title: Functional analysis of a Campylobacter jejuni alkaline phosphatase secreted via the Tat export machinery.
abstract: Bacterial alkaline phosphatases (PhoA) hydrolyse phosphate-containing substrates to provide the preferred phosphorus source inorganic phosphate (P(i)). Campylobacter jejuni does not contain a typical PhoA homologue but contains a phosphatase that is regulated by the two-component system PhosS/PhosR. Here we describe the characterization of the enzyme, its secretion pathway and its function in the bacterium's biology. Phosphatase assays showed that the enzyme utilizes exclusively phosphomonoesters as a substrate, requires Ca(2+) for its activity, and displays maximum activity at a pH of 10. Gene disruption revealed that it is the sole alkaline phosphatase in C. jejuni. The protein contained a twin-arginine motif (RR) at its N terminus, typical of substrates of the Tat secretion system. Substitution of the twin-arginine residues showed that they are essential for enzyme activity. C. jejuni genome analysis indicated the presence of four ubiquitously expressed Tat components that may form a functional Tat secretion system as well as 11 putative Tat substrates, including the alkaline phosphatase (PhoA(Cj)) and the nitrate reductase NapA. Inactivation of tatC caused defects in both PhoA(Cj) and NapA activity as well as a reduction in bacterial growth that were all restored by complementation in trans with an intact tatC copy. The atypical overall features of the PhoA(Cj) compared to Escherichia coli PhoA support the existence in prokaryotes of a separate group of Tat-dependent alkaline phosphatases, classified as the PhoX family.
authors.affiliation: Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
authors.forename: Andries
authors.initials: A
authors.lastname: van Mourik
authors.affiliation: Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
authors.forename: Nancy M C
authors.initials: NMC
authors.lastname: Bleumink-Pluym
authors.affiliation: Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
authors.forename: Linda
authors.initials: L
authors.lastname: van Dijk
authors.affiliation: Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
authors.forename: Jos P M
authors.initials: JPM
authors.lastname: van Putten
authors.affiliation: Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
authors.forename: Marc M S M
authors.initials: MMSM
authors.lastname: Wösten
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001120:Arginine; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D016123:Campylobacter jejuni / Q000201:enzymology*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000378:metabolism; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D001120:Arginine; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli; D002118:Calcium