Multiple Transcriptional Factors Regulate Transcription of the rpoE Gene in Escherichia coli under Different Growth Conditions and When the Lipopolysaccharide Biosynthesis Is Defective

Gracjana Klein,Anna Stupak,Daria Biernacka,Pawel Wojtkiewicz,Buko Lindner,Satish Raina

doi:10.1074/jbc.m116.748954

Abstract

The RpoE σ factor is essential for the viability of Escherichia coli RpoE regulates extracytoplasmic functions including lipopolysaccharide (LPS) translocation and some of its non-stoichiometric modifications. Transcription of the rpoE gene is positively autoregulated by EσE and by unknown mechanisms that control the expression of its distally located promoter(s). Mapping of 5' ends of rpoE mRNA identified five new transcriptional initiation sites (P1 to P5) located distal to EσE-regulated promoter. These promoters are activated in response to unique signals. Of these P2, P3, and P4 defined major promoters, recognized by RpoN, RpoD, and RpoS σ factors, respectively. Isolation of trans-acting factors, in vitro transcriptional and gel retardation assays revealed that the RpoN-recognized P2 promoter is positively regulated by a QseE/F two-component system and NtrC activator, whereas the RpoD-regulated P3 promoter is positively regulated by a Rcs system in response to defects in LPS core biosynthesis, overproduction of certain lipoproteins, and the global regulator CRP. Strains synthesizing Kdo2-LA LPS caused up to 7-fold increase in the rpoEP3 activity, which was abrogated in Δ(waaC rcsB). Overexpression of a novel 73-nucleotide sRNA rirA (RfaH interacting RNA) generated by the processing of 5' UTR of the waaQ mRNA induces the rpoEP3 promoter activity concomitant with a decrease in LPS content and defects in the O-antigen incorporation. In the presence of RNA polymerase, RirA binds LPS regulator RfaH known to prevent premature transcriptional termination of waaQ and rfb operons. RirA in excess could titrate out RfaH causing LPS defects and the activation of rpoE transcription.

Highlights

The cell envelope of Gram-negative bacteria, including Escherichia coli, contains two distinct membranes, an inner (IM)3
During the analyses of signals that induce the RpoE-dependent extracytoplasmic stress response, we showed that strains synthesizing heptoseless LPS due to the absence of either GmhD (RfaD/HtrM) or WaaC exhibited a constitutive induction of RpoE [3,4,5,6]
We addressed the transcriptional regulation of the rpoE gene, encoding the extracytoplasmic function ␴ factor

Summary

Heptosyltransferase I

Heptosyltransferase II LPS glucosyltransferase I LPS core HepI kinase Transcriptional anti-terminator Adenylate cyclase Transcriptional dual regulator RpoE regulon member, DedA family member Regulator of RpoS Transcriptional dual regulator Sensory histidine kinase RpoS ␴ factor RpoN ␴ factor. Several singlecopy chromosomal rpoEP-lacZ promoter fusions, devoid of DNA sequence covering the promoter recognized by E␴E, were constructed to identify trans-acting factors that regulate rpoE transcription, followed by mapping of transcription start sites and verification by in vitro run-off assays. This analysis revealed five new transcription start sites that are utilized by different regulatory factors in response to distinct signals (Fig. 1B)

Results

Discussion

Experimental Procedures