Substrate analysis and molecular cloning of the extracellular alkaline phosphatase of Streptomyces griseus The GenBank accession number for the sequence reported in this paper is AJ278740.

Abstract

Streptomyces species secrete large amounts of alkaline phosphatase (AP) enzymes that have not been characterized so far. An AP has been purified to homogeneity from cultures of Streptomyces griseus IMRU 3570. The enzyme has a monomer size of 62 kDa and is processed in the culture to a 33 kDa protein as shown by immunoblotting. The enzyme was purified by ammonium sulfate precipitation, CM-Sephadex cationic exchange, chromatofocusing and HPLC Sphaerogel 3000SW filtration. The pure enzyme uses a variety of organic phosphorylated compounds as substrates. The N-terminal end of the mature protein was found to be RLREDPFTLGVASGDPHP. The gene phoA has been cloned using as probe an oligomer based on the N-terminal sequence of the S. griseus AP. phoA encodes a protein of 62 678 Da with low homology to the AP of Escherichia coli. The phoA gene was found to be homologous to three alkaline-phosphatase-encoding genes previously identified in the Streptomyces coelicolor genome. On the basis of the optimal pH, substrate specificity and differences in amino acid sequence of motifs defining the active centre of APs, the S. griseus AP uses a wide range of organic phosphate substrates and is different from the phosphatases of Gram-negative bacteria.

Phosphate control of phoA, phoC and phoD gene expression in Streptomyces coelicolor reveals significant differences in binding of PhoP to their promoter regions

“…Interestingly, similar BLAST searches in the genomes of Streptomyces avermitilis and Streptomyces scabies only identified two possible alkaline phosphatases, corresponding to phoA and phoD; so the phoC gene of S. coelicolor seems to be a more recent addition to the S. coelicolor genome and might be involved in the biosynthesis of some S. coelicolor-specific metabolite. PhoA of S. coelicolor corresponds to an authentic alkaline phosphatase with high similarity to the well-known extracellular alkaline phosphatase of S. griseus (Moura et al, 2001) and other micro-organisms. It has been shown to be secreted via the Tat pathway (Widdick et al, 2006) but also contains a typical leader peptide as described for Secdependent secretion (Schaerlaekens et al, 2004).…”

“…Three putative alkaline phosphatase genes, phoA, phoC and phoD, were identified in the genome of S. coelicolor by a comparative search with the phoA gene of S. griseus (corresponding to the sequenced alkaline phosphatase protein; Moura et al, 2001). The encoded proteins PhoA (558 aa, SCO2286), PhoC (553 aa, SCO0828) and PhoD (551 aa, SCO2068) ( Fig.…”

“…Three different alkaline phosphatase genes were initially described in the S. coelicolor genome (Moura et al, 2001;Bentley et al, 2002). Interestingly, similar BLAST searches in the genomes of Streptomyces avermitilis and Streptomyces scabies only identified two possible alkaline phosphatases, corresponding to phoA and phoD; so the phoC gene of S. coelicolor seems to be a more recent addition to the S. coelicolor genome and might be involved in the biosynthesis of some S. coelicolor-specific metabolite.…”

“…Streptomyces species and other soil micro-organisms may use inorganic phosphate, which occurs frequently in the form of partially insoluble phosphate salts, and organic phosphate, which is present in a variety of plant materials such as phytic acid (Martínez-Domínguez et al, 2002), sugar phosphates and nucleotides (Martín & Demain, 1977). To obtain phosphate from organic compounds, Streptomyces and other bacteria use an extracellular alkaline phosphatase encoded by the phoA gene (Moura et al, 2001). When the Streptomyces griseus phoA gene was cloned and compared with the genome of Streptomyces coelicolor, two other putative phosphatase genes (hereafter named phoC and phoD; phoB was avoided because this designation is used for the response regulator in Escherichia coli), in addition to the phoA orthologue, were found (Moura et al, 2001;Bentley et al, 2002).…”

“…To obtain phosphate from organic compounds, Streptomyces and other bacteria use an extracellular alkaline phosphatase encoded by the phoA gene (Moura et al, 2001). When the Streptomyces griseus phoA gene was cloned and compared with the genome of Streptomyces coelicolor, two other putative phosphatase genes (hereafter named phoC and phoD; phoB was avoided because this designation is used for the response regulator in Escherichia coli), in addition to the phoA orthologue, were found (Moura et al, 2001;Bentley et al, 2002). Different phosphatases may work on different phosphorylated substrates.…”