Comparative analysis of eukaryotic-type protein phosphatases in two streptomycete genomes

Liang Shi and Weiwen Zhang

Microbiology Group, Pacific Northwest National Laboratory, 902 Battelle Blvd, PO Box 999, MSIN: P7-50, Richland, WA 99352, USA

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Inspection of the genomes of Streptomyces coelicolor A3(2) and Streptomyces avermitilis reveals that each contains 55 putative eukaryotic-type protein phosphatases (PPs), the largest number ever identified from any single prokaryotic organism. Unlike most other prokaryotic genomes that have only one or two superfamilies of eukaryotic-type PPs, the streptomycete genomes possess the eukaryotic-type PPs that belong to four superfamilies: 2 phosphoprotein phosphatases and 2 low-molecular-mass protein tyrosine phosphatases in each species, 49 Mg2+- or Mn2+-dependent protein phosphatases (PPMs) and 2 conventional protein tyrosine phosphatases (CPTPs) in S. coelicolor A3(2), and 48 PPMs and 3 CPTPs in S. avermitilis. Sixty-four percent of the PPs found in S. coelicolor A3(2) have orthologues in S. avermitilis, indicating that they originated from a common ancestor and might be involved in the regulation of more conserved metabolic activities. The genes of eukaryotic-type PP unique to each surveyed streptomycete genome are mainly located in two arms of the linear chromosomes and their evolution might be involved in gene acquisition or duplication to adapt to the extremely variable soil environments where these organisms live. In addition, 56 % of the PPs from S. coelicolor A3(2) and 65 % of the PPs from S. avermitilis possess at least one additional domain having a putative biological function. These include the domains involved in the detection of redox potential, the binding of cyclic nucleotides, mRNA, DNA and ATP, and the catalysis of phosphorylation reactions. Because they contained multiple functional domains, most of them were assigned functions other than PPs in previous annotations. Although few studies have been conducted on the physiological functions of the PPs in streptomycetes, the existence of large numbers of putative PPs in these two streptomycete genomes strongly suggests that eukaryotic-type PPs play important regulatory roles in primary or secondary metabolic pathways. The identification and analysis of such a large number of putative eukaryotic-type PPs from S. coelicolor A3(2) and S. avermitilis constitute a basis for further exploration of the signal transduction pathways mediated by these phosphatases in industrially important strains of streptomycetes.


Abbreviations: CPTP, conventional protein tyrosine phosphatase; LMMPTP, low-molecular-mass protein tyrosine phosphatase; PK, protein kinase; PP, protein phosphatase; PPM, Mg2+- or Mn2+-dependent protein phosphatase; PPP, phosphoprotein phosphatase


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Reversible protein phosphorylation/dephosphorylation is a universal mechanism used to regulate protein function. Three types of regulatory protein phosphorylation mechanisms, O-, N- and acyl phosphorylation, have been defined, based on the amino acid residue undergoing the covalent modification. Protein O-phosphorylation occurs on the hydroxyl amino acids serine, threonine and tyrosine, while N-and acyl phosphorylation occur on the basic amino acid histidine and the acidic amino acid aspartic acid, respectively (Cozzone, 1988). Protein O-phosphorylation was originally discovered in eukaryotes where it serves as the backbone of the eukaryotic signal transduction network. In contrast, protein N- and acyl phosphorylation were identified initially in prokaryotes where they are involved in the two-component signal transduction network. Results over the last 10 years, however, not only reveal that protein N- and acyl phosphorylation-mediated two-component signal transduction pathways also exist in lower eukaryotic organisms and plants (Stock et al., 2000), but also unequivocally demonstrate that protein O-phosphorylation is a common regulatory mechanism for prokaryotes (reviewed by Kennelly, 2002; Shi, 2004).

In eukaryotes, two classes of enzymes with opposite functions work in concert with each other to regulate the phosphorylation levels of O-phosphoproteins. One is a protein kinase (PK) that adds a phosphate group from ATP to a phosphoprotein (i.e. phosphorylation), which results in a change in the structure and function of the phosphoprotein. Another is a protein phosphatase (PP) which restores the phosphoprotein to its original structure and function by removing the phosphate group (i.e. dephosphorylation) (Cohen, 1989). Five superfamilies of eukaryotic-type PPs have been identified: the phosphoprotein phosphatase (PPP) family, the Mg2+- or Mn2+-dependent protein phosphatase (PPM) family (Cohen, 1994), the conventional protein tyrosine phosphatase (CPTP) family, the Cdc25 family and the low-molecular-mass protein tyrosine phosphatase (LMMPTP) family (Jackson & Denu, 2001). Both PPPs and PPMs possess unique signature sequence motifs that span a region of about 220 or 290 aa, respectively (Fig. 1). CPTP, LMMPTP and Cdc25, however, share a characteristic active site motif, CX5R (X is any amino acid). A catalytic aspartic acid is found anywhere from 25 to 50 residues on the N-terminal side of the active site cysteine in CPTP or from 80 to 110 residues on the C-terminal side of the catalytic cysteine in LMMPTP (Fig. 1). Similarly to CPTP, Cdc25 also possesses a conserved aspartic acid located about 46 residues on the N-terminal side of the active site cysteine. This aspartic acid, however, plays no catalytic role in Cdc25. It is their molecular structures that differentiate the members of these two families (Jackson & Denu, 2001). Thus, based on the relative location of the CX5R motif and conserved aspartic acid, and for the purposes of this study, we combined CPTP and Cdc25 into a single family and denote it as CPTP in this paper (Fig. 1).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1. Schematic representation of signature sequence motifs of the PPP, PPM, CPTP and LMMPTP families of phosphatases. The single-letter amino acid code is used and X can be any amino acid. The individual motifs of each family are indicated by the numbers located above the drawings. The motifs are not drawn to scale.

 
Streptomycetes are among the most numerous and ubiquitous soil bacteria (Hodgson, 2000). Unusually for bacteria, streptomycetes also exhibit complex multicellular development (Hopwood, 1999). The genome sequence of Streptomyces coelicolor A3(2), the best known representative of the genus, was released in 2002 and revealed an 8·7 Mb linear chromosome having a coding density very similar to that of other bacteria. The large size of this genome translates into a higher number of predicted genes than that of a simple eukaryote such as Saccharomyces cerevisiae. This genome also contains an unprecedented proportion of regulatory genes, predominantly those likely to be involved in responses to external stimuli and stresses (Bentley et al., 2002). The second streptomycete genome, Streptomyces avermitilis, was published early last year (Ikeda et al., 2003). This genome, at just over 9 Mb, is larger than that of S. coelicolor A3(2) with slightly fewer protein-encoding sequences (7574 instead of 7825). Comparative analysis of the two streptomycete genomes revealed that nearly 5300 genes were closely related (Ikeda et al., 2003).

Streptomycetes show remarkable morphological differentiation during growth and antibiotic production, which undoubtedly requires various levels of regulation using different signal transduction mechanisms (Chater, 1993). Recent studies show that protein O-phosphorylation is a widespread phenomenon in streptomycetes (Umeyama et al., 2002; Horinouchi, 2003). One well-studied example is the afsK-afsR system from Streptomyces griseus (Ueda et al., 1993). The afsK and afsR genes encode a serine/threonine PK and its targeted protein, respectively. Gene replacement shows that the afsK disruptants do not form aerial mycelium or spores on medium containing glucose at concentrations higher than 1 %, but form spores on mannitol- and glycerol-containing media; this suggests that the afsK gene is essential for morphogenesis in the presence of glucose. Introduction of afsK restores aerial mycelium formation in the disruptants. A few other serine/threonine PKs, Pkg2, Pkg3 and Pkg4, have been cloned from ‘Streptomyces granaticolor’ (Nádvorník et al., 1999). One of them, Pkg2, a transmembrane PK, is involved in the normal morphological development of aerial hyphae (Nádvorník et al., 1999). A recent survey revealed the presence of 34 putative PK genes in the S. coelicolor A3(2) genome, representing about 0·5 % of all coding sequences (Petrickova & Petricek, 2003). This suggests that the occurrence of protein O-phosphorylation in streptomycetes is much more widespread than previously demonstrated.

Meanwhile, the involvement of eukaryotic-type PPs in various metabolic activities has also been found in different streptomycete strains. The first report is a ptpA gene from S. coelicolor A3(2). Functional analysis shows that the disruption of the ptpA gene has no effects on cell growth, formation of aerial mycelium and spores, or secondary metabolism in S. coelicolor A3(2). However, overexpression of the ptpA gene in Streptomyces lividans increases the production of actinorhodin and undecylprodigiosin (Li & Strohl, 1996; Umeyama et al., 1996). Another eukaryotic-type PP gene, sppA of S. coelicolor A3(2), is involved in vegetative growth and formation of hyphae and spores (Umeyama et al., 2000). In contrast to the eukaryotic-type PKs of streptomycetes, whose regulatory functions in primary or secondary metabolism have been well documented, our current understanding of eukaryotic-type PPs in streptomycetes is very limited. It is still unclear how many putative eukaryotic-type PPs are present in the completed streptomycete genomes and at what degree they participate in the regulation of cellular metabolism. The objectives of this study are to use bioinformatic means to search for the answers of the following questions. (1) How many putative eukaryotic-type PPs are present in the S. coelicolor A3(2) and S. avermitilis genomes? (2) Unlike eukaryotic-type PKs which belong to a single superfamily, four superfamilies of eukaryotic-type PPs have been identified. Do streptomycetes possess all four superfamilies of eukaryotic-type PPs and, if so, how are they distributed among the different PP superfamilies? (3) Finally, since most of the genes in S. avermitilis have orthologues in S. coelicolor A3(2), how many eukaryotic-type PP genes found in one of these species have orthologues in another?


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The approach used to identify putative eukaryotic-type PPs from the genome sequences of S. coelicolor A3(2) (www.sanger.ac.uk and www.tigr.org) and S. avermitilis (http://avermitilis.ls.kitasato-u.ac.jp) was similar to that described previously (Shi et al., 1998) with slight modification. Briefly, conserved regions of eukaryotic-type PPs characterized from different prokaryotic organisms and Cdc25 phosphatases from human and yeast were used as templates to search for ORFs whose predicted polypeptide products display similarity to eukaryotic-type PPs by BLAST (E<0·01). Each genome sequence was searched with multiple templates derived from each family of PPs. To confirm that any tentatively identified ORFs indeed possessed the catalytic domain of eukaryotic-type PPs, their DNA-derived amino acid sequences were used to search for conserved domains from the Conserved Domain Databases (CDD) with algorithms of Reverse Position Specific BLAST (E<0·01) provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (Marchler-Bauer et al., 2003). The CDD searched were SMART, PFAM, COG and KOG. Visual inspection was then performed to eliminate those ORFs that lacked the minimum complement of conserved sequence features considered necessary to make a functional enzyme. The specific criteria used for each family were described previously (Shi et al., 1998). After confirmation, the amino acid sequences of the identified putative PPs from each family and their homologues that had been biochemically characterized from other prokaryotic organisms were used to construct a phylogenetic tree with the neighbour-joining-based ALGNX program of Vector NTI from InforMax (Frederick, MD, USA). This method works on a matrix of distance between all pairs of sequences to be analysed. These distances were related to the degree of divergence between the sequences. The phylogenetic tree was calculated after the sequences were aligned. The calculated distance values are displayed in parentheses following the names of sequences (Saitou & Nei, 1987).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Overview
Putative PPs belonging to all four families of protein O-phosphatases, PPP, PPM, CPTP and LMMPTP, were found in both the S. coelicolor A3(2) and S. avermitilis genomes. S. coelicolor A3(2) contained 2 PPPs, 49 PPMs and 2 each of the CPTP and LMMPTP families, while S. avermitilis possessed 2 PPPs, 48 PPMs, 3 CPTPs and 2 LMMPTPs. Thus, the total number of putative eukaryotic-type PPs found was 55 for both S. coelicolor A3(2) and S. avermitilis (Table 1). The arrangement of the identified eukaryotic-type PP genes in these two streptomycete genomes is shown in Fig. 2. Unlike the eukaryotic-type PK genes which were unevenly located mainly in the central conserved region of the chromosome (Petrickova & Petricek, 2003), the streptomycete PP genes were found not only in the central region, which encodes most of the house-keeping genes essential for cell metabolism, but also in both arms that contain loci encoding proteins with probable non-essential functions, such as secondary metabolites and hydrolytic exoenzymes (Bentley et al., 2002) (Fig. 2). Phylogenetic analysis (see following sections for details) showed that each PPP and LMMPTP found in one streptomycete genome had an orthologue in the other. Two out of three CPTPs from S. avermitilis also had orthologues in S. coelicolor A3(2). However, a number of the identified PPM-encoding genes were found to be unique to each genome. Nineteen PPMs from S. avermitilis and 20 PPMs from S. coelicolor A3(2) had no orthologue in the other genome. The distribution of unique PPM and CPTP-encoding genes in the linear chromosomes is shown in Fig. 2.


View this table:
[in this window]
[in a new window]
 
Table 1. Numbers of eukaryotic-type PPs and PKs found in S. coelicolor A3(2), S. avermitilis, B. subtilis 168, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120

The PPs of S. coelicolor A3(2) and S. avermitilis were identified in this study; the PKs of S. coelicolor A3(2) and S. avermitilis were identified by Petrickova & Petricek (2003) and Ikeda et al. (2003), respectively; the numbers of PPs and PKs of B. subtilis 168 were from Shi et al. (1998); the numbers of PPs and PKs of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 were from Shi et al. (1998) and Zhang et al. (1998b), and Wang et al. (2002), respectively.

 


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2. Arrangement of the putative eukaryotic-type PP genes in the S. coelicolor A3(2) and S. avermitilis genomes. The central highly conserved part of the chromosomes, encoding most of the known essential genes, is indicated by double lines. PPP, CPTP and LMMPTP genes are labelled inside the circle and indicated by superscript a, b and c, respectively. PPM genes are labelled outside the chromosome circles. In the S. coelicolor A3(2) genome, 2 PPP, 22 PPM, 2 CPTP and 2 LMMPTP genes are located in the central part of chromosome, while 25 PPM genes are found in both arms. In the S. avermitilis genome, 2 PPP, 19 PPM, 2 CPTP and 2 LMMPTP genes exist in the highly conserved internal region, while 29 PPM and 1 CPTP genes are located in both arms. The names of unique genes in each genome are marked in bold type. Thirteen out of 20 unique PPM genes in S. coelicolor A3(2), 15 out of 19 unique PPM genes and the only 1 unique CPTP gene in S. avermitilis are located in both arms. The scale (in Mb) and the position of the oriC origin of replication are indicated.

 
PPPs
PPPs found in the same streptomycete genome showed only a low degree of sequence similarity to each other: SppA (SCO3941) and SCO5973 were 38 % identical, while SAV4263 and SAV2323 were 35 % identical. However, SppA and SCO5973 of S. coelicolor A3(2) were 81 and 89 % identical to SAV4263 and SAV2323 of S. avermitilis, respectively. Among the PPPs that had been biochemically characterized in other prokaryotic organisms, the identified PPPs from streptomycetes were more similar to PrpE from the Gram-positive bacterium Bacillus subtilis (Iwanicki et al., 2002) and PrpA from the cyanobacterium Anabaena sp. PCC 7120 (Zhang et al., 1998a) (Fig. 3a). While sequence analysis of SppA or SAV4263 revealed no additional functional domains, it was noted that the PPP functional domains of each were located nearly in the middle of their respective peptide sequences. Conversely, for SCO5973 and SAV2323, the PPP functional domains were juxtaposed with a putative kinase domain at their N termini, and a fragment of unknown function of about 400 aa close to their C termini (Fig. 3b). No putative eukaryotic-type PK gene was identified near the putative PPP-encoding genes in either streptomycete genome.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3. (a) Phylogenetic analysis of PPPs. A phylogenetic tree was constructed with the ALGNX program of Vector NTI from InforMax, based on the amino acid sequences of the identified PPPs in S. coelicolor A3(2) and S. avermitilis, and characterized PPPs from other prokaryotes. They include SppA (SCO3941) and SCO5973 of S. coelicolor A3(2), SAV4263 and SAV2323 of S. avermitilis, PP-{lambda} of bacteriophage {lambda} (Cohen & Cohen, 1989), EC-PrpA and EC-PrpB of Escherichia coli (Missiakas & Raina, 1997), ST-PrpA and ST-PrpB of Salmonella typhimurium (Shi et al., 2001), PP1-cyano1 of Microcystis aeruginosa PCC 7820 (Shi et al., 1999b), PP1-cyano2 of Microcystis aeruginosa UTEX 2063 (Shi & Carmichael, 1997; Shi et al., 1999b), Sll1387 of Synechocystis sp. PCC 6803 (Shi et al., 1999a), PP1-arch1 of Sulfolobus solfataricus (Leng et al., 1995), PP1-arch2 of Methanosarcina thermophila TM-1 (Solow et al., 1997), Py-PP1 of Pyrodictium abyssi (Mai et al., 1998), PrpA of Anabaena sp. PCC 7120 (Zhang et al., 1998a) and PrpE of B. subtilis (Iwanicki et al., 2002). The numbers in parentheses after the phosphatase names represent their calculated evolutionary distances. (b) Schematic representation of the functional domains found in the PPPs of S. coelicolor A3(2) and S. avermitilis. K, Kinase domain; PPP, catalytic domain of PPP. The sizes of these domains are not drawn to scale.

 
PPMs
Similarities between various PPMs ranged from 23 to 100 % for those of S. coelicolor A3(2) and 20 to 76 % for those of S. avermitilis. Two ORFs encoding putative PPMs, SCP1.313 and SCP1.41C, were found in the giant linear plasmid SCP1 of S. coelicolor A3(2). SCP1.313 and SCP1.41C encoded two copies of an identical gene product and were both located 903 bp downstream of putative TetR-family regulator genes SCP1.42C and SCP1.312 (SCP1.42C and SCP1.312 were also identical), respectively (Redenbach et al., 1998). Similarities between PPMs from different streptomycete genomes ranged from 31 to 98 %. However, only 59 % of the PPMs identified in S. avermitilis had orthologues in S. coelicolor A3(2) (Fig. 4), while 69 % of the total encoding ORFs had orthologues (Ikeda et al., 2003). Phylogenetic analysis indicated that only 17 of the 97 (18 %) identified PPMs fell into Group A. Members of this group showed close similarity to functionally characterized PPMs from other prokaryotic organisms (Group A, Fig. 4), while the rest of the identified PPMs were clustered distantly from Group A (Group B, Fig. 4).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 4. Phylogenetic analysis of PPMs. A phylogenetic tree was constructed with the ALGNX program of Vector NTI from InforMax, based on the amino acid sequences of the putative PPMs of S. coelicolor A3(2) and S. avermitilis, and characterized PPMs from other prokaryotes. The PPMs of S. coelicolor A3(2) have locus accession numbers starting with ‘SCO’ (annotated by Sanger Sequencing Centre), ‘SCP’ (located in plasmid) and ‘NT’ (annotated by TIGR). The PPMs of S. avermitilis have locus accession numbers starting with ‘SAV’. The PPMs from other prokaryotes include IcfG and PphA of Synechocystis sp. PCC 6803 (Beuf et al., 1994; Shi et al., 1999a; Irmler & Forchhammer, 2001), SpoIIE, RsbP, RsbU RsbX and PrpC of B. subtilis (Duncan et al., 1995; Yang et al., 1996; Obuchowski et al., 2000; Vijay et al., 2000), PA-Stp1 of Pseudomonas aeruginosa (Mukhopadhyay et al., 1999), Pph1 of Myxococcus xanthus (Treuner-Lange et al., 2001), SA-Stp1 of S. agalactiae (Rajagopal et al., 2003) and Pstp of Mycobacterium tuberculosis (Boitel et al., 2003). Different groups of these phosphatases are labelled on the right. Group A contains 17 identified PPMs from streptomycetes and all PPMs characterized from other prokaryotic organisms. Group B contains the remaining 80 identified PPMs of streptomycetes. The numbers in parentheses after the phosphatase names represent their calculated evolutionary distances.

 
In prokaryotes, the genes encoding PPMs are usually parts of operons that also encode cognate PKs and/or their phosphoprotein substrates. Inspection of the area up- or downstream of the streptomycete PPM-encoding genes revealed six and five such gene clusters in the S. coelicolor A3(2) and S. avermitilis genomes, respectively. Depending on the type of PK/phosphoprotein involved, these gene clusters can be divided into two different groups. The clusters of Group I contained genes for SpoII/Rsb-like PKs and phosphoproteins. The clusters from Group II, however, contained genes encoding eukaryotic-type PKs that showed no sequence similarity to SpoII/Rsb-like PKs (Fig. 5).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 5.
 
Many PPMs from both eukaryotes and prokaryotes contain additional domains with distinct functions (Bork et al., 1996). A total of 30 out of 49 (61 %) and 35 out of 48 (73 %) PPMs from S. coelicolor A3(2) and S. avermitilis, respectively, were also found to possess at least one domain, in addition to their catalytic domain, with a putative biological function. The identified functional domains included PAS [PER (period clock protein), ARNT (aryl hydrocarbon receptor nuclear translocator) and SIM (single-minded protein)], GAF (cGMP phosphodiesterases, adenylyl cyclases and bacterial transcription factor FhlA), HATPase (histidine kinase-, DNA gyrase B- and HSP90-like ATPase), ANTAR (AmiR and NasR transcription antitermination regulators), RR (response regulator), TR (transcription regulator) and RsbW (a regulator of sigma B). Based on what additional domain(s) they might contain, 12 groups of PPMs were identified from both S. coelicolor A3(2) and S. avermitilis (Fig. 6). Although SpoII/Rsb-like PKs have frequently been found to work in concert with PPMs in bacterial cells, these proteins are usually encoded by different genes. However, the results from the domain identification work showed that seven PPMs in S. coelicolor A3(2) and nine PPMs in S. avermitilis contained a putative catalytic domain of RsbW, an SpoII/Rsb-like PK (Fig. 6). To our knowledge, this is the first report of such a protein fusion between an SpoII/Rsb-like PK and a PPM.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 6. Schematic representation of the functional domains found in the PPMs of S. coelicolor A3(2) and S. avermitilis. Group 1 includes 19 PPMs from S. coelicolor A3(2) and 13 PPMs from S. avermitilis. Members of this group contain only a PPM catalytic domain. Groups 2–8 consist of PPMs having one additional functional domain, which include PAS in Group 2, GAF in Group 3, HATPase in Group 4, ANTAR in Group 5, RR in Group 6, TR in Group 7 and RsbW in Group 8. Group 9 and 10 include PPMs with two additional functional domains: PAS and GAF in Group 9, and RsbW and GAF in Group 10. Three additional domains are identified in PPMs from Group 11 and 12: PAS, GAF and HATPase in Group 11 and PAS, GAF and RsbW in Group 12. The domains are not drawn to scale. aThe RsbW domain of SAV1097 is located on the N-terminal side of the PPM domain. bThe RsbW domain of SCO7009 is located on the C-terminal side of the PPM domain. cThe RsbW domains of SCO5747 and SAV2513 are located on the N-terminal side of the PAS domain.

 
CPTPs and LMMPTPs
A survey of the areas around the ORFs encoding identified CPTPs and LMMPTPs found no putative PK genes. Among the CPTPs characterized from other prokaryotes, SCO6772, SAV1642 and SAV994 appeared to be closely related to MptpB of Mycobacterium tuberculosis (Koul et al., 2000), while SCO5306 and SAV2948 were most similar to SptP from Salmonella typhimurium and YpoH from Yersinia pseudotuberculosis (Bliska et al., 1991; Kaniga et al., 1996) (Fig. 7a). Phylogenetic analysis also showed that LMMPTPs SC-PtpA (SCO3921) and SAV4272 had a close relationship with MptpA from M. tuberculosis (Koul et al., 2000), while SCO3770 and SAV3756 were closely related to SA-PtpB from Staphylococcus aureus (Soulat et al., 2002) (Fig. 7b). Unlike some of the identified PPPs and PPMs, no additional functional domain was found in any CPTP or LMMPTP from either streptomycete genome.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 7. Phylogenetic analysis of CPTPs (a) and LMMPTPs (b). Phylogenetic trees were constructed with the ALGNX program of Vector NTI from InforMax, based on the amino acid sequences of the putative CPTPs (a) or LMMPTPs (b) of S. coelicolor A3(2) and S. avermitilis, and characterized CPTPs (a) or LMMPTPs (b) from other prokaryotes. (a) CPTPs include SCO5306 and SCO6772 of S. coelicolor A3(2), SAV994, SAV1642 and SAV2948 of S. avermitilis, HopPtoD2 of Pseudomonas syringae (Bretz et al., 2003; Espinosa et al., 2003), IphP of Nostoc commune UTEX 584 (Potts et al., 1993), MptpB of Mycobacterium tuberculosis (Koul et al., 2000), SptP of Salmonella typhimurium (Kaniga et al., 1996) and YpoH of Yersinia pseudotuberculosis (Bliska et al., 1991). (b) LMMPTPs include SC-PtpA (SCO3921) and SCO3770 of S. coelicolor A3(2), SAV4272 and SAV3756 of S. avermitilis, Etp and Wzb of E. coli (Vincent et al., 1999; Wugeditsch et al., 2001), Yor5 of Klebsiella pneumoniae (Preneta et al., 2002), Ptp of Acinetobacter johnsonii (Grangeasse et al., 1998), MptpA of Mycobacterium tuberculosis (Koul et al., 2000), SO-PtpA of Shewanella oneidensis MR-1 (L. Shi, unpublished data), and SA-PtpA and SA-PtpB of Staphylococcus aureus (Soulat et al., 2002). The numbers in parentheses after the phosphatase names represent their calculated evolutionary distances.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although all four families of eukaryotic-type PPs were previously found in other prokaryotic organisms, previous data have showed that only 2 of the 35 surveyed prokaryotic genomes contained all four (Shi et al., 1998; Kennelly, 2002). In addition, most of the surveyed prokaryotic genomes contained fewer than 10 eukaryotic-type PPs (Shi et al., 1998). Only soil bacterium B. subtilis 168 and two aquatic cyanobacteria, Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, were previously found to have >=10 eukaryotic-type PPs (Shi et al., 1998; Zhang et al., 1998b; Wang et al., 2002). Even considering that the genome sizes of S. coelicolor A3(2) (8·7 Mbp) and S. avermitilis (9·02 Mbp) are twice as big as those of B. subtilis 168 (4·2 Mbp) and Synechocystis sp. PCC 6803 (3·57 Mbp) and about 1·5–1·8 Mbp bigger than that of Anabaena sp. PCC 7120 (7·2 Mbp), there are still at least three times more eukaryotic-type PPs present in S. coelicolor A3(2) or S. avermitilis compared to B. subtilis 168, Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120 (Table 1). Therefore, of those prokaryotes surveyed to date, S. coelicolor A3(2) and S. avermitilis have the most abundant putative eukaryotic-type PPs distributed among the four different families, consistent with the notion that streptomycetes contain an unprecedented proportion of regulatory genes, possibly used to regulate the cellular responses to internal or external stimuli and stresses (Bentley et al., 2002).

Many interesting findings in this study are related to the PPMs. In addition to the fact that both surveyed genomes contain a large number of PPM-encoding genes, nearly 40 % of identified PPM-encoding genes are unique to each genome and the majority are located in both arms outside the core conserved region (Fig. 2). Such characteristics suggest that PPM genes may have been recruited by gene acquisition, including horizontal gene transfer, and then subjected to lateral duplication. Although the presumption that streptomycetes acquired most PPMs by gene acquisition still requires more evidence, similar arguments have been proposed for the origination of some eukaryotic-type PKs in bacteria that are thought to have originated by horizontal gene transfer, including YpkA from Yersinia pseudotuberculosis (Galyov et al., 1993). Considering the complex streptomycete life cycle and the fact that differentiation and secondary metabolism are regulated by many environmental factors, it is likely that the acquisition of valuable genes would benefit soil streptomycetes during their adaptation to a new environmental niche. Another interesting finding is that 82 % of identified PPMs are distantly related to PPMs characterized from other prokaryotes (Fig. 4). Although the evolutionary meaning of this finding is not fully understood at this moment, it seems that the ancestors of these two groups of PPMs might have split at an early stage of evolution. Sixty one percent of the PPMs from S. coelicolor A3(2) and 73 % of the PPMs from S. avermitilis possess at least one more domain that appears to have a distinct function [such as the detection of redox potential (PAS), binding to cyclic nucleotides (GAF), mRNA (ANTAR), DNA (RR & TR) and ATP (HATPase), and phosphorylation (RsbW)] (Yang et al., 1996; Taylor & Zhulin, 1999; Ho et al., 2000; Stock et al., 2000; Vijay et al., 2000; Shu & Zhulin, 2002), indicating that these PPMs have diverse biological roles (Fig. 6). Fifteen PPMs of S. coelicolor A3(2) and 17 PPMs of S. avermitilis have at least two additional functional domains (Fig. 6). With multifunction domains evolutionarily installed, these PPMs might serve as more efficient signal proteins with the potential to sense multiple signals and/or act on their phosphoprotein substrates in a coordinated way. Because they contained multiple functional domains, most of them were assigned functions other than those described for PPMs in previous annotations.

The presence of large numbers of eukaryotic-type PPs in S. coelicolor A3(2) and S. avermitilis clearly indicates that the protein O-phosphorylation-mediated signal transduction network, similarly to that in eukaryotic cells, might be one of the major regulatory mechanisms employed by streptomycetes. Although little information is available about the structures of this network, identification and analysis of all putative PPs as well as PKs from streptomycete genomes will provide a starting point to further explore this type of signal transduction network at the molecular level.


   ACKNOWLEDGEMENTS
 
We would like to thank Dr Brian H. Lower from Virginia Polytechnic Institute and State University and Dr Miriam Mayer-Cumblidge from Pacific Northwest National Laboratory for their critical reading of this manuscript. Pacific Northwest National Laboratory is operated by Battelle Memorial Institute for the US Department of Energy through contract DE-AC06-76RLO 1830.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M. & 40 other authors (2002). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147.[CrossRef][Medline]

Beuf, L., Brown, N. P., Hegyi, H. & Schultz, J. (1994). A protein involved in co-ordinated regulation of inorganic carbon and glucose metabolism in the facultative photoautotrophic cyanobacterium Synechocystis PCC 6803. Plant Mol Biol 25, 855–864.[Medline]

Bliska, J. B., Guan, K. L., Dixon, J. E. & Falkow, S. (1991). Tyrosine phosphate hydrolysis of host proteins by an essential Yersinia virulence determinant. Proc Natl Acad Sci U S A 88, 1187–1191.[Abstract]

Boitel, B., Ortiz-Lombardia, M., Duran, R., Pompeo, F., Cole, S. T., Cervenansky, C. & Alzari, P. M. (2003). PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho-Ser/Thr phosphatase, in Mycobacterium tuberculosis. Mol Microbiol 49, 1493–1508.[CrossRef][Medline]

Bork, P., Brown, N. P., Hegyi, H. & Schultz, J. (1996). The protein phosphatase 2C (PP2C) superfamily: detection of bacterial homologues. Protein Sci 5, 1421–1425.[Abstract/Free Full Text]

Bretz, J. R., Mockm, N. M., Charity, J. C., Zeyad, S., Baker, C. J. & Hutcheson, S. W. (2003). A translocated protein tyrosine phosphatase of Pseudomonas syringae pv. tomato DC3000 modulates plant defense response to infection. Mol Microbiol 49, 389–400.[Medline]

Chater, K. F. (1993). Genetics of differentiation in Streptomyces. Annu Rev Microbiol 47, 683–713.

Cohen, P. (1989). The structure and regulation of protein phosphatases. Annu Rev Biochem 58, 453–508.[CrossRef][Medline]

Cohen, P. T. W. (1994). Nomenclature and chromosomal localization of human protein serine/threonine phosphatase genes. Adv Protein Phosphatases 8, 371–376.

Cohen, P. T. W. & Cohen, P. (1989). Discovery of a protein phosphatase activity encoded in the genome of bacteriophage lambda. Probable identity with open reading frame 221. Biochem J 260, 931–934.[Medline]

Cozzone, A. J. (1988). Protein phosphorylation in prokaryotes. Annu Rev Microbiol 42, 97–125.[CrossRef][Medline]

Duncan, L., Alper, S., Arigoni, F., Losick, R. & Stragier, P. (1995). Activation of cell-specific transcription by a serine phosphatase at the site of asymmetric division. Science 270, 641–644.[Abstract]

Espinosa, A., Guo, M., Tam, V. C., Fu, Z. Q. & Alfano, J. R. (2003). The Pseudomonas syringae type III-secreted protein HopPtoD2 possesses protein tyrosine phosphatase activity and suppresses programmed cell death in plants. Mol Microbiol 49, 377–387.[CrossRef][Medline]

Galyov, E. E., Hakansson, S., Forsberg, A. & Wolf-Watz, H. (1993). A secreted protein kinase of Yersinia pseudotuberculosis is an indispensable virulence determinant. Nature 361, 730–732.[CrossRef][Medline]

Grangeasse, C., Doublet, P., Vincent, C., Vaganay, E., Riberty, M., Duclos, B. & Cozzone, A. J. (1998). Functional characterization of the low-molecular-mass phosphotyrosine-protein phosphatase of Acinetobacter johnsonii. J Mol Biol 278, 339–347.[CrossRef][Medline]

Ho, Y. S., Burden, L. M. & Hurley, J. H. (2000). Structure of the GAF domain, a ubiquitous signaling motif and a new class of cyclic GMP receptor. EMBO J 19, 5288–5299.[Abstract/Free Full Text]

Hodgson, D. A. (2000). Primary metabolism and its control in streptomycetes: a most unusual group of bacteria. Adv Microb Physiol 42, 47–238.[Medline]

Hopwood, D. A. (1999). Forty years of genetics with Streptomyces: from in vivo through in vitro to in silico. Microbiology 145, 2183–2202.[Free Full Text]

Horinouchi, S. (2003). AfsR as an integrator of signals that are sensed by multiple serine/threonine kinases in Streptomyces coelicolor A3(2). J Ind Microbiol Biotechnol 20, 462–467.

Ikeda, H., Ishikawa, J., Hanamoto, K. & 7 other authors (2003). Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nature Biotechnol 21, 526–531.[CrossRef][Medline]

Irmler, A. & Forchhammer, K. (2001). A PP2C-type phosphatase dephosphorylates the PII signaling protein in the cyanobacterium Synechocystis PCC 6803. Proc Natl Acad Sci U S A 98, 12978–12983.[Abstract/Free Full Text]

Iwanicki, A., Herman-Antosiewicz, A., Pierchod, M., Seror, S. J. & Obuchowski, M. (2002). PrpE, a PPP protein phosphatase from Bacillus subtilis with unusual substrate specificity. Biochem J 366, 929–936.[Medline]

Jackson, M. D. & Denu, J. M. (2001). Molecular reactions of protein phosphatases – insights from structure and chemistry. Chem Rev 101, 2313–2340.[CrossRef][Medline]

Kaniga, K., Uralil, J., Bliska, J. B. & Galan, J. E. (1996). A secreted protein tyrosine phosphatase with modular effector domains in the bacterial pathogen Salmonella typhimurium. Mol Microbiol 21, 633–641.[Medline]

Kennelly, P. J. (2002). Protein kinases and protein phosphatases in prokaryotes: a genomic perspective. FEMS Microbiol Lett 206, 1–8.[CrossRef][Medline]

Koul, A., Choidas, A., Treder, M., Tyagi, A. K., Drlica, K., Singh, Y. & Ullrich, A. (2000). Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis. J Bacteriol 182, 5425–5432.[Abstract/Free Full Text]

Leng, J., Cameron, A. J. M., Buckel, S. & Kennelly, P. J. (1995). Isolation and cloning a protein-serine/threonine phosphatase from an archaeon. J Bacteriol 177, 6510–6517.[Abstract]

Li, Y. & Strohl, W. R. (1996). Cloning, purification, and properties of a phosphotyrosine protein phosphatase from Streptomyces coelicolor A3(2). J Bacteriol 178, 136–142.[Abstract]

Mai, B., Frey, G., Swanson, R. V., Mathur, E. J. & Stetter, K. O. (1998). Molecular cloning and functional expression of a protein-serine/threonine phosphatase from the hyperthermophilic archaeon Pyrodictium abyssi TAG11. J Bacteriol 180, 4030–4035.[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]

Missiakas, D. & Raina, S. (1997). Signal transduction pathways in response to protein misfolding in the extracytoplasmic compartments of E. coli: role of two new phosphoprotein phosphatases PrpA and PrpB. EMBO J 16, 1670–1685.[Abstract/Free Full Text]

Mukhopadhyay, S., Kapatral, V., Xu, W. & Chakrabarty, A. M. (1999). Characterization of a Hank's type serine/threonine kinase and serine/threonine phosphoprotein phosphatase in Pseudomonas aeruginosa. J Bacteriol 181, 6615–6622.[Abstract/Free Full Text]

Nádvorník, R., Vomastek, T., Janecek, J., Techniková, Z. & Branny, P. (1999). Pkg2, a novel transmembrane protein Ser/Thr kinase of Streptomyces granaticolor. J Bacteriol 181, 15–23.[Abstract/Free Full Text]

Obuchowski, M., Madec, E., Delattre, D., Boel, G., Iwanicki, A., Foulger, D. & Seror, S. J. (2000). Characterization of PrpC from Bacillus subtilis, a member of the PPM phosphatase family. J Bacteriol 182, 5634–5638.[Abstract/Free Full Text]

Petrickova, K. & Petricek, M. (2003). Eukaryotic-type protein kinases in Streptomyces coelicolor: variations on a common theme. Microbiology 149, 1609–1621.[Abstract/Free Full Text]

Potts, M., Sun, H., Mockaitis, K., Kennelly, P. J., Reed, D. & Tonks, N. K. (1993). A protein-tyrosine/serine phosphatase encoded by the genome of the cyanobacterium Nostoc commune UTEX 584. J Biol Chem 268, 7632–7635.[Abstract/Free Full Text]

Preneta, R., Jarraud, S., Vincent, C., Doublet, P., Duclos, B., Etienne, J. & Cozzone, A. J. (2002). Isolation and characterization of a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase from Klebsiella pneumoniae. Comp Biochem Physiol B Biochem Mol Biol 131, 103–112.[CrossRef][Medline]

Rajagopal, L., Clancy, A. & Ruhens, C. E. (2003). A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylates an inorganic pyrophosphatase and affects growth, cell segregation, and virulence. J Biol Chem 278, 14429–14441.[Abstract/Free Full Text]

Redenbach, M., Ikeda, K., Yamasaki, M. & Kinashi, H. (1998). Cloning and physical mapping of the EcoRI fragments of the giant linear plasmid SCP1. J Bacteriol 180, 2796–2799.[Abstract/Free Full Text]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Shi, L. (2004). Manganese-dependent protein O-phosphatases in prokaryotes and their biological functions. Front Biosci 9, 1382–1397.[Medline]

Shi, L. & Carmichael, W. W. (1997). pp1-cyano2, a protein serine/threonine phosphatase 1 gene from the cyanobacterium Microcystis aeruginosa UTEX 2063. Arch Microbiol 168, 528–531.[CrossRef][Medline]

Shi, L., Potts, M. & Kennelly, P. J. (1998). The serine threonine, and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait. FEMS Microbiol Rev 22, 229–253.[CrossRef][Medline]

Shi, L., Bischoff, K. M. & Kennelly, P. J. (1999a). The icfG gene cluster of Synechocystis sp. strain PCC 6803 encodes an Rsb/Spo-like protein kinase, protein phosphatase, and two phosphoproteins. J Bacteriol 181, 4761–4767.[Abstract/Free Full Text]

Shi, L., Carmichael, W. W. & Kennelly, P. J. (1999b). Cyanobacterial PPP-family protein phosphatases possess multifunctional capabilities and are resistant to microcystin-LR. J Biol Chem 274, 10039–10046.[Abstract/Free Full Text]

Shi, L., Kehres, D. G. & Maguire, M. E. (2001). The PPP-family protein phosphatases PrpA and PrpB of Salmonella enterica serovar Typhimurium possess distinct biochemical properties. J Bacteriol 183, 7053–7057.[Abstract/Free Full Text]

Shu, C. J. & Zhulin, I. B. (2002). ANTAR: an RNA-binding domain in transcription antitermination regulatory proteins. Trends Biochem Sci 27, 3–5.[CrossRef][Medline]

Solow, B., Young, J. C. & Kennelly, P. J. (1997). Gene cloning and expression and characterization of toxin-sensitive protein phosphatase from methanogenic archaeon Methanosarcina thermophila TM-1. J Bacteriol 179, 5072–5075.[Abstract]

Soulat, D., Vaganay, E., Duclos, B., Genestier, A. L., Etienne, J. & Cozzone, A. J. (2002). Staphylococcus aureus contains two low-molecular-mass phosphotyrosine protein phosphatases. J Bacteriol 184, 5194–5199.[Abstract/Free Full Text]

Stock, A. M., Robinson, V. L. & Goudreau, P. N. (2000). Two-component signal transduction. Annu Rev Biochem 69, 183–215.[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]

Treuner-Lange, A., Ward, M. J. & Zusman, D. R. (2001). Pph1 from Myxococcus xanthus is a protein phosphatase involved in vegetative growth and development. Mol Microbiol 40, 126–140.[CrossRef][Medline]

Ueda, K., Miyake, K., Horinouchi, S. & Beppu, T. (1993). A gene cluster involved in aerial mycelium formation in Streptomyces griseus encodes proteins similar to the response regulators of two-component regulatory systems and membrane translocators. J Bacteriol 175, 2006–2016.[Abstract]

Umeyama, T., Tanabe, Y., Aigle, B. D. & Horinouchi, S. (1996). Expression of the Streptomyces coelicolor A3(2) ptpA gene encoding a phosphotyrosine protein phosphatase leads to overproduction of secondary metabolites in S. lividans. FEMS Microbiol Lett 144, 177–184.[CrossRef][Medline]

Umeyama, T., Naruka, A. & Horinouchi, S. (2000). Genetic and biochemical characterization of protein phosphatase with dual substrate specificity in Streptomyces coelicolor A3(2). Gene 258, 55–62.[CrossRef][Medline]

Umeyama, T., Lee, P. C. & Horinouchi, S. (2002). Protein serine/threonine kinases in signal transduction for secondary metabolism and morphogenesis in Streptomyces. Appl Microbiol Biotechnol 59, 419–425.[CrossRef][Medline]

Vijay, K., Brody, M. S., Fredlund, E. & Price, C. W. (2000). A PP2C phosphatase containing a PAS domain is required to convey signals of energy stress to the {sigma}B transcription factor of Bacillus subtilis. Mol Microbiol 35, 180–188.[CrossRef][Medline]

Vincent, C., Doublet, P., Grangeasse, C., Vaganay, E., Cozzone, A. J. & Duclos, B. (1999). Cells of Escherichia coli contain a protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb. J Bacteriol 181, 3472–3477.[Abstract/Free Full Text]

Wang, L., Sun, Y.-P., Chen, W.-L., Li, J.-H. & Zhang, C. C. (2002). Genomic analysis of protein kinases, protein phosphatases and two-component regulatory systems of the cyanobacterium Anabaena sp. strain PCC 7120. FEMS Microbiol Lett 217, 155–165.[CrossRef][Medline]

Wugeditsch, T., Paiment, A., Hocking, J., Drummelsmith, J., Forrester, C. & Whitfield, C. (2001). Phosphorylation of Wzc, a tyrosine autokinase, is essential for assembly of group 1 capsular polysaccharides in Escherichia coli. J Biol Chem 276, 2361–2371.[Abstract/Free Full Text]

Yang, X., Kang, C. M., Brody, M. S. & Price, C. W. (1996). Opposing pair of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor. Genes Dev 10, 2265–2275.[Abstract]

Zhang, C. C., Friry, A. & Peng, L. (1998a). Molecular and genetic analysis of two closely linked genes that encode, respectively, a protein phosphatase1/2A/2B homolog and protein kinase homolog in the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 180, 2616–2622.[Abstract/Free Full Text]

Zhang, C. C., Gonzalez, L. & Phalip, C. (1998b). Survey, analysis and genetic organization of genes encoding eukaryotic-like signaling proteins on a cyanobacterial genome. Nucleic Acids Res 26, 3619–3625.[Abstract/Free Full Text]

Received 26 January 2004; revised 15 March 2004; accepted 19 April 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Shi, L.
Articles by Zhang, W.
Articles citing this Article
PubMed
PubMed Citation
Articles by Shi, L.
Articles by Zhang, W.
Agricola
Articles by Shi, L.
Articles by Zhang, W.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2004 Society for General Microbiology.