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. 2016 Oct 28;291(44):22999-23019.
doi: 10.1074/jbc.M116.748954. Epub 2016 Sep 14.

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

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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 et al. J Biol Chem. .
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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.

Keywords: LapB; RNA polymerase; RirA sRNA; RpoE; RpoS; bacterial transcription; glycosyltransferase; heptosyltransferase; lipopolysaccharide (LPS); transcription termination.

Figures

FIGURE 1.
FIGURE 1.
Transcriptional regulation of the rpoE gene. Schematic drawing of previously identified transcriptional start sites of the rpoE gene (3, 16) (A) and those identified in this work (B). Major regulators and factors controlling the rpoE transcription are indicated.
FIGURE 2.
FIGURE 2.
Proposed LPS structure of major glycoforms observed in E. coli K-12. Schematic drawing of LPS glycoforms I (A) and VII (B) with the indicated genes whose products are involved in the LPS core biosynthesis and non-stoichiometric modifications.
FIGURE 3.
FIGURE 3.
Transcriptional regulation of different promoters of the rpoE gene. A, nucleotide sequence of the promoter region of the rpoE gene. Arrows in red indicate the position of transcription start sites. The shared TSS at −327 is utilized by RpoN and RpoD. The P4 start site represents the initiation site recognized by RpoS. The −10 and −35 elements of the RpoD (pink) and RpoS recognized promoter (green) and −12 and −24 elements of RpoN (blue) are indicated. The P6 initiation site corresponds to the EσE-regulated promoter. Nucleotides marked with boxes correspond to IHF, RcsB, NtrC, and CRP recognition sites. Three palindromic regions marked with inverted arrows correspond to UAS1, UAS2, and UAS3 representing QseF-binding sites required for the P2 promoter. Nucleotides marked with asterisks (*) correspond to putative processing sites. B, the alignment of −12 and −24 regions of the rpoEP2 promoter with well characterized RpoN-regulated promoters. C, the alignment of −10 and extended −10 elements of the rpoEP4 promoter with well known RpoS-regulated promoters.
FIGURE 4.
FIGURE 4.
In vitro transcription run-off assays showing selective recruitment of different promoters of the rpoE gene with various forms of RNA polymerase. A, RpoS (σ38) and RpoD complexed with the RNAP core initiate transcription from −218 TSS and TSS at −327, respectively. Lane 1 corresponds to the size standard. For lanes 2–4, a DNA template of 105 bp was used. Lane 2 shows the incubation reaction with Eσ70, lane 3 the incubation with Eσ38 resulting in the synthesis of expected 45-nt product marked with the arrow. Lane 4 was incubated with Eσ70 in the presence of phosphorylated RcsB. Lanes 5 and 6 show RNA transcripts synthesized from DNA template of 170 bp. An expected size of 85-nt RNA product was observed when Eσ70 was incubated with the wild-type DNA template (lane 5), whereas only a weak signal was visible when Eσ70 was used with template with mutation at −7T(C) and −11A(G) in the −10 element (lane 6). Bands marked with an asterisk (*) symbol in lanes 3 and 5 indicate nonspecific end-to-end transcription reaction products. B, RpoN (σ54) complexed with the core RNAP in the presence of either NtrC or QseF can initiate transcription from the rpoEP2 promoter using the −327 TSS. Lane 1 corresponds to size standard, lanes 2 and 4 corresponds to reactions with the wild-type DNA template in the presence of either NtrC or QseF. Lanes 3 and 5 correspond to the RNA transcript produced with DNA template containing mutations at −12 (GC to AT) and −24 (GG to GA).
FIGURE 5.
FIGURE 5.
The positive regulation of the rpoEP2 promoter by the QseF activator. A, four independent cultures of wild-type and its ΔqseF derivative carrying either the wild-type rpoEP2 promoter fusion or with rpoEP2* promoter fusion with mutations in the −12 and −24 regions expressing the cloned qseG gene were analyzed for β-galactosidase activity after different growth intervals in the presence of 50 μm IPTG. B, the QseF-DNA interaction at the P2 promoter. Thirty-five ng of the wild-type DNA fragment covering QseF binding sites UAS1 + 2+3 (lanes 2–5) were incubated with increasing concentrations of QseF and analyzed by EMSA. A DNA probe containing only UAS3 was also incubated with QseF as indicated. Lanes 1 and 6 serve as a control with DNA alone. Samples were analyzed as described under “Experimental Procedures.” C, the RpoN-regulated rpoEP2 promoter also senses LPS defects. Isogenic cultures of SR19089 and its ΔwaaC, Δ(waaC rpoN), and Δ(waaC qseF) were analyzed for β-galactosidase activity after different growth intervals and averages of four independent derivatives are plotted.
FIGURE 6.
FIGURE 6.
The rpoEP4 promoter is regulated positively by RpoS and responds to its modulators. Cultures of SR18874 carrying the rpoEP4 promoter fusion, its ΔrpoS or ΔrssB derivatives, and derivative carrying the cloned pcnB gene were grown in LB medium at 30 °C either in the presence of 50 μm IPTG for strain with plasmid or without IPTG for plasmid-free derivatives. Samples from four independent cultures at different growth intervals in each case were analyzed for the β-galactosidase activity.
FIGURE 7.
FIGURE 7.
The Eσ70-recognized rpoEP3 promoter is positively regulated by the Rcs two-component system in response to LPS defects in an RcsB-dependent manner. A, cultures of SR18987 carrying the rpoEP3 promoter fusion and its derivatives with non-polar deletion in various waa genes were grown in LB medium at 30 °C and analyzed for β-galactosidase activity after different intervals. Four independent derivatives in each case were analyzed and averaged data are presented after a 250-min incubation. B, cultures of SR18987, its ΔwaaC and Δ(waaC rcsB) derivatives were grown in LB medium at 30 °C and analyzed for β-galactosidase activity after different intervals of growth. Averages from four cultures in each case are plotted. C, the RcsB-DNA interaction at the P3 promoter. Thirty-five ng of a 280-bp wild-type DNA fragment that includes a putative RcsB recognition site was incubated with increasing concentrations of phosphorylated RcsB and analyzed by EMSA on a 4% native gel. D, specificity of RcsB-DNA interaction at the P3 promoter. Three DNA probes included a 81-bp DNA fragment with the wild-type RcsB sequence (lanes 2 and 3), a second 78-bp DNA fragment lacking conserved CAT trinucleotide residues of RcsB consensus (lanes 5 and 6), and a third 71-bp probe lacking nucleotides CATGGTTTGG of RcsB consensus (lanes 8 and 9) were incubated with 120 and 180 ng of RcsB for each probe, respectively. Lanes 1, 4, and 7 serve as control with DNA alone. After incubation the reaction mixtures were analyzed on a 6% native gel.
FIGURE 8.
FIGURE 8.
The LPS composition of derivatives with specific mutations causing LPS truncation leading to the induction of the rpoEP3 transcription. Charge deconvoluted ES FT-ICR mass spectra in the negative ion mode of native LPS obtained from isogenic deletion derivatives of the wild-type strain carrying the ΔwaaC mutation (A), ΔwaaF (B), ΔwaaG (C), and ΔwaaP (D) grown in phosphate-limiting medium at 30 °C. Mass numbers refer to monoisotopic peaks. The mass peaks corresponding to additional substitutions with P-EtN and/or l-Ara4N are indicated.
FIGURE 9.
FIGURE 9.
Defects in the lipid A biogenesis of Δwaa mutants exhibiting increased rpoEP3 transcriptional activity. Charge deconvoluted ESI FT-ICR mass spectra of isogenic ΔwaaC (A), ΔwaaF (B), ΔwaaG (C), and ΔwaaP (D) in the negative ion mode depicting the lipid A composition and its modifications from the LPS obtained from cultures grown in phosphate-limiting medium at 30 °C. Part of the negative ion mass spectra of the native LPS after unspecific fragmentation, leading to the cleavage of the labile lipid A-Kdo linkage, is presented. The mass peaks corresponding to the hexaacylated lipid A part and substitutions with P-EtN and/or l-Ara4N are indicated.
FIGURE 10.
FIGURE 10.
The absence of LapAB proteins or overexpression of lipoprotein encoded by the yhdV gene induces the rpoEP3 transcription. A, overnight cultures of SR7917 (carrying chromosomal rpoEP1-P5-lacZ fusion), its Δ(lapA lapB) and Δ(rcsB lapA lapB) derivatives, and B, SR18987 (rpoEP3-lacZ), its isogenic Δ(lapA lapB) and Δ(rcsB lapA lapB) derivatives, were grown in M9 medium at 30 °C and analyzed for β-galactosidase activity after different intervals. Data averaged from four independent samples are presented for panels A and B. C, cultures of SR18987 carrying rpoEP3-lacZ fusion with empty vector, its derivative with the cloned yhdV gene, and its ΔrcsB derivative with the yhdV gene on the plasmid were analyzed for β-galactosidase activity after the addition of 50 μm IPTG. Data from four replicates are plotted.
FIGURE 11.
FIGURE 11.
A novel sRNA rirA in multicopy induces the rpoE transcriptional activity and controls LPS biosynthesis. A, nucleotide sequence of the gene encoding the rirA sRNA and its promoter region. The arrow indicates the TSS of the rirA sRNA. The −10 and extended −10 promoter elements, the conserved JUMPstart, and ops sites are depicted. B, either overexpression of the rirA gene or the absence of RfaH induces the rpoEP3 promoter activity. Cultures of SR18987 with the vector pRS551, its ΔrfaH derivative, or when the rirA gene is expressed from its own promoter in pRS551 were grown in LB medium at 30 °C and analyzed for the β-galactosidase activity after different growth intervals. Average of four independent samples are presented. C, model of RirA generated using M-fold.
FIGURE 12.
FIGURE 12.
RirA sRNA binds to RfaH and its overexpression causes defects in the LPS synthesis. A, interaction of RirA with RfaH in the presence or absence of RNAP. Fifty ng of RirA were incubated at 37 °C with 150, 300, and 450 ng of RfaH in the presence of 50 μg of RNA polymerase core (lanes 2–4) or in the absence of RNAP (lanes 5–7). Lane 1 corresponds to RirA alone. Samples were analyzed on a 6% native polyacrylamide gel. Arrows indicate the position of complex and free RirA. B, the replacement of the 8-nt ops site by 8 A nt in RirA abolishes interaction with RfaH + RNAP core. Fifty ng of wild-type RirA (lanes 2–4) and RirA with mutated ops site (lanes 5–7) were analyzed for the complex formation with RfaH in the presence of RNAP and resolved by native gel electrophoresis. C, a portion of whole cell lysate obtained from the wild-type E. coli K-12 strain BW25113 with vector pRS551 (lane 1), its derivative expressing the wild-type rirA gene in pRS551 (lane 2), and rirA with 8-nt ops site replaced by 8 A residues rirA* (lane 3) were applied on a 16.5% SDS-Tricine gel and LPS was revealed after silver staining. The arrow indicates the position of the LPS core. D, a portion of whole cell lysates after proteinase K treatment obtained from isogenic strains with pMF19 and vector pRS551 alone (lane 1) and its derivative expressing the rirA gene from its own promoter in pRS551 with pMF19 (lane 2) were resolved on a 14% SDS-Tricine gel and LPS was revealed after silver staining. Arrows indicate the position of the lipid A-core, lipid A-core + 1 O-antigen unit, and lipid A-core + polymeric O-antigen. E, isogenic cultures carrying vector pRS551 alone (lane 1) or expressing the wild-type rirA gene (lane 2) in the strain with chromosomal waaO, rfbB, or waaC genes epitoped at the C-terminal end with 3× FLAG were grown in LB medium at 30 °C up to an A600 of 0.2. An equivalent amount of total protein was analyzed on 12% SDS-PAGE, followed by immunoblotting using anti-FLAG monoclonal antibody.
FIGURE 13.
FIGURE 13.
The rpoEP3 promoter is positively regulated by the CRP activator protein. A, cultures of the wild-type strain carrying the rpoEP3 promoter fusion and its four independent Δcrp transductants were grown in LB medium at 30 °C and analyzed for β-galactosidase activity after different intervals. B, interaction of CRP with DNA containing the P3 promoter and upstream DNA region. A 118-bp DNA fragment covering the P3 promoter region was incubated with increasing concentrations of CRP activated by cAMP (lanes 2–5) and complexes were resolved on a 6% native gel. Lane 1 serves as a negative control with DNA alone. Arrows show the position of free dsDNA and CRP-DNA complex.
FIGURE 14.
FIGURE 14.
The rpoEP3 promoter activity is induced in the absence of ecfLM genes. Isogenic cultures of the wild-type and its Δ(ecfL ecfM) derivative carrying the rpoEP3 promoter fusion were grown in LB medium at 37 °C and analyzed for the β-galactosidase activity.
FIGURE 15.
FIGURE 15.
Co-integration of multiple signaling pathways and recruitment of different transcriptional factors in the regulation of the transcription of the rpoE gene in response to specific stimuli. A schematic drawing of the promoter region of the rpoE gene, depicting the organization of six promoters designated P1 to P6. The transcription from rpoEP6 is initiated by EσE and responds to OM protein defects via the RseA. P2 and P3 promoters utilize the same TSS. The rpoEP2 is recognized by σ54 and can recruit either NtrC or QseF as activators. The QseE/F system can be activated by QseG. QseE/F-regulated transcription of the rpoEP2 and glmY sRNA can co-integrate signals of cell envelope constituents like LPS and peptidoglycan synthesis to rpoE transcription. The rpoEP3 is recognized by σ70 and its transcription is specifically induced when LPS biosynthesis is compromised (lack of assembly protein LapB, titration of RfaH by RirA sRNA, or when the inner core of LPS is truncated). LPS defects could the transmit signal from the RcsF OM lipoprotein leading to the activation of the RcsB response regulator. The P2 promoter can also be induced in response to LPS defects albeit to a lower extent than the rpoEP3 promoter. The global regulator CRP protein in response to cAMP levels also positively regulates the rpoEP3 promoter. The rpoEP4 promoter is recognized by the stationary phase σ factor RpoS and is activated in response to diverse stresses like challenge with high osmolarity and factors that regulate transcription of the rpoS gene and the stability of σS.

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