U482649.pdf (34.25 MB)
Regulation of Escherichia coli K5 capsular polysaccharide expression.
thesisposted on 2015-11-19, 09:10 authored by Mark Paul. Stevens
The role of known, regulatory genes m expression of the Escherichia coli K5 (group II) capsular polysaccharide was investigated. Capsule expression was found to require the rfaH gene, which regulates several virulence and fertility genes in E. coli. None of the mutations studied induced capsule production at 18 °C. By using chromosomal reporter gene fusions and by direct detection of RNA, RfaH was shown to activate transcription of the serotype-specific region 2 genes. Expression of region 1 and 3 genes, which are conserved in group II capsule gene clusters, was not regulated by RfaH. A model is presented in which RfaH prevents the termination of region 3 transcripts such that they continue into region 2. Transcription antitermination as this is called, may serve to boost the amount of region 2 products to a level at which efficient polysaccharide biosynthesis can occur. By reverse transcriptase-PCR a transcript spanning the gap between regions 2 and 3 was detected. RNase protection and primer extension analysis were used to locate the promoter upstream of region 3. Transcription was found to initiate from the same site in an rfaH mutant. Approximately 1.2 kb of DNA sequence 5' of region 3 was obtained and the end of the capsule gene cluster defined. A short nucleotide sequence termed JUMPstart, located upstream of region 3 in group II capsule gene clusters, was found to be homologous to sequences 5' of RfaH-regulated operons. A deletion of this sequence abolished expression of the K5 antigen at 37 C and produced a pattern of transcription of the capsule genes similar to that seen in an rfaH mutant. This suggests that the JUMPstart sequence may be needed for the action of RfaH. The JUMPstart sequence is also found upstream of many other polysaccharide biosynthesis genes in enteric bacteria and this may indicate a common mode of regulation of these genes by RfaH.
Date of award1995-01-01
Awarding institutionUniversity of Leicester