Common and divergent features in transcriptional control of the homologous small RNAs GlmY and GlmZ in Enterobacteriaceae

Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from σ54-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional σ70-promoters overlap the σ54-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single σ70-promoter. Thus, transcription of glmY and glmZ is controlled by σ54 and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of σ70-dependent transcription. In addition, we show that activity of the σ54-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression

Interaction of lipoprotein QseG with sensor kinase QseE in the periplasm controls the phosphorylation state of the two-component system QseE/QseF in Escherichia coli.

Histidine kinase QseE and response regulator QseF compose a two-component system in Enterobacteriaceae. In Escherichia coli K-12 QseF activates transcription of glmY and of rpoE from Sigma 54-dependent promoters by binding to upstream activating sequences. Small RNA GlmY and RpoE (Sigma 24) are important regulators of cell envelope homeostasis. In pathogenic Enterobacteriaceae QseE/QseF are required for virulence. In enterohemorrhagic E. coli QseE was reported to sense the host hormone epinephrine and to regulate virulence genes post-transcriptionally through employment of GlmY. The qseEGF operon contains a third gene, qseG, which encodes a lipoprotein attached to the inner leaflet of the outer membrane. Here, we show that QseG is essential and limiting for activity of QseE/QseF in E. coli K-12. Metabolic 32P-labelling followed by pull-down demonstrates that phosphorylation of the receiver domain of QseF in vivo requires QseE as well as QseG. Accordingly, QseG acts upstream and through QseE/QseF by stimulating activity of kinase QseE. 32P-labelling also reveals an additional phosphorylation in the QseF C-terminus of unknown origin, presumably at threonine/serine residue(s). Pulldown and two-hybrid assays demonstrate interaction of QseG with the periplasmic loop of QseE. A mutational screen identifies the Ser58Asn exchange in the periplasmic loop of QseE, which decreases interaction with QseG and concomitantly lowers QseE/QseF activity, indicating that QseG activates QseE by interaction. Finally, epinephrine is shown to have a moderate impact on QseE activity in E. coli K-12. Epinephrine slightly stimulates QseF phosphorylation and thereby glmY transcription, but exclusively during stationary growth and this requires both, QseE and QseG. Our data reveal a three-component signaling system, in which the phosphorylation state of QseE/QseF is governed by interaction with lipoprotein QseG in response to a signal likely derived from the cell envelope

Drastic Differences in Crh and HPr Synthesis Levels Reflect Their Different Impacts on Catabolite Repression in Bacillus subtilis

In Bacillus subtilis, carbon catabolite repression (CCR) of catabolic genes is mediated by ATP-dependent phosphorylation of HPr and Crh. Here we show that the different efficiencies with which these two proteins contribute to CCR may be due to the drastic differences in their synthesis rates under conditions that cause CCR

QseG and QseE are required for phosphorylation of the QseF receiver domain.

<p><b>A</b>. StrepTactin pull-down of QseF-NTD following metabolic [³²P] labeling. Strains Z197 (<i>wild-type</i>), Z477 (Δ<i>qseG</i>) and Z970 (Δ<i>qseE</i>) harboring plasmid pYG279 (encoding QseF-NTD-Strep) were grown in LB and synthesis of QseF-NTD-Strep following addition of IPTG was verified by SDS-PAGE of total protein extracts (left panel). Cells were labelled using [³²P] and QseF-NTD-Strep was subsequently isolated by pull-down and separated by 15% SDS-PAGE. Gels were analyzed by Western blotting (middle panel) using an antibody directed against the Strep-tag and autoradiography (right panel). <b>B</b>. Pulse-chase experiment to assess QseE phosphatase activity <i>in vivo</i>. The transformants used in (A) were labelled using [³²P] and subsequently chased with “cold” phosphorus for the indicated times. Synthesis of QseF-NTD-Strep was confirmed by SDS-PAGE of total protein extracts (left panel). Following chase, the QseF-NTD-Strep was pulled down and analyzed by Western blotting (top panel, right) and autoradiography (bottom panel, right). Obtained phosphorylation signals were quantified and quantifications are displayed below the autoradiographs. Phosphorylation signal intensities are expressed in percentage of the signal obtained in the <i>wild-type</i> following pulse-labeling (no chase).</p

QseF is phosphorylated at multiple sites <i>in vivo</i>.

<p><b>A</b>. <i>In vivo</i> H₃[³²P]O₄ labeling of strains Z197 (<i>wild-type</i>) and Z970 (Δ<i>qseE</i>) carrying either the empty expression vector pKES170 (lanes 1, 6) or overexpressing <i>wild-type qseF</i> from plasmid pYG253 (lanes 2–3, 7–8) or <i>qseF</i>-D56A from plasmid pYG254 (lanes 4–5, 9–10). For induction of <i>qseF</i> expression IPTG was added as indicated. After metabolic [³²P] labeling total protein extracts were analyzed by SDS-PAGE and autoradiography. <b>B</b>. StrepTactin pull-down assay of QseF after metabolic [³²P] labeling. Strains Z196 (Δ<i>qseF</i>, <i>qseG</i>⁺) and Z955 (Δ<i>qseGF</i>) were transformed with plasmids pYG269, expressing <i>qseF-strep</i> and pYG269-D56A encoding <i>qseF-D56A-strep</i>. Transformants were grown to mid log phase (OD₆₀₀ ~0.8), 1 mM IPTG was added where indicated and whole cell extracts were analyzed by western blotting using α-Strep antiserum (input, top). H₃[³²P]O₄ metabolic labeling was followed by StrepTactin pull-down and pull-down fractions were analyzed by Western blotting with α-Strep antiserum (middle) and by SDS-PAGE and autoradiography (bottom).</p

Impact of epinephrine on the QseE/QseF TCS.

<p><b>A</b>. Transcription of a chromosomal <i>glmY’-lacZ</i> fusion in <i>wild-type</i> strain Z197 during growth in absence and presence of 150 μM epinephrine (EPI). Cells were inoculated in LB with or without EPI to an OD₆₀₀ = 0.1. Following the indicated times of incubation, samples were harvested and the β-galactosidase activity was determined. <b>B</b>. Expression of <i>glmY</i> in <i>wild-type</i> (Z197), Δ<i>qseG</i> (Z477) and Δ<i>qseE</i> (Z970) strains following 16 h of growth overnight in absence and presence of 150 μM Epi. Subsequently, the β-galactosidase activities were determined to assess expression of the <i>glmY’-lacZ</i> fusion carried on the chromosomes of these strains. In addition, total RNA was extracted from the <i>wild-type</i> strain and GlmY amounts were analyzed by Northern Blotting (right panel, top). Detection of 5S rRNA served as loading control (right panel, bottom). <b>C</b>. Effect of epinephrine on phosphorylation of the QseF receiver domain <i>in vivo</i>. Strains Z197 (<i>wild-type</i>), Z477 (Δ<i>qseG</i>) and Z970 (Δ<i>qseE</i>) harboring plasmid pYG279 (encoding QseF-NTD-Strep) were grown in the presence of IPTG for induction of QseF-NTD-Strep and subsequently subjected to metabolic ³²P labeling and StrepTactin pull-down for isolation of QseF-NTD. Where indicated 150 μM Epi was added to the cells prior to addition of H₃[³²P]O₄. Proper synthesis of QseF-NTD-Strep was confirmed before labelling by analysis of total protein extracts by SDS-PAGE and Coomassie blue staing (left panel). Pull-down fractions were analyzed by Western blotting using α-Strep antiserum for successful isolation of the QseF-NTD-Strep (middle panel) and by autoradiography (right panel). Obtained phosphorylation signals were quantified from at least three independent experiments and quantifications are displayed in the diagram (right). Phosphorylation signal intensities are expressed in percentage of the signal obtained in the <i>wild-type</i> in the absence of EPI.</p

Mutations in the QseE N-terminus impairing interaction with QseG concomitantly decrease QseE activity.

<p><b>A</b>. Schematic representation of the domain architecture of sensor kinase QseE. Amino acid residues encompassing the respective domains are given in parenthesis and the phosphorylated histidine residue H259 is depicted by a circle. Positions are according to the EcoCyc database [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007547#pgen.1007547.ref006" target="_blank">6</a>]. The HAMP domain has been predicted by Pfam [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007547#pgen.1007547.ref068" target="_blank">68</a>]. The sequence coding for amino acid residues 1–258 was randomly mutagenized and the resulting QseE mutant library was phenotypically screened for loss of interaction with QseG in the context of BACTH. The position of the thereby identified S58N substitution is indicated with an arrow. <b>B</b>. Quantitative BACTH analysis of the interaction potential of T25-QseE variants identified in the screen for loss of interaction with T18-TM-QseG. The following plasmid combinations were tested in reporter strain BTH101 (left to right): pYG242/pYG199; pYG242/pYG199_TM1; pYG242/pYG199_1.6; pYG246/pYG199; pYG246/pYG199_TM1; pYG246/pYG199_1.6. <b>C</b>. Complementation analysis assessing the ability of QseE variants to activate transcription from promoter <i>P</i>_<i>glmY</i>. The following plasmids encoding the proteins under <i>P</i>_<i>tac</i> control were introduced into the Δ<i>qseE</i> mutant Z970 carrying a <i>glmY’-lacZ</i> reporter fusion and the β-galactosidase activities were determined: pKESK23 (empty vector, column 3), pYG221 (wt-QseE, column 4), pYG221-H259A (QseE-H259A, column 5), pYG221-TM1 (QseE-M5, column 6), pYG221-S58N (QseE-S58N, column 7). As controls, un-transformed <i>wild-type</i> (Z197) and Δ<i>qseE</i> (Z970) strains were used (first two columns).</p

Model for control of sRNA <i>glmY</i> transcription by the QseE/QseG/QseF three-component system in <i>E</i>. <i>coli</i> K-12.

<p>The model summarizes data obtained in the current and in previous studies [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007547#pgen.1007547.ref008" target="_blank">8</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007547#pgen.1007547.ref009" target="_blank">9</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007547#pgen.1007547.ref016" target="_blank">16</a>]. In the absence of QseG, kinase QseE is inactive and unable to activate response regulator QseF. QseG is a lipoprotein attached to the outer membrane and binds the periplasmic loop of kinase QseE. Interaction with QseG may activate kinase QseE to phosphorylate response regulator QseF at residue Asp56 in the receiver domain. Whether this interaction occurs with membrane-attached or soluble QseG remains unclear. The host hormone epinephrine moderately stimulates phosphorylation of QseF by QseE in a QseG-dependent manner when cells reside in the stationary growth phase. In addition, QseF is phosphorylated by an unknown activity in the C-terminus, presumably at Thr or Ser residue(s). Assisted by the integration host factor IHF, phosphorylated QseF binds to conserved sites upstream of <i>glmY</i> and activates <i>glmY</i> transcription from a σ⁵⁴-dependent promoter. The sRNA GlmY in turn counteracts degradation of the homologous sRNA GlmZ through sequestration of protein RapZ, which is required for GlmZ decay. Through a base-pairing mechanism GlmZ activates synthesis of glucosamine-6-phosphate synthase, which generates glucosamine-6-phosphate—the first dedicated metabolite for synthesis of peptidoglycan and lipopolysaccharides.</p