title: Contribution of Phe-7 to Tat-dependent export of β-lactamase in Xanthomonas campestris.
abstract: Strains of Xanthomonas campestris pv. campestris isolated in Taiwan are commonly resistant to ampicillin owing to the constitutive expression of a chromosomally encoded β-lactamase that is secreted into the periplasm. In this study, we found that levels of β-lactamase vary among X. campestris pv. campestris strains, a difference that can be attributed to amino acid substitutions at least at positions 7 and 206, with the former having the major impact. Bioinformatic and PCR analyses indicated that X. campestris pv. campestris possesses tatABC genes and that the signal peptide of X. campestris pv. campestris pre-Bla contains the typical twin-arginine motif (N-R-R-Q-F-L at amino acid residues 3 to 8 in strain X. campestris pv. campestris strain 11), suggesting that Bla is secreted via the Tat pathway. To assess the importance of Phe(7) in the efficient export of X. campestris pv. campestris Bla, we prepared mutant constructs containing amino acid substitutions and monitored their expression by measuring enzyme activity and detecting Bla protein by Western blotting. The results indicate that replacement of Phe(7) with Leu severely inhibited Bla export whereas replacement with Pro almost abolished it. Although a change to Arg caused moderate inhibition of export, replacement with Tyr had no effect. These results suggest that for efficient export of Bla by X. campestris pv. campestris, the aromatic-aromatic interactions and stability of protein structure around the twin-arginine motif are important, since only proteins that can attain a folded state in the cytoplasm are competent for export via the Tat pathway.
authors.affiliation: Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Chen-Wei
authors.initials: CW
authors.lastname: Lee
authors.forename: Yi-Hsuan
authors.initials: YH
authors.lastname: Tseng
authors.forename: Fu-Seng
authors.initials: FS
authors.lastname: Deng
authors.forename: Juey-Wen
authors.initials: JW
authors.lastname: Lin
authors.forename: Yi-Hsiung
authors.initials: YH
authors.lastname: Tseng
authors.forename: Shu-Fen
authors.initials: SF
authors.lastname: Weng
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D017422:Sequence Analysis, DNA; D013624:Taiwan; D016959:Xanthomonas campestris / Q000201:enzymology* / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D010649:Phenylalanine; D001618:beta-Lactamases