Yi-Ping Wang

C4-dicarboxylates sensing mechanism revealed by the crystal structures of DctB sensor domain.

C(4)-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C(4)-dicarboxylates depend on typical two-component systems (TCS) consisting of a transmembrane sensor histidine kinase and a cytoplasmic response regulator. The DctB-DctD system is the first identified TCS for C(4)-dicarboxylates sensing. Direct ligand binding to the sensor domain of DctB is believed to be the first step of the sensing events. In this report, the water-soluble periplasmic sensor domain of Sinorhizobium meliloti DctB (DctBp) was studied, and three crystal structures were solved: the apo protein, a complex with C(4) succinate, and a complex with C(3) malonate. Different from the two structurally known CitA family of carboxylate sensor proteins CitA and DcuS, the structure of DctBp consists of two tandem Per-Arnt-Sim (PAS) domains and one N-terminal helical region. Only the membrane-distal PAS domain was found to bind the ligands, whereas the proximal PAS domain was empty. Comparison of DctB, CitA, and DcuS suggests a detailed stereochemistry of C(4)-dicarboxylates ligand perception. The structures of the different ligand binding states of DctBp also revealed a series of conformational changes initiated upon ligand binding and propagated to the N-terminal domain responsible for dimerization, providing insights into understanding the detailed mechanism of the signal transduction of TCS histidine kinases.

Transcriptome profiling defines a novel regulon modulated by the LysR-type transcriptional regulator MexT in Pseudomonas aeruginosa

The LysR-family regulator MexT modulates the expression of the MexEF-OprN efflux system in the human pathogen Pseudomonas aeruginosa. Recently, we demonstrated that MexT regulates certain virulence phenotypes, including the type-three secretion system and early attachment independent of its role in regulating MexEF-OprN. In this study, transcriptome profiling was utilized to investigate the global nature of MexT regulation in P. aeruginosa PAO1 and an isogenic mexEF mutant. Twelve genes of unknown function were highly induced by overexpressing MexT independent of MexEF-OprN. A well-conserved DNA motif was identified in the upstream regulatory region of nine of these genes and upstream of mexE. Reporter fusion analysis demonstrated that the expression of the genes was significantly induced by MexT in P. aeruginosa and a heterogenous Escherichia coli strain and that the conserved sequence was required for this induction. The conserved DNA motif was further characterized as the MexT binding site by site-directed mutagenesis and electrophoretic mobility shift assays. Genes containing this conserved regulatory sequence were identified across other Pseudomonas species, and their expression was activated by MexT. Thus, a novel regulon directly modulated by MexT, that includes but is independent of mexEF-oprN, has been identified.

The CRP–cAMP complex and downregulation of the glnAp2 promoter provides a novel regulatory linkage between carbon metabolism and nitrogen assimilation in Escherichia coli

In Escherichia coli, glnA (encoding glutamine synthetase) is transcribed from two promoters (glnAp1 and glnAp2). The glnAp1 is a σ70‐dependent promoter that is activated by the cAMP receptor protein (CRP). Under nitrogen‐deficient growth conditions, glnAp1 is repressed by NtrC‐phosphate. The downstream glnAp2 promoter is σ54‐dependent and is activated by NtrC‐phosphate. Here, we show that glnAp2 expression is affected by different carbon sources and that the CRP–cAMP complex inhibits the glnAp2 promoter activity. Primer extension and KMnO4 footprinting analysis indicate that the inhibitory effect is at the transcriptional level in vivo. When glnAp2 is activated by NifA, a similar inhibitory effect by CRP–cAMP is observed. Site‐directed mutagenesis and deletion analysis indicate that the characterized and putative CRP‐binding sites located in the upstream region of the glnAp2 promoter are not essential for the inhibitory effect. CRP–cAMP inhibits σ54‐dependent glnAp2 strongly, by 21‐fold. By activating glnAp1 and downregulating glnAp2, the overall effect of CRP–cAMP on glnA expression is an approximately fourfold reduction, which correlates with the reduction of γ‐glutamyl transferase activities in the cells. We propose therefore that a physiological role of CRP–cAMP activation of glnAp1 is to partially compensate for CRP–cAMP downregulation of glnAp2, allowing a low but non‐negligible level of expression of the important genes transcribed from it. A novel regulatory linkage between carbon and nitrogen regulons is proposed.

Protein‐induced DNA bending clarifies the architectural organization of the σ54‐dependent glnAp2 promoter

σ54‐RNA polymerase (Eσ54) predominantly contacts one face of the DNA helix in the closed promoter complex, and interacts with the upstream enhancer‐bound activator via DNA looping. Up to date, the precise face of Eσ54 that contacts the activator to convert the closed complex to an open one remains unclear. By introducing protein‐induced DNA bends at precise locations between upstream enhancer sequences and the core promoter of the σ54‐dependent glnAp2 promoter without changing the distance in‐between, we observed a strong enhanced or decreased promoter activity, especially on linear DNA templates in vitro. The relative positioning and orientations of Eσ54, DNA bending protein and enhancer‐bound activator on linear DNA were determined by in vitro footprinting analysis. Intriguingly, the locations from which the DNA bending protein exerted its optimal stimulatory effects were all found on the opposite face of the DNA helix compared with the DNA bound Eσ54 in the closed complex. Therefore, these results provide evidence that the activator must approach the Eσ54 closed complexes from the unbound face of the promoter DNA helix to catalyse open complex formation. This proposal is further supported by the modelling of activator‐promoter DNA‐Eσ54 complex.

Structures of the RNA polymerase-σ54 reveal new and conserved regulatory strategies

Keeping gene transcription in check Transcription of all genes is carried out by RNA polymerase (RNAP). The enzyme is thus a pivotal regulation point for many cell and developmental processes. In bacteria, sigma factors play a vital role in transcription regulation, with σ54 being critical for transcription of many stress response genes. Yang et al. determined the x-ray crystal structure of RNAP bound to σ54, as well as promoter DNA. In the initial inhibited state of the RNAP-σ54 complex, the σ54 blocks the template DNA from entering the RNAP active site and the downstream DNA channel. Science, this issue p. 882 A regulatory protein factor in bacteria ensures that RNA polymerase stays switched off until it receives the signal to go. Transcription by RNA polymerase (RNAP) in bacteria requires specific promoter recognition by σ factors. The major variant σ factor (σ54) initially forms a transcriptionally silent complex requiring specialized adenosine triphosphate–dependent activators for initiation. Our crystal structure of the 450-kilodalton RNAP-σ54 holoenzyme at 3.8 angstroms reveals molecular details of σ54 and its interactions with RNAP. The structure explains how σ54 targets different regions in RNAP to exert its inhibitory function. Although σ54 and the major σ factor, σ70, have similar functional domains and contact similar regions of RNAP, unanticipated differences are observed in their domain arrangement and interactions with RNAP, explaining their distinct properties. Furthermore, we observe evolutionarily conserved regulatory hotspots in RNAPs that can be targeted by a diverse range of mechanisms to fine tune transcription.

CRP interacts with promoter‐bound σ54 RNA polymerase and blocks transcriptional activation of the dctA promoter

The cAMP receptor protein (CRP) is an activator of σ70‐dependent transcription. Analysis of the σ54‐dependent dctA promoter reveals a novel negative regulatory function for CRP. CRP can bind to two distant sites of the dctA promoter, sites which overlap the upstream activator sequences for the DctD activator. CRP interacts with Eσ54 bound at the dctA promoter via DNA loop formation. When the CRP‐binding sites are deleted, CRP still interacts in a cAMP‐dependent manner with the stable Eσ54 closed complex via protein–protein contacts. CRP is able to repress activation of the dctA promoter, even in the absence of specific CRP‐binding sites. CRP affects both the final level and the kinetics of activation. The establishment of the repression and its release by the NtrC activator proceed via slow processes. The kinetics suggest that CRP favours a new form of closed complex which interconverts slowly with the classical closed intermediate. Only the latter is capable of interacting with an activator to form an open promoter complex. Thus, Eσ54 promoters are responsive to CRP, a protein unrelated to σ54 activators, and the repression exerted is the direct result of an interaction between Eσ54 and the CRP–cAMP complex.

From signal perception to signal transduction: ligand‐induced dimeric switch of DctB sensory domain in solution

Sinorhizobium meliloti DctB is a typical transmembrane sensory histidine kinase, which senses C4‐dicarboxylic acids (DCA) and regulates the expression of DctA, the DCA transporter. We previously reported the crystal structures of its periplasmic sensory domain (DctBp) in apo and succinate‐bound states, and these structures showed dramatic conformational changes at dimeric level. Here we show a ligand‐induced dimeric switch in solution and a strong correlation between DctBps dimerization states and the in vivo activities of DctB. Using site‐directed mutagenesis, we identify important determinants for signal perception and transduction. Specifically, we show that the ligand‐binding pocket is essential for DCA‐induced ‘on’ activity of DctB. Mutations at different sections of DctBps dimerization interface can lock full‐length DctB at either ‘on’ or ‘off’ state, independent of ligand binding. Taken together, these results suggest that DctBps signal perception and transduction occur through a ‘ligand‐induced dimeric switch’, in which the changes in the dimeric conformations upon ligand binding are responsible for the signal transduction in DctB.

IHF-binding sites inhibit DNA loop formation and transcription initiation

Transcriptional activation of enhancer and σ54-dependent promoters requires efficient interactions between enhancer-binding proteins (EBP) and promoter bound σ54-RNA polymerase (Eσ54) achieved by DNA looping, which is usually facilitated by the integration host factor (IHF). Since the lengths of the intervening region supporting DNA-loop formation are similar among IHF-dependent and IHF-independent promoters, the precise reason(s) why IHF is selectively important for the frequency of transcription initiation remain unclear. Here, using kinetic cyclization and in vitro transcription assays we show that, in the absence of IHF protein, the DNA fragments containing an IHF-binding site have much less looping-formation ability than those that lack an IHF-binding site. Furthermore, when an IHF consensus-binding site was introduced into the intervening region between promoter and enhancer of the target DNA fragments, loop formation and DNA-loop-dependent transcriptional activation are significantly reduced in a position-independent manner. DNA-looping-independent transcriptional activation was unaffected. The binding of IHF to its consensus site in the target promoters clearly restored efficient DNA looping formation and looping-dependent transcriptional activation. Our data provide evidence that one function for the IHF protein is to release a communication block set by intrinsic properties of the IHF DNA-binding site.

Identification and solution structures of a single domain biotin/lipoyl attachment protein from Bacillus subtilis.

Protein biotinylation and lipoylation are post-translational modifications, in which biotin or lipoic acid is covalently attached to specific proteins containing biotin/lipoyl attachment domains. All the currently reported natural proteins containing biotin/lipoyl attachment domains are multidomain proteins and can only be modified by either biotin or lipoic acid in vivo. We have identified a single domain protein with 73 amino acid residues from Bacillus subtilis strain 168, and it can be both biotinylated and lipoylated in Escherichia coli. The protein is therefore named as biotin/lipoyl attachment protein (BLAP). This is the first report that a natural single domain protein exists as both a biotin and lipoic acid receptor. The solution structure of apo-BLAP showed that it adopts a typical fold of biotin/lipoyl attachment domain. The structure of biotinylated BLAP revealed that the biotin moiety is covalently attached to the side chain of Lys35, and the bicyclic ring of biotin is folded back and immobilized on the protein surface. The biotin moiety immobilization is mainly due to an interaction between the biotin ureido ring and the indole ring of Trp12. NMR study also indicated that the lipoyl group of the lipoylated BLAP is also immobilized on the protein surface in a similar fashion as the biotin moiety in the biotinylated protein.

CpxR Activates MexAB-OprM Efflux Pump Expression and Enhances Antibiotic Resistance in Both Laboratory and Clinical nalB-Type Isolates of Pseudomonas aeruginosa

Resistance-Nodulation-Division (RND) efflux pumps are responsible for multidrug resistance in Pseudomonas aeruginosa. In this study, we demonstrate that CpxR, previously identified as a regulator of the cell envelope stress response in Escherichia coli, is directly involved in activation of expression of RND efflux pump MexAB-OprM in P. aeruginosa. A conserved CpxR binding site was identified upstream of the mexA promoter in all genome-sequenced P. aeruginosa strains. CpxR is required to enhance mexAB-oprM expression and drug resistance, in the absence of repressor MexR, in P. aeruginosa strains PA14. As defective mexR is a genetic trait associated with the clinical emergence of nalB-type multidrug resistance in P. aeruginosa during antibiotic treatment, we investigated the involvement of CpxR in regulating multidrug resistance among resistant isolates generated in the laboratory via antibiotic treatment and collected in clinical settings. CpxR is required to activate expression of mexAB-oprM and enhances drug resistance, in the absence or presence of MexR, in ofloxacin-cefsulodin-resistant isolates generated in the laboratory. Furthermore, CpxR was also important in the mexR-defective clinical isolates. The newly identified regulatory linkage between CpxR and the MexAB-OprM efflux pump highlights the presence of a complex regulatory network modulating multidrug resistance in P. aeruginosa.