Submitted to: Microbial Pathogenesis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/29/2008
Publication Date: 3/8/2009
Citation: Ducey, T.F., Jackson, L., Orvis, J., Dyer, D.W. 2009. Transcript analysis of nrrF, a Fur repressed sRNA of Neisseria gonorrhoeae. Microbial Pathogenesis 46:166-170. Interpretive Summary: Iron is an element essential for most life, and most microorganisms have developed methods to acquire it. These methods are often tightly controlled by a variety of genetic regulatory factors. One such regulatory factor is via non-coding small RNAs (sRNA). Here we report the identification of a sRNA, nrrF, from Neisseria gonorrhoeae that is regulated by iron availability. Bioinformatics analysis reveals that nrrF resembles other iron regulatory sRNA’s from other organisms. Quantitative Real Time PCR analysis demonstrates that nrrF is regulated by iron and may also be temporally expressed. We also report the development of a new fluorescent primer extension protocol which allows for the rapid identification of transcriptional start sites. This protocol is also ideally suited for high-throughput analysis.
Technical Abstract: Like most microorganisms, Neisseria gonorrhoeae alters gene expression in response to iron availability. The ferric uptake regulator Fur has been shown to be involved in controlling this response, but the extent of this involvement remains unknown. It is known that in addition to working directly to repress gene expression, Fur may also work indirectly by controlling additional regulatory elements. Using in silico analysis, we identified a putative small RNA (sRNA) homolog of the meningococcal nrrF locus, and demonstrate that this sRNA is iron-repressible, suggesting that this is the gonococcal analog of the rhyB locus in E. coli. Quantitative real-time RT-PCR analysis indicates that this transcript may also be temporally regulated. Transcript analysis identified the 5’ start of the transcript, using a new single reaction, fluorescent-based, primer extension assay. This protocol allows for the rapid identification of transcriptional start sites of RNA transcripts, and could be used for high-throughput transcript mapping.