THE CYANOBACTERIUM SYNECHOCOCCUS SP STRAIN PCC-7942 CONTAINS A 2ND ALKALINE-PHOSPHATASE ENCODED BY PHOV

A gene (phoV) encoding an alkaline phosphatase from Synechococcus sp. strain PCC 7942 was isolated by screening a plasmid gene bank for expr ession of alkaline phosphatase activity in Escherichia coli JM103. Two independent clones carrying the same alkaline-phosphatase-encoding ge ne were isolated. One of these clones (pKW1) was further analysed and the nucleotide sequence of a contiguous 3234 bp DNA fragment was deter mined. Two complete open reading frames (ORF1 and phoV) and an incompl ete ORF3 were identified reading in the same direction. The deduced ph oV gene product showed 34% identity to the alkaline phosphatase PhoA f rom Zymomonas mobilis, and the N-terminal part of the putative ORF3 pr otein exhibited 57% identity to a protein of unknown function from Fra nkia sp. Insertional inactivation of the Synechococcus PCC 7942 phoV g ene failed, indicating an essential role for either the phoV or the OR F3 gene product. PhoV consists of 550 amino acid residues, resulting i n a molecular mass of 61.3 kDa. To overexpress the Synechococcus PCC 7 942 phoV gene in E. coli, plasmid pKW1 was transformed into a phoA mut ant of E. coli (CC118). In E. coli strain CC118(pKW1) PhoV was express ed constitutively with high rates of activity, and was shown to be mem brane associated in the periplasmic space. After partial purification of the recombinant PhoV, it was shown that, like other alkaline phosph atases, the Synechococcus PhoV had a broad pH optimum in the alkaline region and a broad substrate specificity for phosphomonoesters, requir ed Zn2+ for activity, and was inhibited by phosphate. In contrast to s everal other alkaline phosphatases, PhoV was inhibited by Mn2+. Due to the lack of a Synechococcus PCC 7942 phoV mutant strain, the function of PhoV remains uncertain. However, the present results show that Syn echococcus PCC 7942 has a second, probably phosphate-irrepressible, al kaline phosphatase (PhoV, 61.3 kDa) in addition to the phosphate-repre ssible enzyme (PhoA, 145 kDa) already described.