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A putative twin-arginine translocation system in the phytopathogenic bacterium Xylella fastidiosa

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Abstract:

The twin-arginine translocation (Tat) pathway of the xylem-limited phytopathogenic bacterium Xylella fastidiosa strain 9a5c, responsible for citrus variegated chlorosis, was explored. The presence of tatA, tatB, and tatC in the X. fastidiosa genome together with a list of proteins harboring 2 consecutive arginines in their signal peptides suggested the presence of a Tat pathway. The functional Tat dependence of X. fastidiosa OpgD was examined. Native or mutated signal peptides were fused to the -lactamase. Expression of fusion with intact signal peptides mediated high resistance to ampicillin in Escherichia coli tat+ but not in the E. coli tat null mutant. The replacement of the 2 arginines by 2 lysines prevented the export of -lactamase in E. coli tat+, demonstrating that X. fastidiosa OpgD carries a signal peptide capable of engaging the E. coli Tat machinery. RT-PCR analysis revealed that the tat genes are transcribed as a single operon. tatA, tatB, and tatC genes were cloned. Complementation assays in E. coli devoid of all Tat or TatC components were unsuccessful, whereas X. fastidiosa Tat components led to a functional Tat translocase in E. coli TatB-deficient strain. Additional experiments implicated that X. fastidiosa TatB component could form a functional heterologous complex with the E. coli TatC component.

Le système de transport appelé Tat (twin-arginine translocation) a été exploré chez la bactérie phytopathogène Xylella fastidiosa souche CVC 9a5c. La présence des gènes tatA, tatB et tatC dans le génome de X. fastidiosa ainsi qu’une liste de protéines présentant 2 arginines consécutives (appelées twin-arginine) dans leur peptide signal ont suggéré la présence d’un transport Tat fonctionnel chez Xylella. Le transport d’OpgD de X. fastidiosa par le système Tat a été examiné. La séquence signal native ou mutée a été fusionnée à la -lactamase. L’expression de la fusion avec la séquence signal native conduit à une forte résistance à l’ampicilline chez la souche d’Escherichia coli tat+ mais pas chez la souche mutante. Le remplacement des 2 arginines par 2 lysines supprime le transport de la -lactamase dans le périplasme de la souche E. coli tat+ démontrant que le substrat OpgD de X. fastidiosa porte une séquence signal reconnue par la machinerie Tat d’E. coli. L’analyse des transcrits par RT-PCR a révélé que les gènes tatA, tatB et tatC sont transcrits en opéron chez X. fastidiosa. Ces gènes ont été clonés. Les tentatives de complémentation chez les mutants nul ou tatC d’E. coli sont restées infructueuses. Par contre, l’expression de l’opéron tat de X. fastidiosa chez le mutant tatB d’E. coli a permis une restauration du transport de la -lactamase. Des expériences additionnelles indiquent que TatB de X. fastidiosa peut former un complexe hétérologue et fonctionnel avec TatC d’E. coli.

Document Type: Research Article

Publication date: February 1, 2011

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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