Detection and quantification of intergenic transcription in Mycoplasma hyopneumoniae

Abstract

Mycoplasmas are thought to control gene expression through simple mechanisms. The switching mechanisms needed to regulate transcription during significant environmental shifts do not seem to be required for these host-adapted organisms. Mycoplasma hyopneumoniae, a swine respiratory pathogen, undergoes differential gene expression, but as for all mycoplasmas, the mechanisms involved are still unknown. Since mycoplasmas contain only a single sigma factor and few regulator-type proteins, it is likely that other mechanisms control gene regulation, possibly involving intergenic (IG) regions. To study this further, we investigated whether IG regions are transcribed in M. hyopneumoniae, and measured transcription levels across five specific regions. Microarrays were constructed with probes covering 343 IG regions of the M. hyopneumoniae genome, and RNA isolated from laboratory-grown cells was used to interrogate the arrays. Transcriptional signals were identified in 321 (93.6 %) of the IG regions. Five large (>500 bp) IG regions were chosen for further analysis by qRT-PCR by designing primer sets whose products reside in flanking ORFs, bridge flanking ORFs and the IG region, or reside solely within the IG region. The results indicate that no single transcriptional start site can account for transcriptional activity within IG regions. Transcription can end abruptly at the end of an ORF, but this does not seem to occur at high frequency. Rather, transcription continues past the end of the ORF, with RNA polymerase gradually releasing the template. Transcription can also be initiated within IG regions in the absence of accepted promoter-like sequences.