Mutations affecting mRNA processing and fimbrial biogenesis in the Escherichia coli pap operon

Abstract

The Escherichia coli pap genetic determinant includes 11 genes and encodes expression of Pap pili on the bacterial surface. An RNase E-dependent mRNA-processing event in the intercistronic papB-papA region results in the accumulation of a papA-gene-specific mRNA in considerable excess of the primary papB-papA mRNA transcription product. We have introduced mutations in the intercistronic region and studied the effect in vivo of these mutations on the processing event, PapA protein expression, and the biogenesis of fimbriae on the bacterial surface. Our studies establish that mRNA processing is an important event in the mechanism resulting in differential gene expression of the major pap operon. The deletion of sequences corresponding to the major cleavage site abolished processing, reduced expression of PapA protein, and resulted in ''crew-cut'' bacteria with short fimbrial structures on the bacterial surface. Only a limited part of the intercistronic region appeared to be required as the recognized target for the processing to occur. Upstream sequences to a position within 10 nucleotides of the major RNase E-dependent cleavage site could be deleted without any detectable effect on papB-papA mRNA processing, PapA protein expression, or fimbria formation. Substitution mutations of specific bases at the cleavage site by site-directed mutagenesis showed that there were alternative positions at which cleavage could be enhanced, and tests with an in vitro processing assay showed that such cleavages were also RNase E dependent. Our findings are discussed in relation to other fimbrial operons and other known targets of the RNase E endoribonuclease.Several genetic determinants encoding different fimbrial structures have been identified in pathogenic strains of Escherichia coli. Expression of fimbriae (also denoted pili) on the bacteria is thought to play an essential role in the pathogenic process leading to infection by enabling the bacteria to adhere to epithelial surfaces. A common property of many of these determinants is the organization of the genes into polycistronic operons. These operons determine the production of one major structural protein (the pilin), some minor pilins, and other proteins involved in the assembly process of the fimbriae (37). The major structural proteins of the different determinants are expressed at levels markedly higher than those of the other proteins of the operons. Differential mRNA stability and mRNA processing have been suggested to be mechanisms to achieve the observed differential expression of genes in some E. coli pili determinants (3,7,17,24,28,39).pap gene clusters are commonly found in uropathogenic E. coli isolates. Studies of pap genes (papBAHCDJKEFG) from E. coli J96 suggest that expression includes mRNA processing, differential mRNA stability, and partial termination or attenuation (3, 28, 39). The two first cistrons expressed from the papB promoter, papB and papA, are cotranscribed to mRNA (mRNA-BA). A minor fraction of transcription proceeds past a terminator signal in...

Transcriptional Analysis of the sfa Determinant Revealing Multiple mRNA Processing Events in the Biogenesis of S Fimbriae in Pathogenic Escherichia coli

“…However, it was evident that the activity of the pA promoter was very low and that most of the sfa transcription was initiated at pB under the conditions used (23). It was proposed previously that the sfaA transcript could be produced primarily by endoribonucleic processing of mRNA precursors that initiated at pB, similar to what was described in the transcriptional studies of the sfaA analog papA (1,24,26).…”

“…The high steady-state level of the sfaA transcript seems to be achieved by mechanisms similar to those described previously for other stable transcripts. For the gene analogous to sfaA, papA, it was determined that a stable transcript corresponding to only this gene is generated by RNase E-mediated cleavage at the intercistronic region between the papB and papA genes (24,26). Recently, it was demonstrated that the secondary structure of the resulting stable transcript after the RNase E cleavage includes a stemloop near the 5Ј end that stabilizes the transcript by protecting it from degradation (6).…”

Regulation of P-Fimbrial Phase Variation Frequencies in Escherichia coli CFT073

“…Colonies of the pyelonephritis isolate E. coli J96 (Fig. 1A to C) were shown by flow cytometry to contain an average of 18% Ϯ 2% P-fimbriate bacteria labeled with a polyclonal anti-PapA antiserum (32). In E. coli CFT073, the positive proportion determined by flow cytometry was found to be 4.8% Ϯ 0.4% using an anti-P polyclonal serum (Fig.…”

A Secondary Structure in the 5' Untranslated Region ofadhEmRNA Required for RNase G-Dependent Regulation

“…42,43) For example, suppression of mRNA degradation has been achieved by ribosome occupation due to enhancement of translation efficiency. 38,44,45) The introduction of hairpin structures into untranslated regions of mRNA, [46][47][48][49] removal of RNase recognition sequences, [50][51][52][53][54] and mutations of RNase 55) have been reported. A combinatorial approach employing these techniques to tune relative gene expression in an operon has been also reported.…”