Dataset Information

Contribution of Phe-7 to Tat-dependent export of ?-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 ?-lactamase that is secreted into the periplasm. In this study, we found that levels of ?-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.

SUBMITTER: Lee CW

PROVIDER: S-EPMC3393447 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Publications

Contribution of Phe-7 to Tat-dependent export of β-lactamase in Xanthomonas campestris.

Lee Chen-Wei CW Tseng Yi-Hsuan YH Deng Fu-Seng FS Lin Juey-Wen JW Tseng Yi-Hsiung YH Weng Shu-Fen SF

Antimicrobial agents and chemotherapy 20120423 7

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 tha ...[more]

PMID: 22526303

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Project description:Xanthomonas campestris pv. campestris is an epiphytic bacterium that can become a vascular pathogen responsible for black rot disease of crucifers. To adapt gene expression in response to ever-changing habitats, phytopathogenic bacteria have evolved signal transduction regulatory pathways, such as extracytoplasmic function (ECF) σ factors. The alternative sigma factor σ(E), encoded by rpoE, is crucial for envelope stress response and plays a role in the pathogenicity of many bacterial species. Here, we combine different approaches to investigate the role and mechanism of σ(E)-dependent activation in X. campestris pv. campestris. We show that the rpoE gene is organized as a single transcription unit with the anti-σ gene rseA and the protease gene mucD and that rpoE transcription is autoregulated. rseA and mucD transcription is also controlled by a highly conserved σ(E)-dependent promoter within the σ(E) gene sequence. The σ(E)-mediated stress response is required for stationary-phase survival, resistance to cadmium, and adaptation to membrane-perturbing stresses (elevated temperature and ethanol). Using microarray technology, we started to define the σ(E) regulon of X. campestris pv. campestris. These genes encode proteins belonging to different classes, including periplasmic or membrane proteins, biosynthetic enzymes, classical heat shock proteins, and the heat stress σ factor σ(H). The consensus sequence for the predicted σ(E)-regulated promoter elements is GGAACTN(15-17)GTCNNA. Determination of the rpoH transcription start site revealed that rpoH was directly regulated by σ(E) under both normal and heat stress conditions. Finally, σ(E) activity is regulated by the putative regulated intramembrane proteolysis (RIP) proteases RseP and DegS, as previously described in many other bacteria. However, our data suggest that RseP and DegS are not only dedicated to RseA cleavage and that the proteolytic cascade of RseA could involve other proteases.

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Dataset Information

Contribution of Phe-7 to Tat-dependent export of ?-lactamase in Xanthomonas campestris.

Publications

Contribution of Phe-7 to Tat-dependent export of β-lactamase in Xanthomonas campestris.

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