25 research outputs found

    Expression capable library for studies of Neisseria gonorrhoeae, version 1.0

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    Background The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system. Results The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci. Conclusion This archived set of Gateway® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome

    Two ABC Transporter Operons and the Antimicrobial Resistance Gene mtrF Are pilT Responsive in Neisseria gonorrhoeae▿

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    Retraction of type IV pili is mediated by PilT. We show that loss of pilT function leads to upregulation of mtrF (multiple transferable resistance) and two operons encoding putative ABC transporters in Neisseria gonorrhoeae MS11. This effect occurs indirectly through the transcriptional regulator FarR, which until now has been shown to regulate only farAB. l-Glutamine can reverse pilT downregulation of the ABC transporter operons and mtrF

    Journal Of Bacteriology,

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    this report, we show that PilA does not regulate transcription of a pilE-lacZ fusion in E. coli, regardless of the conditions used. We present evidence to suggest that PilA plays a role in protein maturation. We show that overproduction of PilA in E. coli causes the accumulation of a presecretory protein and that this accumulation appears to be independent of SecY. We also demonstrate that pilA can complement an ftsY conditional mutation in E. coli and restore normal growth to this strain under nonpermissive conditions and that a PilA mutant defective in GTPase activity can no longer perform this function. Finally, we show that PilA can partially replace FtsY in an in vitro translocation assay. We conclude from these data that PilA is the gonococcal Fts
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