| Literature DB >> 16046166 |
Sandra McKean1, John Davies, Robert Moore.
Abstract
Corynebacterium pseudotuberculosis is the etiological agent of the sheep disease caseous lymphadenitis. We have developed a promoter reporter system for this organism based on expression of the green fluorescent protein (gfp) gene from Aequorea victoria. A promoterless vector, pSM20, containing the gfp gene was constructed, and promoters were inserted upstream of the gfp gene. Upon transformation into C. pseudotuberculosis, fluorescence could be visualised by fluorescence microscopy, and relative promoter strength measured by flow cytometry. The usefulness of this system for measuring changes in gene expression was demonstrated by measuring fluorescence levels of heat shocked C. pseudotuberculosis carrying a dnaK promoter construct. Replication of C. pseudotuberculosis within J774 macrophages could be monitored by fluorescence microscopy. The establishment of the system allowed the use of differential fluorescence to identify genes that showed up-regulation following macrophage infection. Genes coding for a non-ribosomal peptide synthetase and the beta chain of a propionyl CoA carboxylase were identified as possessing promoters that demonstrated enhanced activity following macrophage infection by C. pseudotuberculosis.Entities:
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Year: 2005 PMID： 16046166 DOI： 10.1016/j.micinf.2005.05.002
Source DB: PubMed Journal: Microbes Infect ISSN： 1286-4579 Impact factor: 2.700