspartate conserved in conventional phosphorylation pockets (Ainsa et al., 1999). This prompted the suggestion that whiI is regulated via another means, such as small ligand binding, or interaction with another protein. Ainsa and co-workers speculated that the presence of the conserved Asp-69 in the putative phosphorylation pocket might permit phosphorylation as part of a phospho-relay signal transduction system similar to that of the Spo0A sporulation cascade of B. subtilis (Hoch, 1993).

RedZ.
RedZ, the pathway-specific activator for undecylprodigiosin (also known as Red), lacks the paired aspartate residues and the lysine residue conserved in the phosphorylation pocket of conventional response regulators (Guthrie et al., 1998). Despite this, there is experimental evidence to suggest that RedZ is regulated post-translationally (J. White & M. J. Bibb, personal communication).

Not just RedZ.
The atypical nature of several S. coelicolor RRs, either lacking the conventional phosphorylation pocket, or having a pseudo-phosphorylation pocket, raises the possibility that these are regulated by other means. DnrN, from Streptomyces. peucetius, is a response regulator required for daunorubicin biosynthesis. It has a typical phosphorylation domain, but DnrN with an Asp-55N mutation is active in vivo, and the mutant protein binds its target site with equal affinity to the wild-type protein (Otten et al., 1995; Furuya & Hutchinson, 1996). The possibility of post-translational regulation mediated by a co-regulator interacting with the phosphorylation pocket of the atypical response regulator has been suggested for both BldM and RedZ (Molle & Buttner, 2000; J. White & M. J. Bibb, personal communication). Interestingly, all the above examples of atypical RRs lack a paired, cognate sensor kinase. This suggests that lack of the cognate kinase might result in regulation of these proteins by a means other than Asp-54 type phosphorylation.

Typical response regulators
RamR.
The ram (rapid aerial mycelium) cluster plays an important role in the development of S. coelicolor. The ram genes encode a membrane-bound kinase (RamC), a small protein (RamS), the subunits of an ATP binding cassette (ABC) transporter (RamAB), and a RR (RamR). The ramC and ramR genes are required for production of aerial hyphae but are dispensable for vegetative growth (O'Connor et al., 2002), and over-expression of RamR overcomes the bald phenotype of all tested bld mutants of S. coelicolor (Nguyen et al., 2002). A homologous gene cluster amf is found in Streptomyces griseus and Streptomyces avermitilis. In S. coelicolor, the ramR gene product has a typical phosphorylation pocket and is essential for transcription of the ramCSAB cluster (Keijser et al., 2002). In S. griseus, the integrity of the phosphorylation pocket and the conserved aspartate residue have been shown to be essential for the function of the RamR homologue, AmfR, suggesting that phosphorylation of the aspartate is required for activity (Ueda et al., 1993). However, this has not been demonstrated experimentally in S. coelicolor. A caveat to this is that the sequence similarity between the S. coelicolor ram cluster and the amf cluster of S. griseus is lower than would be expected for orthologous genes (Keijser et al., 2002).

GlnR.
The S. coelicolor genome contains five glutamine synthetase (GS) homologues: a GSI of the {beta}-subtype, three of the GSI-{alpha}-subtype, and a GSII-type enzyme, a type normally associated with eukaryotes (Fink et al., 2002; for a full discussion of GS types see Brown et al., 1994). The OmpR-like RR, GlnR, is required for transcription of the gene encoding the GSI-like enzyme, glnA, in S. coelicolor (Fink et al., 2002; Wray & Fisher, 1991), and a glnR null mutant exhibits glutamine auxotrophy. A close homologue of GlnR, named GlnRII, is required for GSII transcription. GlnRII shows significant similarity to GlnR in the C-terminal DNA binding domain but lacks the N-terminal CheY domain for SK interaction. In addition, the conserved phosphorylatable aspartate, and the conserved serine/threonine, and tyrosine residues in the phosphorylation pocket, also postulated to be involved in phosphotransfer, are all absent in GlnRII (Fink et al., 2002). Thus it would appear that GlnRII is not a functional homologue of GlnR and is not a true member of the RR family.

The remaining five orphan RRs with typical phosphorylation pockets have yet to be characterized (Table 2). However, BldM is an example of a RR that has a typical phosphorylation pocket and does not require D54 for activity. Therefore, while Table 2 lists these RR domains as typical or atypical, only experimental analysis will determine whether or not these proteins are true members of the RR family.

Accessory proteins
It has recently become apparent that in addition to protein–protein interactions with each other the RR or SK proteins of many two-component systems also interact with accessory proteins that either positively or negatively regulate their activity. In some cases this has been shown simply through the use of two-hybrid systems to screen libraries for interaction with either the SK or RR (e.g. Steyn et al., 2003), while in an increasing number of cases both negative and positive regulators of two-component systems have been reported. Thus, what once appeared to be relatively simple signalling pathways are likely, in some cases, to be more complex and to involve more than just two components. Regulation of these systems can occur at several levels: modulation of the SK sensor domain, for example CpxP of E. coli (DiGiuseppe & Silhavy, 2003); inhibition of SK autophosphorylation, for example KipI of B. subtilis (Wang et al., 1997); phosphatase activity against the SK, for example SixA of E. coli (Ogino et al., 1998); or phosphatase activity against the RR, for example CheZ of E. coli (Parkinson, 2003). In the sporulation signal transduction cascade of B. subtilis the response regulator Spo0F is subject to regulation by the RapA, B and C phosphatases while Spo0A is regulated by the Spo0E, YnzD and YisI phosphatases (Perego, 2001).

In S. coelicolor, the genes encoding six of the group IIIa SKs, including CseC and AfsQ2, lie in operons with genes encoding predicted membrane proteins, secreted proteins or lipoproteins. The cseA gene encodes a membrane protein, whereas afsQ3, SCO3011, SCO5305 and SCO7535 encode putative lipoproteins, and SCO7232 encodes a putative secreted protein. Five of the six genes also lie in operons with a RR gene, while a single operon encodes only a SK (SCO5304) and a putative lipoprotein (SCO5305). Analysis of the primary sequences of the six gene products using TopPred II predicts that each protein has a single N-terminal transmembrane helix. Furthermore, the C-terminus of each protein is predicted to be extra-cytoplasmic (Claros & von Heijne, 1994; http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html). Although these proteins have little homology to one another at the primary sequence level, this analysis suggests that they have similar structures and may therefore have similar functions.

The best-studied of the six SKs in this subgroup is CseC, which is encoded by the last gene in the sigE operon. The sigE gene encodes a well-studied extra-cytoplasmic sigma factor, {sigma}E (Hong et al., 2002), and the sigE operon promoter is positively regulated by the CseB/C two-component system (Paget et al., 1999). Recent work has shown that CseA is a negative regulator of sigE transcription, that it is localized to the plasma membrane by the first N-terminal 21 amino acids, and that the C-terminus is extracytoplasmic (M. I. Hutchings, H.-J. Hong, E. Leibowitz & M. J. Buttner, unpublished). Thus the TopPred II predictions, for this protein at least, are correct and imply that CseA must interact with the sensor domain of CseC, or another component of the signal transduction system acting upstream of CseC, in order to negatively regulate transcription of the sigE operon. It is interesting that the CseA protein has no homologues in the databases, although it seems possible that the lipoproteins encoded by the other operons discussed here are structural homologues, i.e. they also function as accessory proteins to their linked TCS. Studies are under way to analyse the AfsQ proteins and to determine if CseA interacts with CseC.

Future prospects
The recent development of a rapid knockout strategy in S. coelicolor (Gust et al., 2003) will make it possible to disrupt each of the TCSs in turn in an effort to determine which are essential for survival and to gain some clues as to their function through characterization of the mutant phenotypes, as was recently reported for E. coli (Zhou et al., 2003). Coupled with the availability of S. coelicolor whole-genome microarrays, rapid progress should now be possible in further characterizing the 67 TCSs, 13 orphan RRs and 17 unpaired SKs. Since the streptomycetes produce around 80 % of the commercially available antibiotics, understanding the TCSs of this model organism may also provide insights into increasing antibiotic production in closely related, industrially important actinomycetes.


   ACKNOWLEDGEMENTS
 
We are grateful to Stephen Bentley, Marie Elliot, David Hopwood, Keith Chater, Ray Dixon and Justin Nodwell for comments on the manuscript and useful discussions, to Paul Herron for critical reading of the manuscript and to Amy Bishop, Paul Dyson and Paul Herron for communication of results prior to publication.


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