The Escherichia coli acid phosphatase gene appA is expressed in response to oxygen deprivation and is positively controlled by the product of appR (katF) which encodes a putative new sigma transcription-initiation factor. However, transcription of appA from its nearest promoter (P1) did not account for total pH 2.5 acid phosphatase expression and was not subject to regulation. The cloned region upstream of appA was extended and analyzed by insertions of transposon TnphoA and by fusions with lacZ. It contains two new genes, appC and appB, which both encode extracytoplasmic proteins. appC and appB are expressed from a promoter (P2) lying just upstream of appC. Both genes are regulated by oxygen, as is appA, and by appR gene product exactly as previously shown for appA. Analysis of the nucleotide sequence and of the origins of transcription have confirmed that the P2-appC-appB- (ORFX)-P1-appA region is organized on the chromosome as an operon transcribed clockwise from P2 and that P1 is a minor promoter for appA alone. Genes appC and appB encode proteins of Mr 58,133 and 42,377, respectively, which have the characteristics of integral membrane proteins. The deduced amino acid sequences of appC and appB show 60% and 57% homology, respectively, with subunits I and II of the E. coli cytochrome d oxidase (encoded by genes cydA and cydB). The notion that the AppC and AppB proteins constitute a new cytochrome oxidase or a new oxygen-detoxifying system is supported by the observation of enhanced sensitivity to oxygen of mutants lacking all three genes, cyo (cytochrome o oxidase), cyd (cytochrome d oxidase) and appB, compared to that of cyo cyd double mutants.

The whole nucleotide sequence of Escherichia coli gene appA, which encodes periplasmic phosphoanhydride phosphohydrolase (optimum pH, 2.5), and its flanking regions was determined. The AppA protein is significantly homologous to the product of the nearby gene agp, acid glucose-1-phosphatase. Because identical amino acids are distributed over the whole lengths of the proteins, it is likely that appA and agp originate from the same ancestor gene.

No abstract

Several acid phosphatases in Escherichia coli have been described (27), but the exact number of distinct enzyme species, their physiological role, and their mode of regulation are not known precisely. Enzymatic activities promoting the hydrolysis of the synthetic substrate p-nitrophenylphosphate (pNPP) in acidic conditions have been found in osmotic shock fluids (4,16,26). Three such genes have been identified: (i) gene ush specifies UDP glucose hydrolase (25) and lies in the 11-min region of the chromosome (6); (ii) appA, the structural gene for an acid phosphoanhydride phosphohydrolase, which also cleaves pNPP (14), maps at min 22 (13); and (iii) gene cpdB encodes the 2'-3' cyclic phosphodiesterase (1-3), which is also active against pNPP (16) and is located at min 96 (5). The three genes have now been cloned, their restriction maps are known, and their nucleotide sequences have been partly or entirely elucidated (9,11,22,29).At least two other pNPP-hydrolyzing enzymes with pH optima ranging from 4.0 to 6.0 have been described and partially purified, namely an acid hexose phosphatase and a nonspecific acid phosphatase (16,27,31

The structural gene (appA) for the periplasmic acid phosphatase (optimum pH 2.5) of Escherichia coli was cloned into a plasmid by using a combination of in vivo and in vitro techniques. The position and orientation of the appA gene within the cloned DNA fragment were identified by using fusions to the alkaline phosphatase gene (phoA) generated by Tn5 IS50L::phoA (TnphoA) insertions. For TnphoA-generated hybrid proteins to have high enzymatic activity, it appears that the phoA gene must be fused to a target gene coding for a signal which promotes protein export. The approach used to identify the appA gene thus appears to provide a simple general means of selectively identifying genes encoding membrane and secreted proteins.