Regulation of Transcription of the Escherichia coli Group 2 Capsule Gene Clusters

Abstract

Submitted by Hasan Abdulhadi Hussein Aal Owaif for the Degree of Doctor ofPhilosophy in The University of Manchester and entitled 'Regulation ofTranscription of the Escherichia coli Group 2 Capsule Gene Clusters' inSeptember, 2016.Capsular polysaccharides are expressed by many bacteria and in the case ofpathogens are believed to act as essential virulence factors conferring resistanceto host defenses. Escherichia coli strains that express Group 2 capsules areassociated with a number of infections including urinary tract infections, and lifethreatening infections such as neonatal meningitis and septicemia. Theexpression of Group 2 capsular polysaccharide is controlled by two temperatureregulated promoters PR1 and PR3 that allow the transcription at 37°C but not at20°C. The 5' untranslated regions of these promoters are involved in theregulation of transcription by interacting with global regulators.In this study, PR1-lacZ and PR3-lacZ transcriptional fusions and transposonmutagenesis was used to identify new regulators of Group 2 E. coli capsuleexpression. The cAMP-CRP complex was identified to be a new transcriptionalregulator of E. coli Group 2 capsular polysaccharide expression. Deletion of cyaAor crp gene led to significant decrease in the transcriptional activity of PR1 andPR3. It was found that PR1 activity is affected by different carbon sources,glucose reduces the transcriptional activity of PR1, while the addition of glycerolincreases PR1 activity. The results showed that the cAMP-CRP complex indirectlyactivates the transcription at PR1 while it directly upregulates the transcriptionfrom PR3 by binding to the CRP binding site at +240 bp relative to thetranscription start site. Site-directed mutagenesis of the CRP binding siteabolished the binding capability of CRP and led to significant decrease in thetranscription from PR3. This regulation of transcription at both PR1 and PR3promoters by the cAMP-CRP complex is growth phase dependent, thetranscription from both promoters at mid-log phase is not affected by CRP but atlate exponential or stationary phase CRP is required for maximal transcriptionfrom these promoters. In addition, this study demonstrated that the transcriptionfrom the promoter on the antisense strand of kpsF, occurring mainly at stationaryphase, is not regulated by RpoS and does not regulate the transcription from PR1.In conclusion, this study identified a new regulator of E. coli Group 2 capsule geneexpression and added more to the current model of the complex regulation of E.coli capsular polysaccharide expression. By understanding capsule expression, itis hoped to detect therapeutic targets in pathogenic E. coli to block capsularpolysaccharide expression in the future.

Date of Award	1 Aug 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ian Roberts (Supervisor) & Jennifer Cavet (Supervisor)