|BONO, ASHLEY - University Of Georgia|
|PORWOLLIK, STEFFEN - University Of California|
|MCCLELLAND, MICHAEL - University Of California|
|MRAZEK, JAN - University Of Georgia|
|HOOVER, TIMOTHY - University Of Georgia|
|KARLS, ANNA - University Of Georgia|
Submitted to: American Society for Microbiology Conference
Publication Type: Abstract Only
Publication Acceptance Date: 10/5/2013
Publication Date: 10/5/2013
Citation: Bono, A.C., Frye, J.G., Porwollik, S., Mcclelland, M., Mrazek, J., Hoover, T.R., Karls, A.C. 2013. The sigma54 global regulon in Salmonella enterica serovar Typhimurium 14028s: an extensive array of intragenic sigma54 regulatory sites revealed. 4th American Society for Microbiology Conference on Salmonella: The Bacterium, the Host and the Environment. October 5-9, 2013. Boston, Massachusetts.
Technical Abstract: An essential determinant of a transcriptional regulon is the sigma factor that associates with core RNA polymerase (E) to direct promoter-specific binding and transcription initiation by the holoenzyme (Esigma). In addition to the primary sigma factor, sigma70, S. Typhimurium has five alternative sigma factors, sigmaS, sigmaH, sigmaE, sigmaF and sigma54, controlling specialized regulons that are expressed in response to specific environmental or developmental signals. The sigma70 global regulon and the transcriptomes of sigmaS, sigmaH, and sigmaE have been characterized for S. Typhimurium. In order to gain a complete picture of global transcriptional networks involved in the pathogenesis process, we have defined the sigma54 (RpoN) global regulon of the virulent S. Typhimurium strain 14028s. Definition of the S. Typhimurium sigma54 global regulon is complicated by the requirement for activation of the E sigma54-promoter closed complex by one of 13 known or predicted bacterial enhancer-binding proteins (bEBPs) that each respond to a different environmental stimulus. We previously showed that the isolated AAA+ ATPase regulatory domain of a bEBP from Sinorhizobium meliloti, DctD250, is constitutively active and stimulates transcription initiation by E sigma54 at promoters that are normally responsive to different bEBPs in S. Typhimurium LT2. The sigma54 transcriptome and genomic binding sites were examined in wild type (wt) and deleted rpoN S. Typhimurium 14028 strains that contained a DctD250 expression plasmid, using high-density tiled microarrays for cDNA hybridization and ChIP-chip (hybridization after chromatin immunoprecipitation with anti-sigma54 polyclonal antibody). Twenty-one operons were sigma54-dependent (wt/deleted rpoN signal ratio>3) and ChIP-chip results confirmed Esigma54 binding to typical sigma54-dependent promoter elements upstream of these operons. A total of 278 E sigma54 (or sigma54) binding sites were identified by ChIP-chip, including all previously identified sigma54-dependent promoters. Approximately 80% of the binding sites are located within annotated coding sequences. Intragenic Esigma54/sigma54 binding sites were associated with down-regulation of multiple operons and with intragenic transcription. Selected Esigma54 intragenic binding sites and their associated transcripts have been confirmed by 5’ RACE and qRT-PCR. These results suggest that an extensive array of Esigma54/sigma54 binding sites is involved in regulating gene expression through multiple mechanisms, such as transcription interference/repression, anti-sense RNA, or other sRNA regulatory pathways. Since these studies were performed with S. Typhimurium 14028s that expressed DctD250, we are currently evaluating the physiological relevance of intragenic sites associated with novel transcripts or repression of gene expression.