Structure of a bacterial ribonucleoprotein complex central to the control of cell envelope biogenesis.

Biogenesis of the essential precursor of the bacterial cell envelope, glucosamine-6-phosphate (GlcN6P), is controlled by intricate post-transcriptional networks mediated by GlmZ, a small regulatory RNA (sRNA). GlmZ stimulates translation of the mRNA encoding GlcN6P synthtase in Escherichia coli, but when bound by RapZ protein, the sRNA becomes inactivated through cleavage by the endoribonuclease RNase E. Here, we report the cryoEM structure of the RapZ:GlmZ complex, revealing a complementary match of the RapZ tetrameric quaternary structure to structural repeats in the sRNA. The nucleic acid is contacted by RapZ mostly through a highly conserved domain that shares an evolutionary relationship with phosphofructokinase and suggests links between metabolism and riboregulation. We also present the structure of a precleavage intermediate formed between the binary RapZ:GlmZ complex and RNase E that reveals how GlmZ is presented and recognised by the enzyme. The structures provide a framework for understanding how other encounter complexes might guide recognition and action of endoribonucleases on target transcripts, and how structured substrates in polycistronic precursors may be recognised for processing by RNase E.

Multiple regulatory inputs including cell envelope stress orchestrate expression of the Escherichia coli rpoN operon.

The rpoN operon, an important regulatory hub in Enterobacteriaceae, includes rpoN encoding sigma factor σ54, hpf involved in ribosome hibernation, rapZ regulating glucosamine-6-phosphate levels, and two genes encoding proteins of the nitrogen-related phosphotransferase system. Little is known about regulatory mechanisms controlling the abundance of these proteins. This study employs transposon mutagenesis and chemical screens to dissect the complex expression of the rpoN operon. We find that envelope stress conditions trigger read-through transcription into the rpoN operon from a promoter located upstream of the preceding lptA-lptB locus. This promoter is controlled by the envelope stress sigma factor E and response regulator PhoP is required for its full response to a subset of stress signals. σE also stimulates ptsN-rapZ-npr expression using an element downstream of rpoN, presumably by interfering with mRNA processing by RNase E. Additionally, we identify a novel promoter in the 3' end of rpoN that directs transcription of the distal genes in response to ethanol. Finally, we show that translation of hpf and ptsN is individually regulated by the RNA chaperone Hfq, perhaps involving small RNAs. Collectively, our work demonstrates that the rpoN operon is subject to complex regulation, integrating signals related to envelope stress and carbon source quality.

Role of the 5' end phosphorylation state for small RNA stability and target RNA regulation in bacteria.

In enteric bacteria, several small RNAs (sRNAs) including MicC employ endoribonuclease RNase E to stimulate target RNA decay. A current model proposes that interaction of the sRNA 5' monophosphate (5'P) with the N-terminal sensing pocket of RNase E allosterically activates cleavage of the base-paired target in the active site. In vivo evidence supporting this model is lacking. Here, we engineered a genetic tool allowing us to generate 5' monophosphorylated sRNAs of choice in a controllable manner in the cell. Four sRNAs were tested and none performed better in target destabilization when 5' monophosphorylated. MicC retains full activity even when RNase E is defective in 5'P sensing, whereas regulation is lost upon removal of its scaffolding domain. Interestingly, sRNAs MicC and RyhB that originate with a 5' triphosphate group are dramatically destabilized when 5' monophosphorylated, but stable when in 5' triphosphorylated form. In contrast, the processing-derived sRNAs CpxQ and SroC, which carry 5'P groups naturally, are highly stable. Thus, the 5' phosphorylation state determines stability of naturally triphosphorylated sRNAs, but plays no major role for target RNA destabilization in vivo. In contrast, the RNase E C-terminal half is crucial for MicC-mediated ompD decay, suggesting that interaction with Hfq is mandatory.

Small RNA-binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli.

The RNA-binding protein RapZ cooperates with small RNAs (sRNAs) GlmY and GlmZ to regulate the glmS mRNA in Escherichia coli. Enzyme GlmS synthesizes glucosamine-6-phosphate (GlcN6P), initiating cell envelope biosynthesis. GlmZ activates glmS expression by base-pairing. When GlcN6P is ample, GlmZ is bound by RapZ and degraded through ribonuclease recruitment. Upon GlcN6P depletion, the decoy sRNA GlmY accumulates through a previously unknown mechanism and sequesters RapZ, suppressing GlmZ decay. This circuit ensures GlcN6P homeostasis and thereby envelope integrity. In this work, we identify RapZ as GlcN6P receptor. GlcN6P-free RapZ stimulates phosphorylation of the two-component system QseE/QseF by interaction, which in turn activates glmY expression. Elevated GlmY levels sequester RapZ into stable complexes, which prevents GlmZ decay, promoting glmS expression. Binding of GlmY also prevents RapZ from activating QseE/QseF, generating a negative feedback loop limiting the response. When GlcN6P is replenished, GlmY is released from RapZ and rapidly degraded. We reveal a multifunctional sRNA-binding protein that dynamically engages into higher-order complexes for metabolite signaling.

Adaptor protein RapZ activates endoribonuclease RNase E by protein-protein interaction to cleave a small regulatory RNA.

In Escherichia coli, endoribonuclease RNase E initiates degradation of many RNAs and represents a hub for post-transcriptional regulation. The tetrameric adaptor protein RapZ targets the small regulatory RNA GlmZ to degradation by RNase E. RapZ binds GlmZ through a domain located at the carboxyl terminus and interacts with RNase E, promoting GlmZ cleavage in the base-pairing region. When necessary, cleavage of GlmZ is counteracted by the homologous small RNA GlmY, which sequesters RapZ through molecular mimicry. In the current study, we addressed the molecular mechanism employed by RapZ. We show that RapZ mutants impaired in RNA-binding but proficient in binding RNase E are able to stimulate GlmZ cleavage in vivo and in vitro when provided at increased concentrations. In contrast, a truncated RapZ variant retaining RNA-binding activity but incapable of contacting RNase E lacks this activity. In agreement, we find that tetrameric RapZ binds the likewise tetrameric RNase E through direct interaction with its large globular domain within the catalytic amino terminus, independent of RNA. Although RapZ stimulates cleavage of at least one non-cognate RNA by RNase E in vitro, its activity is restricted to GlmZ in vivo as revealed by RNA sequencing, suggesting that certain features within the RNA substrate are also required for cleavage. In conclusion, RapZ boosts RNase E activity through interaction with its catalytic domain, which represents a novel mechanism of RNase E activation. In contrast, RNA-binding has a recruiting role, increasing the likelihood that productive RapZ/GlmZ/RNase E complexes form.

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.

Targeted decay of a regulatory small RNA by an adaptor protein for RNase E and counteraction by an anti-adaptor RNA.

Bacterial small RNAs (sRNAs) are well established to regulate diverse cellular processes, but how they themselves are regulated is less understood. Recently, we identified a regulatory circuit wherein the GlmY and GlmZ sRNAs of Escherichia coli act hierarchically to activate mRNA glmS, which encodes glucosamine-6-phosphate (GlcN6P) synthase. Although the two sRNAs are highly similar, only GlmZ is a direct activator that base-pairs with the glmS mRNA, aided by protein Hfq. GlmY, however, does not bind Hfq and activates glmS indirectly by protecting GlmZ from RNA cleavage. This complex regulation feedback controls the levels of GlmS protein in response to its product, GlcN6P, a key metabolite in cell wall biosynthesis. Here, we reveal the molecular basis for the regulated turnover of GlmZ, identifying RapZ (RNase adaptor protein for sRNA GlmZ; formerly YhbJ) as a novel type of RNA-binding protein that recruits the major endoribonuclease RNase E to GlmZ. This involves direct interaction of RapZ with the catalytic domain of RNase E. GlmY binds RapZ through a secondary structure shared by both sRNAs and therefore acts by molecular mimicry as a specific decoy for RapZ. Thus, in analogy to regulated proteolysis, RapZ is an adaptor, and GlmY is an anti-adaptor in regulated turnover of a regulatory small RNA.

Structural basis for the regulatory interaction of the methylglyoxal synthase MgsA with the carbon flux regulator Crh in Bacillus subtilis.

Utilization of energy-rich carbon sources such as glucose is fundamental to the evolutionary success of bacteria. Glucose can be catabolized via glycolysis for feeding the intermediary metabolism. The methylglyoxal synthase MgsA produces methylglyoxal from the glycolytic intermediate dihydroxyacetone phosphate. Methylglyoxal is toxic, requiring stringent regulation of MgsA activity. In the Gram-positive bacterium Bacillus subtilis, an interaction with the phosphoprotein Crh controls MgsA activity. In the absence of preferred carbon sources, Crh is present in the nonphosphorylated state and binds to and thereby inhibits MgsA. To better understand the mechanism of regulation of MgsA, here we performed biochemical and structural analyses of B. subtilis MgsA and of its interaction with Crh. Our results indicated that MgsA forms a hexamer (i.e. a trimer of dimers) in the crystal structure, whereas it seems to exist in an equilibrium between a dimer and hexamer in solution. In the hexamer, two alternative dimers could be distinguished, but only one appeared to prevail in solution. Further analysis strongly suggested that the hexamer is the biologically active form. In vitro cross-linking studies revealed that Crh interacts with the N-terminal helices of MgsA and that the Crh-MgsA binding inactivates MgsA by distorting and thereby blocking its active site. In summary, our results indicate that dimeric and hexameric MgsA species exist in an equilibrium in solution, that the hexameric species is the active form, and that binding to Crh deforms and blocks the active site in MgsA.

Feedback regulation of small RNA processing by the cleavage product.

Many bacterial small RNAs (sRNAs) are processed resulting in variants with roles potentially distinct from the primary sRNAs. In Enterobacteriaceae sRNA GlmZ activates expression of glmS by base-pairing when the levels of glucosamine-6-phosphate (GlcN6P) are low. GlmS synthesizes GlcN6P, which is required for cell envelope biosynthesis. When dispensable, GlmZ is cleaved by RNase E in the base-pairing sequence. Processing requires protein RapZ, which binds GlmZ and recruits RNase E by interaction. Cleavage is counteracted by the homologous sRNA GlmY, which accumulates upon GlcN6P scarcity and sequesters RapZ. Here, we report a novel role for a processed sRNA. We observed that processing of GlmZ is never complete in vivo. Even upon RapZ overproduction, a fraction of GlmZ remains full-length, while the 5' cleavage product (GlmZ*) accumulates. GlmZ* retains all elements required for RapZ binding. Accordingly, GlmZ* can displace full-length GlmZ from RapZ and counteract processing in vitro. To mimic GlmZ* in vivo, sRNA chimeras were employed consisting of foreign 3' ends including a terminator fused to the 3' end of GlmZ*. In vitro, these chimeras perform indistinguishable from GlmZ*. Expression of the chimeras in vivo inhibited processing of endogenous GlmZ, causing moderate upregulation of GlmS synthesis. Hence, accumulation of GlmZ* prevents complete GlmZ turnover. This mechanism may serve to adjust a robust glmS basal expression level that is buffered against fluctuations in RapZ availability.

Structural insights into RapZ-mediated regulation of bacterial amino-sugar metabolism.

In phylogenetically diverse bacteria, the conserved protein RapZ plays a central role in RNA-mediated regulation of amino-sugar metabolism. RapZ contributes to the control of glucosamine phosphate biogenesis by selectively presenting the regulatory small RNA GlmZ to the essential ribonuclease RNase E for inactivation. Here, we report the crystal structures of full length Escherichia coli RapZ at 3.40 Å and 3.25 Å, and its isolated C-terminal domain at 1.17 Å resolution. The structural data confirm that the N-terminal domain of RapZ possesses a kinase fold, whereas the C-terminal domain bears closest homology to a subdomain of 6-phosphofructokinase, an important enzyme in the glycolytic pathway. RapZ self-associates into a domain swapped dimer of dimers, and in vivo data support the importance of quaternary structure in RNA-mediated regulation of target gene expression. Based on biochemical, structural and genetic data, we suggest a mechanism for binding and presentation by RapZ of GlmZ and the closely related decoy sRNA, GlmY. We discuss a scenario for the molecular evolution of RapZ through re-purpose of enzyme components from central metabolism.

Non-canonical activation of histidine kinase KdpD by phosphotransferase protein PtsN through interaction with the transmitter domain.

The two-component system KdpD/KdpE governs K+ homeostasis by controlling synthesis of the high affinity K+ transporter KdpFABC. When sensing low environmental K+ concentrations, the dimeric kinase KdpD autophosphorylates in trans and transfers the phosphoryl-group to the response regulator KdpE, which subsequently activates kdpFABC transcription. In Escherichia coli, KdpD can also be activated by interaction with the non-phosphorylated form of the accessory protein PtsN. PtsN stimulates KdpD kinase activity thereby increasing phospho-KdpE levels. Here, we analyzed the interplay between KdpD/KdpE and PtsN. PtsN binds specifically to the catalytic DHp domain of KdpD, which is also contacted by KdpE. Accordingly, PtsN and KdpE compete for binding, providing a paradox. Low levels of non-phosphorylated PtsN stimulate, whereas high amounts reduce kdpFABC expression by blocking access of KdpE to KdpD. Ligand fishing experiments provided insight as they revealed ternary complex formation of PtsN/KdpD2 /KdpE in vivo demonstrating that PtsN and KdpE bind different protomers in the KdpD dimer. PtsN may bind one protomer to stimulate phosphorylation of the second KdpD protomer, which then phosphorylates bound KdpE. Phosphorylation of PtsN prevents its incorporation in ternary complexes. Interaction with the conserved DHp domain enables PtsN to regulate additional kinases such as PhoR.

Domain swapping between homologous bacterial small RNAs dissects processing and Hfq binding determinants and uncovers an aptamer for conditional RNase E cleavage.

In E. coli, small RNA GlmZ activates the glmS mRNA by base-pairing in an Hfq dependent manner. When not required, GlmZ is bound by adaptor protein RapZ and recruited to RNase E, which cleaves GlmZ in its base-pairing sequence. Small RNA GlmY counteracts cleavage of GlmZ by sequestration of RapZ. Although both sRNAs are highly homologous, only GlmZ specifically binds Hfq and undergoes cleavage by RNase E. We used domain swapping to identify the responsible modules. Two elements, the 3' terminal oligo(U) stretch and the base-pairing region enable GlmZ to interact with Hfq. Accordingly, Hfq inhibits cleavage of GlmZ, directing it to base-pairing. Intriguingly, the central stem loop of GlmZ is decisive for cleavage, whereas the sequence comprising the actual cleavage site is dispensable. Assisted by RapZ, RNase E cleaves any RNA fused to the 3' end of this module. These results suggest a novel mode for RNase E recognition, in which one of the required handholds in the substrate is replaced by an RNA binding protein. This device can generate RNAs of interest in their 5' monophosphorylated form on demand. As these species are rapidly degraded, this tool allows to regulate gene expression post-transcriptionally by modulation of RapZ levels.

Lies and deception in bacterial gene regulation: the roles of nucleic acid decoys.

Bacteria use intricately interconnected mechanisms acting at the transcriptional and post-transcriptional level to adjust gene expression to their needs. An intriguing example found in the chitosugar utilization systems of Escherichia coli and Salmonella is uncovered in a study by Plumbridge and colleagues. Three transcription factors (TFs), a small regulatory RNA (sRNA) and a sRNA trap cooperate to set thresholds and dynamics in regulation of chitosugar utilization. Specifically, under inducing conditions a decoy site on the polycistronic chitobiose (chbBCARFG) mRNA sequesters sRNA ChiX, which represses synthesis of the separately encoded chitoporin ChiP. Base-pairing of ChiX with its decoy has no role for the chb genes themselves when the mRNA is in excess. In the absence of substrate, however, this base-pairing tightly represses chbC encoding a subunit of the chitosugar transporter. Thus, one and the same sRNA/mRNA interaction serves different regulatory functions under different environmental conditions. The employment of RNA decoys to control the activities of post-transcriptional regulators themselves is an increasingly recognized mechanism in gene regulation. Another observation in the current study highlights the possibility that decoy sites might even exist on the DNA controlling the availability of TFs for their target promoters.

Activation of Escherichia coli antiterminator BglG requires its phosphorylation.

Transcriptional antiterminator proteins of the BglG family control the expression of enzyme II (EII) carbohydrate transporters of the bacterial phosphotransferase system (PTS). In the PTS, phosphoryl groups are transferred from phosphoenolpyruvate (PEP) via the phosphotransferases enzyme I (EI) and HPr to the EIIs, which phosphorylate their substrates during transport. Activity of the antiterminators is negatively controlled by reversible phosphorylation catalyzed by the cognate EIIs in response to substrate availability and positively controlled by the PTS. For the Escherichia coli BglG antiterminator, two different mechanisms for activation by the PTS were proposed. According to the first model, BglG is activated by HPr-catalyzed phosphorylation at a site distinct from the EII-dependent phosphorylation site. According to the second model, BglG is not activated by phosphorylation, but solely through interaction with EI and HPr, which are localized at the cell pole. Subsequently BglG is released from the cell pole to the cytoplasm as an active dimer. Here we addressed this discrepancy and found that activation of BglG requires phosphorylatable HPr or the HPr homolog FruB in vivo. Further, we uniquely demonstrate that purified BglG protein becomes phosphorylated by FruB as well as by HPr in vitro. Histidine residue 208 in BglG is essential for this phosphorylation. These data suggest that BglG is in fact activated by phosphorylation and that there is no principal difference between the PTS-exerted mechanisms controlling the activities of BglG family proteins in Gram-positive and Gram-negative bacteria.

Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16.

EIIA(Ntr) is a member of a truncated phosphotransferase (PTS) system that serves regulatory functions and exists in many Proteobacteria in addition to the sugar transport PTS. In Escherichia coli, EIIA(Ntr) regulates K(+) homeostasis through interaction with the K(+) transporter TrkA and sensor kinase KdpD. In the ß-Proteobacterium Ralstonia eutropha H16, EIIA(Ntr) influences formation of the industrially important bioplastic poly(3-hydroxybutyrate) (PHB). PHB accumulation is controlled by the stringent response and induced under conditions of nitrogen deprivation. Knockout of EIIA(Ntr) increases the PHB content. In contrast, absence of enzyme I or HPr, which deliver phosphoryl groups to EIIA(Ntr), has the opposite effect. To clarify the role of EIIA(Ntr) in PHB formation, we screened for interacting proteins that co-purify with Strep-tagged EIIA(Ntr) from R. eutropha cells. This approach identified the bifunctional ppGpp synthase/hydrolase SpoT1, a key enzyme of the stringent response. Two-hybrid and far-Western analyses confirmed the interaction and indicated that only non-phosphorylated EIIA(Ntr) interacts with SpoT1. Interestingly, this interaction does not occur between the corresponding proteins of E. coli. Vice versa, interaction of EIIA(Ntr) with KdpD appears to be absent in R. eutropha, although R. eutropha EIIA(Ntr) can perfectly substitute its homologue in E. coli in regulation of KdpD activity. Thus, interaction with KdpD might be an evolutionary 'ancient' task of EIIA(Ntr) that was subsequently replaced by interaction with SpoT1 in R. eutropha. In conclusion, EIIA(Ntr) might integrate information about nutritional status, as reflected by its phosphorylation state, into the stringent response, thereby controlling cellular PHB content in R. eutropha.

Ménage à trois: post-transcriptional control of the key enzyme for cell envelope synthesis by a base-pairing small RNA, an RNase adaptor protein, and a small RNA mimic.

In Escherichia coli, small RNAs GlmY and GlmZ feedback control synthesis of glucosamine-6-phosphate (GlcN6P) synthase GlmS, a key enzyme required for synthesis of the cell envelope. Both small RNAs are highly similar, but only GlmZ is able to activate the glmS mRNA by base-pairing. Abundance of GlmZ is controlled at the level of decay by RNase adaptor protein RapZ. RapZ binds and targets GlmZ to degradation by RNase E via protein-protein interaction. GlmY activates glmS indirectly by protecting GlmZ from degradation. Upon GlcN6P depletion, GlmY accumulates and sequesters RapZ in an RNA mimicry mechanism, thus acting as an anti-adaptor. As a result, this regulatory circuit adjusts synthesis of GlmS to the level of its enzymatic product, thereby mediating GlcN6P homeostasis. The interplay of RNase adaptor proteins and anti-adaptors provides an elegant means how globally acting RNases can be re-programmed to cleave a specific transcript in response to a cognate stimulus.

Evaluation of 5'-End Phosphorylation for Small RNA Stability and Target Regulation In Vivo.

Bacterial small RNAs (sRNAs) can be equipped at the 5' end with triphosphate (5'PPP) or monophosphate (5'P) groups, depending on whether they are primary transcripts, undergo dephosphorylation or originate via processing. Often, 5' groups hallmark RNAs for rapid decay, but whether this also applies to sRNAs is little explored. Moreover, the sRNA 5'P group could activate endoribonuclease RNase E to cleave the base-paired target RNA, but a tool for investigation in vivo was lacking. Here, we describe a two-plasmid system suitable for the generation of 5' monophosphorylated RNAs on demand inside the cell. The sRNA gene of interest is fused to the 3' end of a fragment of sRNA GlmZ and transcribed from a plasmid in an IPTG-inducible manner. The fusion RNA gets cleaved upon arabinose-controlled expression of rapZ, provided on a compatible plasmid. Adaptor protein RapZ binds the GlmZ aptamer and directs RNase E to release the sRNA of choice with 5'P ends. An isogenic plasmid generating the same sRNA with a 5'PPP end allows for direct comparison. The fates of the sRNA variants and target RNA(s) are monitored by Northern blotting. This tool is applicable to E. coli and likely other enteric bacteria.

Dynamic localization of a transcription factor in Bacillus subtilis: the LicT antiterminator relocalizes in response to inducer availability.

Bacillus subtilis transports ß-glucosides such as salicin by a dedicated phosphotransferase system (PTS). The expression of the ß-glucoside permease BglP is induced in the presence of the substrate salicin, and this induction requires the binding of the antiterminator protein LicT to a specific RNA target in the 5' region of the bglP mRNA to prevent the formation of a transcription terminator. LicT is composed of an N-terminal RNA-binding domain and two consecutive PTS regulation domains, PRD1 and PRD2. In the absence of salicin, LicT is phosphorylated on PRD1 by BglP and thereby inactivated. In the presence of the inducer, the phosphate group from PRD1 is transferred back to BglP and consequently to the incoming substrate, resulting in the activation of LicT. In this study, we have investigated the intracellular localization of LicT. While the protein was evenly distributed in the cell in the absence of the inducer, we observed a subpolar localization of LicT if salicin was present in the medium. Upon addition or removal of the inducer, LicT rapidly relocalized in the cells. This dynamic relocalization did not depend on the binding of LicT to its RNA target sites, since the localization pattern was not affected by deletion of all LicT binding sites. In contrast, experiments with mutants affected in the PTS components as well as mutations of the LicT phosphorylation sites revealed that phosphorylation of LicT by the PTS components plays a major role in the control of the subcellular localization of this RNA-binding transcription factor.

Killing two birds with one stone: an ABC transporter regulates gene expression through sequestration of a transcriptional regulator at the membrane.

Transcriptional regulators are controlled through various, mostly well-understood, principles. In the study of Richet et al., published in this issue of Molecular Microbiology, fluorescence microscopy was used to uncover an unorthodox mechanism that relies on the dynamic shuttling of a gene regulator between the membrane and the chromosome. When not occupied with transport, the maltose-specific ABC transporter sequesters and thereby inactivates its cognate transcriptional regulator MalT. Upon maltose transport, MalT is released from the membrane and activates the maltose utilization and transport genes. This mechanism prevents induction of MalT by endogenously produced maltotriose, which is the inducer. Thus, the maltose uptake system is a trigger transporter with a bi-functional role in transport and regulation.

The phosphotransferase protein EIIA(Ntr) modulates the phosphate starvation response through interaction with histidine kinase PhoR in Escherichia coli.

Many Proteobacteria possess the paralogous PTS(Ntr), in addition to the sugar transport phosphotransferase system (PTS). In the PTS(Ntr) phosphoryl-groups are transferred from phosphoenolpyruvate to protein EIIA(Ntr) via the phosphotransferases EI(Ntr) and NPr. The PTS(Ntr) has been implicated in regulation of diverse physiological processes. In Escherichia coli, the PTS(Ntr) plays a role in potassium homeostasis. In particular, EIIA(Ntr) binds to and stimulates activity of a two-component histidine kinase (KdpD) resulting in increased expression of the genes encoding the high-affinity K(+) transporter KdpFABC. Here, we show that the phosphate (pho) regulon is likewise modulated by PTS(Ntr). The pho regulon, which comprises more than 30 genes, is activated by the two-component system PhoR/PhoB under conditions of phosphate starvation. Mutants lacking EIIA(Ntr) are unable to fully activate the pho genes and exhibit a growth delay upon adaptation to phosphate limitation. In contrast, pho expression is increased above the wild-type level in mutants deficient for EIIA(Ntr) phosphorylation suggesting that non-phosphorylated EIIA(Ntr) modulates pho. Protein interaction analyses reveal binding of EIIA(Ntr) to histidine kinase PhoR. This interaction increases the amount of phosphorylated response regulator PhoB. Thus, EIIA(Ntr) is an accessory protein that modulates the activities of two distinct sensor kinases, KdpD and PhoR, in E. coli.