Use of TnphoA to detect genes for exported proteins in Escherichia coli: identification of the plasmid-encoded gene for a periplasmic acid phosphatase

Boquet, P; Manoil, Colin; Beckwith, Jonathan

doi:10.1128/jb.169.4.1663-1669.1987

Cited by 53 publications

(45 citation statements)

References 24 publications

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“…Restriction analysis shows that this gene is different from cpdB, ush, or appA (8,9,11,22). Comparison of our results with previous data from Dvorak et al (16) suggests that the product of gene agp could be different from the acid hexose phosphatase purified earlier from wild-type cells and also reported to be active as a dimer.…”

Section: Discussionsupporting

confidence: 63%

“…Colonies of bacteria from the two related clones pEP1339 and pEP1341 were noticeably stained by the chromogenic substrate XP, even when grown on a high-phosphate medium, suggesting that the enzyme specified could contribute to the known background staining of wild-type strains or AphoA mutants by XP. The 1.7-kb-long BamHI-SphI chromosomal DNA fragment common to these two clones and subcloned into pBR322 promoted the overexpression of a (8,24).…”

Section: Discussionmentioning

confidence: 99%

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Acid phosphatases of Escherichia coli: molecular cloning and analysis of agp, the structural gene for a periplasmic acid glucose phosphatase

Pradel

Boquet

1988

J Bacteriol

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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

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Section: Discussionsupporting

confidence: 63%

Section: Discussionmentioning

confidence: 99%

Acid phosphatases of Escherichia coli: molecular cloning and analysis of agp, the structural gene for a periplasmic acid glucose phosphatase

Pradel

Boquet

1988

J Bacteriol

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“…The precise cause of this degradation is unknown, but cleavage of other PhoA protein fusions has been observed in both R. sphaeroides (36,48) and E. coli (1,3,30,45). In fact, we see similar degradation of the CycA-PhoA hybrids in E. coli CC118 (data not shown), so it is apparently not related to the physiological function of cyt c2 in electron transport.…”

Section: Discussionsupporting

confidence: 52%

Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function

et al. 1991

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Cytochrome c2 is a periplasmic redox protein involved in both the aerobic and photosynthetic electron transport chains of Rhodobacter sphaeroides. The process of cytochrome c2 maturation has been analyzed in order to understand the protein sequences involved in attachment of the essential heme moiety to the cytochrome c2 polypeptide and localization of the protein to the periplasm. To accomplish this, five different translational fusions which differ only in the cytochrome c2 fusion junction were constructed between cytochrome c2 and the Escherichia coli periplasmic alkaline phosphatase. All five of the fusion proteins are exported to the periplasmic space. The four fusion proteins that contain the NH2-terminal site of covalent heme attachment to cytochrome c2 are substrates for heme binding, suggesting that the COOH-terminal region of the protein is not required for heme attachment. Three of these hybrids possess heme peroxidase activity, which indicates that they are functional as electron carriers. Biological activity is possessed by one hybrid protein constructed five amino acids before the cytochrome c2 COOH terminus, since synthesis of this protein restores photosynthetic growth to a photosynthetically incompetent cytochrome c2-deficient derivative of R. sphaeroides. Biochemical analysis of these hybrids has confirmed CycA polypeptide sequences sufficient for export of the protein (A. R. Varga and S. Kaplan, J. Bacteriol. 171:5830-5839, 1989), and it has allowed us to identify regions of the protein sufficient for covalent heme attachment, heme peroxidase activity, docking to membrane-bound redox partners, or the capability to function as an electron carrier.

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“…Degradation products with a size about the same as that of alkaline phosphatase (47 kDa) were found from both hybrid proteins. Similar proteolytic processing occurring in vivo or during the preparation of cell fractions has been reported for several other PhoA fusion proteins [10,38,39]. Fig.…”

Section: Topology Of Cytochrome C-550 In the Membranementioning

confidence: 72%

Bacillussubtilis holo‐cytochrome c‐550 can be synthesised in aerobic Escherichia coli

Wachenfeldt

Hederstedt

1990

FEBS Letters

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Bacillus subtilis membrane‐bound holo‐cytochrome c‐550 was found to be expressed from the structural gene cloned on a plasmid vector in aerobically grown Escherichia coli and exhibited normal biochemical properties. This occurs despite the lack of endogenous eytochrome c and suggests that eytochrome c‐heme lyase activity is also present in aerobic E. coli. The membrane topology of B. subtilis eytochrome c‐550 was studied using fusions to alkaline phosphatase (PhoA). The results show that the heme domain (at least when fused to PhoA) can be translocated as apo‐cytochrome and confirm that the N‐terminal part of the cytochrome functions as both export signal and membrane anchor for the C‐tenninal heme domain. A model for the organisation of B. subtilis cytochrome c‐550 in the cytoplasmic membrane is presented.

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Use of TnphoA to detect genes for exported proteins in Escherichia coli: identification of the plasmid-encoded gene for a periplasmic acid phosphatase

Cited by 53 publications

References 24 publications

Acid phosphatases of Escherichia coli: molecular cloning and analysis of agp, the structural gene for a periplasmic acid glucose phosphatase

Acid phosphatases of Escherichia coli: molecular cloning and analysis of agp, the structural gene for a periplasmic acid glucose phosphatase

Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function

Bacillussubtilis holo‐cytochrome c‐550 can be synthesised in aerobic Escherichia coli

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