1887

Abstract

A gene () encoding an alkaline phosphatase from sp. strain PCC 7942 was isolated by screening a plasmid gene bank for expression of alkaline phosphatase activity in JM103. Two independent clones carrying the same alkaline-phosphatase-encoding gene were isolated. One of these clones (pKW1) was further analysed and the nucleotide sequence of a contiguous 3234 bp DNA fragment was determined. Two complete open reading frames (ORF1 and ) and an incomplete ORF3 were identified reading in the same direction. The deduced gene product showed 34% identity to the alkaline phosphatase PhoA from , and the N-terminal part of the putative ORF3 protein exhibited 57% identity to a protein of unknown function from sp. Insertional inactivation of the PCC 7942 gene failed, indicating an essential role for either the or the ORF3 gene product. PhoV consists of 550 amino acid residues, resulting in a molecular mass of 61.3 kDa. To overexpress the PCC 7942 gene in , plasmid pKW1 was transformed into a mutant of (CC118). In strain CC118(pKW1) PhoV was expressed constitutively with high rates of activity, and was shown to be membrane associated in the periplasmic space. After partial purification of the recombinant PhoV, it was shown that, like other alkaline phosphatases, the PhoV had a broad pH optimum in the alkaline region and a broad substrate specificity for phosphomonoesters, required Zn† for activity, and was inhibited by phosphate. In contrast to several other alkaline phosphatases, PhoV was inhibited by Mn†. Due to the lack of a PCC 7942 mutant strain, the function of PhoV remains uncertain. However, the present results show that PCC 7942 has a second, probably phosphate-irrepressible, alkaline phosphatase (PhoV, 61.3 kDa) in addition to the phosphate-repressible enzyme (PhoA, 145 kDa) already described.

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1995-12-01
2021-05-18
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