Genetic regulation of the yefM-yoeB and pezAT toxin-antitoxin loci of Streptococcus pneumoniae / Chan Wai Ting.

Chan, Wai Ting (2011) Genetic regulation of the yefM-yoeB and pezAT toxin-antitoxin loci of Streptococcus pneumoniae / Chan Wai Ting. PhD thesis, University of Malaya.

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Abstract

The genome of Streptococcus pneumoniae harbours at least eight putative toxinantitoxin (TA) loci. Two of these TA loci, pezAT and yefM-yoeBSpn, have previously been shown to be functional and the regulation mechanism of both TA loci was investigated in this study. Like most TA loci investigated, both the pezAT and yefMyoeBSpn loci were co-transcribed from σ70-type promoters. Transcriptional fusion assays demonstrated that the PezA and YefMSpn antitoxins served as repressors whereas their respective cognate toxins, PezT and YoeBSpn, served as co-repressors to further repress the activities of their promoters. DNAse I footprinting results indicated that the PezA and YefMSpn antitoxins bind to palindromic operator sites which overlap their respective promoter regions where they may hinder the binding of RNA polymerase thus resulting in the observed transcriptional repression. On the other hand, the PezA-PezT and YefMYoeBSpn TA protein complexes were able to bind to their respective operator sites with lesser amounts, which indicated that the PezT and YoeBSpn toxins served as corepressors to enhance the binding affinity of their cognate antitoxins, in agreement with the results from the transcription fusion assays and findings from investigations of other TA systems. However, the regulation of yefM-yoeBSpn appeared to be more complex than pezAT or other common TA loci. A BOX mobile element was found within the intergenic region between yefMSpn and the upstream gene in the S. pneumoniae R6 genome. The insertion of the BOX element, termed boxA-C, led to the incorporation of an additional promoter, PyefM1, upstream of its original promoter, PyefM2. Transcriptional fusion assays indicated that PyefM1 is a much weaker promoter compared to PyefM2. Footprinting assays showed that either the YefMSpn antitoxin or the YefM-YoeBSpn TA complex binds only to a palindromic sequence that overlapped the –35 region of the PyefM2 promoter but not to any regions overlapping or surrounding the PyefM1 promoter. With just the PyefM2 promoter alone, the YefMSpn antitoxin repressed transcription from PyefM2 both in trans and in cis and this repression was augmented by YoeBSpn. However, in the presence of the entire upstream regulatory region, which included boxA-C, PyefM1 and PyefM2, slight repression was observed when yefMSpn was expressed in trans, but no further repression was observed when yefMSpn and yoeBSpn were expressed in trans. Interestingly, when yefMSpn was constructed in cis along with the entire promoter region, transcriptional activation was observed, and the activation persisted even in the presence of the entire yefM-yoeBSpn reading frames in cis. This indicated that the regulation of yefM-yoeBSpn may involve cis-acting elements which include the entire promoter region along with the yefMSpn reading frame and/or host factors that have yet to be determined. As the boxA-C element is conserved in the genome of all sequenced S. pneumoniae strains in the database, it is suggested that the boxA-C element may provide a selective advantage to the host. It is also postulated that PyefM1 is a constitutive promoter that provided a basal level of transcription to the yefM-yoeBSpn locus to enable a faster response to any drastic changes in the environment.

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