Microbiology Department, Pacific Northwest National Laboratory, 902 Battelle Blvd, PO Box 999, Mail Stop: P7-50, Richland, WA 99352, USA
Correspondence
Weiwen Zhang
weiwen.zhang{at}pnl.gov
Liang Shi
liang.shi{at}pnl.gov
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ABSTRACT |
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INTRODUCTION |
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In our previous study (Shi & Zhang, 2004), we identified 55 eukaryotic-type PPs in each of the S. coelicolor and Streptomyces avermitilis genomes. Considering that most of the previously surveyed prokaryotic genomes contained fewer than ten eukaryotic-type PPs (Shi et al., 1998
), the number of PPs found in Streptomyces was unexpectedly large. Analysis of the catalytic domains of the Streptomyces PPs showed that 49 of the PPs in S. coelicolor and 48 of the PPs in S. avermitilis belong to the PPM family. Only 29 of these PPMs have orthologues in both species (Shi & Zhang, 2004
). As these streptomycetes have the largest number of PPMs ever identified from any single prokaryotic organism, the existence of PPMs in Streptomyces genomes raises questions of how they originated. Comparison of the S. coelicolor and S. avermitilis genomes revealed that the chromosomes appear to have expanded by internal duplication of DNA, and gene acquisition by horizontal transfer (Bentley et al., 2002
). This expansion has presumably allowed these organisms to adapt to a wider range of environmental conditions, and exploit a greater variety of nutrient sources. The preferential incorporation (and subsequent maintenance) of occasionally beneficial sequences outside the conserved ancestral core region of the chromosome has created arms comprising mostly non-essential functions (Bentley et al., 2002
; Ikeda et al., 2003
). Consistent with this hypothesis, we have found that the majority of Streptomyces PPM genes that are unique to each genome are located outside of the core conserved region (Shi & Zhang, 2004
). This suggests that recent gene acquisition or duplication events might be responsible, at least to a certain degree, for the presence of such a large number of PPMs in Streptomyces genomes. In this study, we have performed a detailed phylogenetic and domain structural analysis of the Streptomyces PPMs to show that these genes were divided into two major subfamilies after their establishment in the Streptomyces genome. Our analysis indicates that one PPM subfamily (designated subfamily I) has retained the original eukaryotic PPM domain architecture through the course of evolution. In contrast, the other PPM subfamily (designated subfamily II) has been subjected to significant evolutionary modification, including gene duplication and additional sensory domain recruitment. These observations suggest a mechanism by which Streptomyces species may have acquired a diverse population of PPMs to provide the robust signal transduction systems required for survival in the extremely diverse soil environments in which they are found.
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METHODS |
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Sequence alignment and phylogenetic reconstruction.
The protein sequences of a total of 148 PPMs (146 from prokaryotes and 2 from eukaryotes) were retrieved from the NCBI or the TIGR database (http://www.ncbi.nlm.nih.gov or http://www.tigr.org). The domain structures were identified and analysed using the molecular architecture research tools provided by SMART (http://smart.embl-heidelberg.de) with E value <0·01 (Letunic et al., 2002). The sequence alignments were performed using default parameters of the CLUSTALW program available from the LaserGene software package (DNAStar), and PAUP* 4.0 beta version (Blumenberg, 1988
). Confidence levels were determined by analysing 100 bootstrap replicates.
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RESULTS |
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Phylogenetic analysis of the catalytic domains of Streptomyces PPMs
To infer the evolutionary histories of Streptomyces PPMs, a phylogenetic analysis was performed using sequences of PPM catalytic domains rather than complete coding sequences of whole proteins, in order to increase the confidence level of sequence alignment. The protein sequences of the 49 identified microbial PPMs, 2 eukaryotic PPMs, and 97 PPMs identified from the S. coelicolor and S. avermitilis genomes, were subjected to domain identification using the molecular architecture research tools provided by SMART. The PPM catalytic domain sequences, which were about 220 aa with all the conserved residues (Fig. 1), were then extracted and used for phylogenetic tree construction. A phylogenetic tree was generated from the aligned catalytic domain sequences, and the confidence of the tree topology was evaluated by bootstrap analysis (Fig. 2
).
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Another interesting observation that can be made from our phylogenetic analysis is that there was strong bootstrap support for the division of Streptomyces PPMs into two major subfamilies. Subfamily I contained only 6 Streptomyces PPMs (three pairs of homologues from genomes of S. coelicolor and S. avermitilis), and 31 other microbial PPMs from 24 different microbial species. The catalytic domains of the PPM subfamily I all shared the same domain architecture as eukaryotic PPMs, in that they contained 11 conserved motifs, with 8 absolutely conserved residues. The diversification of the PPM subfamily I functional domain between species might be due to the subsequent bacterial speciation and functional specialization. No obvious sign of gene duplication was found for the PPMs in this subfamily. Subfamily II, however, contained 91 Streptomyces PPMs and 18 PPMs from other microbial sources, mainly from cyanobacteria and Bacillus. PPMs from this subfamily showed a relatively high degree of similarity, indicating that some might have arisen by gene duplication events occurring after the original acquisition of the progenitor PPM gene. Comparison of the architecture of catalytic domains showed that they all lacked the 5a and 5b motifs, and showed much less sequence similarity to eukaryotic PPMs. These results suggested that the diversification of architecture in the PPM subfamily II may be due more to functional specialization than to bacterial speciation.
Phylogenetic analysis of additional sensory domains of Streptomyces PPMs
Our previous study reported that 63 % of PPMs from S. coelicolor and 75 % of the PPMs from S. avermitilis possess at least one extra sensory domain, in addition to their PP catalytic domain (Shi & Zhang, 2004). Interestingly, all of the PPMs with additional sensory domains were from subfamily II (Fig. 2
), while none of the PPMs from subfamily I contained any additional sensory domain. Two types of sensory domains were most frequently found to be added in these Streptomyces subfamily II PPMs. The first was a PAS domain that binds the chromophore 4-hydroxycinnamyl, and is involved in sensing energy-related environmental factors such as oxygen, redox potential or light; the second is the GAF domain involved in binding cyclic nucleotides (Shi & Zhang, 2004
; Taylor & Zhulin, 1999
; Taylor et al., 1999
; Aravind & Ponting, 1997
). Five of the Streptomyces PPMs with an additional sensory domain were found to contain the PAS domain only, 14 contained the GAF domain only, and 29 contained both PAS and GAF domains (Fig. 2
). Outside of the Streptomyces PPMs, a PAS domain was also found in the B. subtilis RsbP PPM gene, and GAF domains were found in PPMs from Synechocystis sp., Thermosynechococcus elongatus and Synechococcus sp. WH8102. In addition to the PAS and GAF domains, all four of the PPMs identified in Nostoc sp. PCC 7120 contained REC domains. The REC domain encodes a cheY-homologous receiver domain involved in receiving signals from the sensor partner in bacterial two-component signal transduction systems. The observation that these REC domains were primarily present in subfamily II PPMs in Nostoc suggests that there may be species preferences in recruiting additional sensory domains, possibly due to different environmental selective pressures.
For Streptomyces spp. living in particularly complex and variable soil environments, having multiple sensory domains added to the PPMs may provide a selective advantage by allowing these PPMs to sense multiple environmental signals and/or interact with multiple regulatory proteins simultaneously. The observation that the additional sensory domains in Streptomyces PPMs are found exclusively in subfamily II raises the possibility that the additional sensory domains were recruited into the subfamily II PPM molecules at a late evolutionary stage. To test this hypothesis, we performed a detailed phylogenetic analysis of PAS and GAF domains to see whether they cluster with genes of unrelated lineage. Forty-two PAS domain sequences (mean of 65100 aa) and 45 sequences of GAF domain (mean of 150200 aa) were identified from Streptomyces PPMs using the SMART program, and were used to construct phylogenetic trees, along with a few dozen PAS or GAF domains from other bacterial sources. The bootstrap analysis was performed to check the reliability of the cluster assignments (Fig. 3). Analysis of PAS domains showed seven recognizable clusters, each with strong bootstrap support. Although individual exceptions were present, significant clustering based on their putative physiological function, rather than taxonomic relationship, was observed within each PAS cluster (Fig. 3A
). This suggests that PAS domains with different functional specialities were either recruited into Streptomyces PPMs directly from other species, or that they were already present in Streptomyces and were later recruited into PPM molecules.
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Further evidence for lateral recruitment of additional sensory domains was provided by the phylogenetic trees of catalytic domains and PAS or GAF domains. An obvious incongruity is observed when comparing these trees, in that two PPMs with homologous catalytic domains do not necessarily possess the same additional sensory domains. For example, the SAV4009 PPM (from S. avermitilis) contains both PAS and GAF domains, while its homologous PPM SCO4201 in S. coelicolor has no additional sensory domain, suggesting that the additional domain recruitment occurred after the Streptomyces speciation. These results suggest that both PAS and GAF domain recruitment in Streptomyces PPMs may not have originated from a simple scheme of vertical transmission occurring concurrently with speciation, but may be the result of domain recruitment in later evolutionary stages. This domain recruitment may have occurred as a result of adaptive function specialization occurring after duplication of the catalytic domain of another PPM gene.
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DISCUSSION |
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PPMs are functionally diverse enzymes, and they have been found to be involved in regulation of cellular responses to environmental stress in mammalian and yeast cells, and to the growth regulator abscisic acid in plant cells. The functions of the microbial PPMs that have been characterized include regulation of spore formation, stress response, cell density during stationary phase, carbon and nitrogen assimilation, vegetative growth, development of fruiting bodies and cell segregation (Beuf et al., 1994; Duncan et al., 1995
; Gaidenko et al., 2002
; Irmler & Forchhammer, 2001
; Rajagopal et al., 2003
; Shi et al., 1999
; Treuner-Lange et al., 2001
; Yang et al., 1996
). It is notable that most bacterial species containing relatively large numbers of PPM-encoding genes, such as Anabaena sp. PCC 7120, Bacillus spp. and Streptomyces spp., are able to undergo complex morphological differentiation, similar to that seen in multicellular eukaryotes. Although it still needs proof, preliminary analysis points to a possible link between the complexity of a micro-organism's life style, and the distribution of PPM-encoding genes in its genome. In the past several years, extensive knowledge has been accumulating on the cellular roles of PAS-domain-based signalling systems and, on the basis of this knowledge, it is often possible to propose a role for a particular PAS domain based on known functions in similar signalling systems (Taylor & Zhulin, 1999
). Our observation that most of the PAS domains from Streptomyces PPMs are clustered with PAS domains of known function from other species might thus provide some guidance for further investigations of the physiological functions of Streptomyces PPMs.
Although the physiological function of most Streptomyces PPMs is still unknown, studies in B. subtilis suggest that sigma factors might be one of the predominant targets regulated by PPM-mediated signal transduction pathways (Mittenhuber, 2002). In support of this, a B. subtilis PPM containing a PAS domain was recently found to be necessary for conveying signals of energy stress to the sigma B transcription factor in this organism (Vijay et al., 2000
). A possible functional link between PPMs and sigma factors is also suggested by the correlation between the number of PPMs and sigma factors in each genome, with most PPM-rich micro-organisms also containing a large number of sigma factors. For example, B. subtilis contains 18 sigma factors, Synechocystis sp. PCC 6803 contains 7, and Anabaena sp. PCC 7120 contains 9 (Gruber & Gross, 2003
). A recent survey has identified an astonishing 63 sigma-factor-encoding genes from the S. coelicolor genome, and phylogenetic analysis suggested that gene duplications might be responsible for the origin of some of these sigma factors. The gene duplication in group 4, which contains 49 out of the total of 63 sigma factors, was particularly evident. Among this group are the sigma factors
BldN and
E involved in regulation of aerial hyphae formation and the integrity of the cell envelope (Gruber & Gross, 2003
). Although still requiring biochemical and genetic verification, the correlation between the numbers of PPMs and sigma factors, and their apparently similar evolutionary histories, strongly suggests that the PPMs in Streptomyces, especially those from the second subfamily, are most likely to be involved in regulating the activities of at least some sigma factors.
Like the PPMs previously characterized from other bacteria (Shi, 2004), Streptomyces PPMs were clearly divided into two major subfamilies. However, the unbalanced distribution of these Streptomyces PPMs into the two subfamilies is unusual. The first subfamily contains only six Streptomyces PPMs, each with a conserved catalytic domain architecture similar that of eukaryotic PPMs, and showing no sign of either gene duplication or additional domain recruitment. One possible explanation could be that these six PPMs are involved in the regulation of critical house-keeping functions, and their regulatory functions were specialized at a very early stage of evolution. If this were the case, any gene duplication and mutation in this subfamily would be likely to have an adverse effect on the fitness of the cell. In contrast, the PPM genes in the second subfamily might be involved in the regulation of less essential metabolic activities, and would therefore be flexible enough to serve as a basis for gene duplication and domain recruitment to generate novel sensory and regulatory activities to deal with highly variable environments. Several recent studies comparing multiple complete genomes from phylogenetically distant species have concluded that the number of universally conserved gene families is very small, and that multiple events of horizontal gene transfer and domain recruitment within or across species constitute a major evolutionary path to the generation of novel genes (Copley et al., 2003
; Koonin & Galperin, 1997
). This is especially applicable in the case of the non-essential genes that tend to be recruited to deal with the survival needs in new niches. The phenomenon of horizontal gene transfer between streptomycetes and other bacteria and eukaryotes has been suggested to be responsible for genomic changes ranging from acquisition of individual genes, to entire antibiotic gene clusters (Egan et al., 2001
; Coque et al., 1991
). Such gene transfers have been demonstrated to occur at high frequencies, even in the soil environment (Wellington et al., 1992
). It is especially noteworthy that the large-scale domain recruitment we observed in Streptomyces PPMs represents regulatory rather than structural genes, since it has been proposed that regulatory functions are not routinely acquired by means of heterogeneous acquisition (Jain et al., 1999
; Ma & Zeng, 2004
). Our study provides an insight into how Streptomyces spp. may have expanded their PPM-based signal transduction networks to enable them to respond to a greater range of environmental changes.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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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, 141147.[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, 855864.[Medline]
Blumenberg, M. (1988). Concerted gene duplications in the two keratin gene families. J Mol Evol 27, 203211.[Medline]
Bork, P., Brown, N. P., Hegyi, H. & Schultz, J. (1996). The protein phosphatase 2C (PP2C) superfamily: detection of bacterial homologues. Protein Sci 5, 14211425.
Chater, K. F. (1993). Genetics of differentiation in Streptomyces. Annu Rev Microbiol 47, 683713.
Cohen, P. T. W. (1994). Nomenclature and chromosomal localization of human protein serine/threonine phosphatase genes. Adv Protein Phosphatases 8, 371376.
Copley, R. R., Goodstadt, L. & Ponting, C. (2003). Eukaryotic domain evolution inferred from genome comparisons. Curr Opin Genet Dev 13, 623628.[CrossRef][Medline]
Coque, J. J., Martin, J. F., Calzada, J. G. & Liras, P. (1991). The cephamycin biosynthetic genes pcbAB, encoding a large multidomain peptide synthetase, and pcbC of Nocardia lactamdurans are clustered together in an organization different from the same genes in Acremonium chrysogenum and Penicillium chrysogenum. Mol Microbiol 5, 11251133.[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, 641644.[Abstract]
Egan, S., Wiener, P., Kallifidas, D. & Wellington, E. M. (2001). Phylogeny of Streptomyces species and evidence for horizontal transfer of entire and partial antibiotic gene clusters. Antonie Van Leeuwenhoek 79, 127133.[CrossRef][Medline]
Gaidenko, T., Kim, T. J. & Price, C. W. (2002). The PrpC serine-threonine phosphatase and PrkC kinase have opposing physiological roles in stationary-phase Bacillus subtilis cells. J Bacteriol 184, 61096114.
Gruber, T. M. & Gross, C. A. (2003). Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol 57, 441466.[CrossRef][Medline]
Hopwood, D. A. (1999). Forty years of genetics with Streptomyces: from in vivo through in vitro to in silico. Microbiology 145, 21832202.[Medline]
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, 462467.
Ikeda, H., Ishikawa, J., Hanamoto, K. & 7 other authors (2003). Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nature Biotechnol21, 526531.[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, 1297812983.
Jain, R., Rivera, M. C. & Lake, J. A. (1999). Horizontal gene transfer among genomes: the complexity hypothesis. Proc Natl Acad Sci U S A 96, 38013806.
Kennelly, P. J. (2001). Protein phosphatase a phylogenetic perspective. Chem Rev 101, 22912312.[CrossRef][Medline]
Kennelly, P. J. (2002). Protein kinases and protein phosphatases in prokaryotes: a genomic perspective. FEMS Microbiol Lett 206, 18.[CrossRef][Medline]
Koonin, E. V. & Galperin, M. Y. (1997). Prokaryotic genomes: the emerging paradigm of genome-based microbiology. Curr Opin Genet Dev 7, 757763.[CrossRef][Medline]
Letunic, I., Goodstadt, L., Dickens, N. J. & 7 other authors (2002). Recent improvements to the SMART domain-based sequence annotation resource. Nucleic Acids Res 30, 242244.
Li, Y. & Strohl, W. R. (1996). Cloning, purification, and properties of a phosphotyrosine protein phosphatase from Streptomyces coelicolor A3(2). J Bacteriol 178, 136142.[Abstract]
Ma, H. W. & Zeng, A. P. (2004). Phylogenetic comparison of metabolic capacities of organisms at genome level. Mol Phylogenet Evol 31, 204213.[CrossRef][Medline]
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, 383387.
Mittenhuber, G. (2002). A phylogenomic study of the general stress response sigma factor sigmaB of Bacillus subtilis and its regulatory proteins. J Mol Microbiol Biotechnol 4, 427452.[CrossRef][Medline]
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, 1523.
Ponting, C. P., Aravind, L., Schultz, J., Bork, P. & Koonin, E. V. (1999). Eukaryotic signalling domain homologues in archaea and bacteria. Ancient ancestry and horizontal gene transfer. J Mol Biol 1289, 729745.[CrossRef]
Rajagopal, L., Clancy, A. & Ruhens, C. E. (2003). A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylate an inorganic pyrophosphatase and affect growth, cell segregation, and virulence. J Biol Chem 278, 1442914441.
Shi, L. (2004). Manganese-dependent protein O-phosphatases in prokaryotes and their biological functions. Front Biosci 9, 13821397.[Medline]
Shi, L. & Zhang, W. (2004). Comparative analysis of eukaryotic-type protein phosphatases in two streptomycete genomes. Microbiology 150, 22472256.[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, 229253.[CrossRef][Medline]
Shi, L., Bischoff, K. M. & Kennelly, P. J. (1999). 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, 47614767.
Taylor, B. L. & Zhulin, I. B. (1999a). PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol Mol Biol Rev 63, 479506.
Taylor, B. L., Zhulin, I. B. & Johnson, M. S. (1999b). Aerotaxis and other energy-sensing behavior in bacteria. Annu Rev Microbiol 53, 103128.[CrossRef][Medline]
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, 126140.[CrossRef][Medline]
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, 177184.[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, 5562.[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, 419425.[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 sigmaB transcription factor of Bacillus subtilis. Mol Microbiol 35, 180188.[CrossRef][Medline]
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, 155165.[CrossRef][Medline]
Wellington, E. M., Cresswell, N. & Herron, P. R. (1992). Gene transfer between streptomycetes in soil. Gene 115, 193198.[CrossRef][Medline]
Woese, C. R., Kandler, O. & Wheelis, M. L. (1990). Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A 87, 45764579.[Abstract]
Yang, X., Kang, C. M., Brody, M. S. & Price, C. W. (1996). Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor. Genes Dev 10, 22652275.[Abstract]
Zhang, C. C., Gonzalez, L. & Phalip, C. (1998). Survey, analysis and genetic organization of genes encoding eukaryotic-like signaling proteins on a cyanobacterial genome. Nucleic Acids Res 26, 36193625.
Received 9 July 2004;
revised 29 August 2004;
accepted 3 September 2004.
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