LysR-type Regulator Research Articles

A LysR-type regulator influencing swimming motility, galactose utilization, and virulence in Ralstonia solanacearum GMI1000

BackgroundLysR-type transcriptional regulators (LTTRs) are one of the largest families of regulators in prokaryotic organism, which help the bacterium adapt to diverse conditions by controlling a wide array of regulons, encompassing genes responsible for nitrogen and carbon fixation, oxidative stress response, bacterial virulence, and the breakdown of diverse compounds. Ralstonia solanacearum strain GMI1000 possesses 80 LTTR genes, yet the precise roles and functional contributions of only three of these LTTRs have been conclusively established among the total. In this work, our group reveal a novel LTTR member LysR7 (RS_RS02375) that exerts multiple regulatory roles in motility, carbon metabolism and virulence.ResultsIn this investigation, an in-frame deletion mutant ΔlysR7 and a complemented strain CΔlysR7 were prepared. The mutant ΔlysR7 had increased swimming motility on semi-soft medium and showed a reduced replication rate in nutrient-rich medium and in planta. Moreover, ΔlysR7 was unable to grow on nutrient-limited medium, supplemented with galactose as a single carbon resource. RT-qPCR analysis and GUS activity detection indicated that the expression of lysR7 was induced in the presence of galactose. The mutant ΔlysR7 caused weaker wilt disease on either Solanum lycopersicum or Capsicum annuum plants compared to both wild type GMI1000 and CΔlysR7. Transcriptome analysis revealed that 12 upregulated and 8 downregulated differentially expressed genes (DEGs) in ΔlysR7 were restored in CΔlysR7 relative to wild type. In particular, the expression of hrpG, a key gene responsible for type III secretion system, was downregulated. KEGG analysis revealed that, except for lysR7 gene, the 19 DEGs were most enriched in microbial metabolism in diverse environments and metabolic pathways.ConclusionsThe data indicate that LysR7 regulates multiple processes in association with motility, galactose metabolism and virulence in R. solanacearum. The study offers valuable evidence to understand comprehensive regulatory mechanisms mediated by LTTR family members in R. solanacearum.Graphical

Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Is Required in Bradyrhizobium diazoefficiens for Efficient Soybean Root Colonization and Competition for Nodulation.

The Hyphomicrobiales (Rhizobiales) order contains soil bacteria with an irregular distribution of the Calvin-Benson-Bassham cycle (CBB). Key enzymes in the CBB cycle are ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), whose large and small subunits are encoded in cbbL and cbbS, and phosphoribulokinase (PRK), encoded by cbbP. These genes are often found in cbb operons, regulated by the LysR-type regulator CbbR. In Bradyrhizobium, pertaining to this order and bearing photosynthetic and non-photosynthetic species, the number of cbbL and cbbS copies varies, for example: zero in B. manausense, one in B. diazoefficiens, two in B. japonicum, and three in Bradyrhizobium sp. BTAi. Few studies addressed the role of CBB in Bradyrhizobium spp. symbiosis with leguminous plants. To investigate the horizontal transfer of the cbb operon among Hyphomicrobiales, we compared phylogenetic trees for concatenated cbbL-cbbP-cbbR and housekeeping genes (atpD-gyrB-recA-rpoB-rpoD). The distribution was consistent, indicating no horizontal transfer of the cbb operon in Hyphomicrobiales. We constructed a ΔcbbLS mutant in B. diazoefficiens, which lost most of the coding sequence of cbbL and has a frameshift creating a stop codon at the N-terminus of cbbS. This mutant nodulated normally but had reduced competitiveness for nodulation and long-term adhesion to soybean (Glycine max (L.) Merr.) roots, indicating a CBB requirement for colonizing soybean rhizosphere.

A Sb(III)-specific efflux transporter from Ensifer adhaerens E-60

Antimony is pervasive environmental toxic substance, and numerous genes encoding mechanisms to resist, transform and extrude the toxic metalloid antimony have been discovered in various microorganisms. Here we identified a major facilitator superfamily (MFS) transporter, AntB, on the chromosome of the arsenite-oxidizing bacterium Ensifer adhaerens E-60 that confers resistance to Sb(III) and Sb(V). The antB gene is adjacent to gene encoding a LysR family transcriptional regulator termed LysRars, which is an As(III)/Sb(III)-responsive transcriptional repressor that is predicted to control expression of antB. Similar antB and lysRars genes are found in related arsenic-resistant bacteria, especially strains of Ensifer adhaerens, and the lysRars gene adjacent to antB encodes a member of a divergent subgroup of putative LysR-type regulators. Closely related AntB and LysRars orthologs contain three conserved cysteine residues, which are Cys17, Cys99, and Cys350 in AntB and Cys81, Cys289 and Cys294 in LysRars, respectively. Expression of antB is induced by As(III), Sb(III), Sb(V) and Rox(III) (4-hydroxy-3-nitrophenyl arsenite). Heterologous expression of antB in E. coli AW3110 (Δars) conferred resistance to Sb(III) and Sb(V) and reduced the intracellular concentration of Sb(III). The discovery of the Sb(III) efflux transporter AntB enriches our knowledge of the role of the efflux transporter in the antimony biogeochemical cycle.

The LysR-type transcription factor LsrB regulates beta-lactam resistance in Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a plant pathogen, broadly known as the causal agent of the crown gall disease. The soil bacterium is naturally resistant to beta-lactam antibiotics by utilizing the inducible beta-lactamase AmpC. Our picture on the condition-dependent regulation of ampC expression is incomplete. A known regulator is AmpR controlling the transcription of ampC in response to unrecycled muropeptides as a signal for cell wall stress. In our study, we uncovered the global transcriptional regulator LsrB as a critical player acting upstream of AmpR. Deletion of lsrB led to severe ampicillin and penicillin sensitivity, which could be restored to wild-type levels by lsrB complementation. By transcriptome profiling via RNA-Seq and qRT-PCR and by electrophoretic mobility shift assays, we show that ampD coding for an anhydroamidase involved in peptidoglycan recycling is under direct negative control by LsrB. Controlling AmpD levels by the LysR-type regulator in turn impacts the cytoplasmic concentration of cell wall degradation products and thereby the AmpR-mediated regulation of ampC. Our results substantially expand the existing model of inducible beta-lactam resistance in A. tumefaciens by establishing LsrB as higher-level transcriptional regulator.

A double-negative feedback loop between NtrBC and a small RNA rewires nitrogen metabolism in legume symbionts.

Root nodule endosymbioses between diazotrophic rhizobia and legumes provide the largest input of combined N to the biosphere, thus representing an alternative to harmful chemical fertilizers for sustainable crop production. Rhizobia have evolved intricate strategies to coordinate N assimilation for their own benefit with N2 fixation to sustain plant growth. The rhizobial N status is transduced by the NtrBC two-component system, the seemingly ubiquitous form of N signal transduction in Proteobacteria. Here, we show that the regulatory sRNA NfeR1 (nodule formation efficiency RNA) of the alfalfa symbiont Sinorhizobium meliloti is transcribed from a complex promoter repressed by NtrC in a N-dependent manner and feedback silences ntrBC by complementary base-pairing. These findings unveil a more prominent role of NtrC as a transcriptional repressor than hitherto anticipated and a novel RNA-based mechanism for NtrBC regulation. The NtrBC-NfeR1 double-negative feedback loop accurately rewires symbiotic S. meliloti N metabolism and is likely conserved in α-rhizobia.

Membrane-enclosed Pseudomonas quinolone signal attenuates bacterial virulence by interfering with quorum sensing.

Outer membrane vesicle (OMV)-delivered Pseudomonas quinolone signal (PQS) plays a critical role in cell-cell communication in Pseudomonas aeruginosa. However, the functions and mechanisms of membrane-enclosed PQS in interspecies communication in microbial communities are not clear. Here, we demonstrate that PQS delivered by both OMVs from P. aeruginosa and liposome reduces the competitiveness of Burkholderia cenocepacia, which usually shares the same niche in the lungs of cystic fibrosis patients, by interfering with quorum sensing (QS) in B. cenocepacia through the LysR-type regulator ShvR. Intriguingly, we found that ShvR regulates the production of the QS signals cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) by directly binding to the promoters of signal synthase-encoding genes. Perception of PQS influences the regulatory activity of ShvR and thus ultimately reduces QS signal production and virulence in B. cenocepacia. Our findings provide insights into the interspecies communication mediated by the membrane-enclosed QS signal among bacterial species residing in the same microbial community. IMPORTANCE Quorum sensing (QS) is a ubiquitous cell-to-cell communication mechanism. Previous studies showed that Burkholderia cenocepacia mainly employs cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) QS systems to regulate biological functions and virulence. Here, we demonstrate that Pseudomonas quinolone signal (PQS) delivered by outer membrane vesicles from Pseudomonas aeruginosa or liposome attenuates B. cenocepacia virulence by targeting the LysR-type regulator ShvR, which regulates the production of the QS signals BDSF and AHL in B. cenocepacia. Our results not only suggest the important roles of membrane-enclosed PQS in interspecies and interkingdom communications but also provide a new perspective on the use of functional nanocarriers loaded with QS inhibitors for treating pathogen infections.

An underlying mechanism for MleR activating the malolactic enzyme pathway to enhance acid tolerance in Lacticaseibacillus paracasei L9.

Tolerance to acid stress is a crucial property of probiotics against gastric acids. The malolactic enzyme pathway is one of the most important acid resistance systems in lactic acid bacteria. It has been reported that the malolactic enzyme pathway was regulated by the transcriptional regulator, MleR. However, regulatory mechanisms underlying malolactic enzyme pathway to cope with acid stress remain unknown. In this study, the acid tolerance ability of the ΔmleR deletion strain was significantly lower than that of the wild-type strain, and the complementation of the mleR gene into the ΔmleR strain restored the acid tolerance of the ΔmleR strain, indicating that MleR was involved in acid tolerance response of Lacticaseibacillus paracasei L9. Real-time quantitative PCR and transcriptional fusion experiments confirmed MleR-activated transcription of the mleST gene cluster. Furthermore, MleR was confirmed to directly bind to the promoter region of the mleST operon using ChIP assays and EMSAs. The transcription start site G of the mleST operon was located at position -198 relative to the start codon of the mleS gene. The region from -80 to -61 upstream of the transcription start site was determined to be essential for MleR binding. Moreover, L-malic acid acted as an effector for MleR to activate the transcription of the mleST operon in a dose-dependent manner. These results revealed the regulatory mechanism behind MleR-mediated activation of the malolactic enzyme pathway to enhance acid tolerance in Lc. paracasei L9. IMPORTANCE Lacticaseibacillus paracasei is extensively used as probiotics in human health and fermented dairy production. Following consumption, Lc. paracasei is exposed to a variety of physico-chemical stresses, such as low pH in the stomach and bile salts in the intestines. The high acidity of the stomach severely inhibits bacterial metabolism and growth. Therefore, the acid tolerance response is critical for Lc. paracasei to survive. It has been reported that the malolactic enzyme (MLE) pathway plays an important role for LAB to resist acid stress. However, the regulatory mechanism has not yet been investigated. In this study, we determined that the LysR-type regulator MleR positively regulated the MLE pathway to enhance acid tolerance by binding -80 to -61 upstream of the transcription start site of the mleST operon. Further, L-malic acid acts as a co-inducer for MleR transcriptional regulation. Our study provides novel insights into acid tolerance mechanisms in LAB.

The XylR/NtrC-type regulator CbsR positively regulates upstream pathway of chlorobenzene degradation in Pandoraea pnomenusa.

Pandoraea pnomenusa MCB032 completely degrades chlorobenzene, whose metabolic pathway is encoded by cbs and clc gene clusters. The putative regulatory factors ClcR and CbsR are predicted to regulate the cbs and clc gene clusters. This research aims to understand the function of ClcR and CbsR. RT-PCR analyses demonstrated that the cbsFAaAbAcAdB operon that encodes catabolic pathways for the degradation of chlorobenzene to chlorocatechol is located on an operon. Moreover, the clcABCDE operon is involved in the 3-chlorocatechol pathway. Gene knockout and transcriptional analysis showed that the transcription of the cbsFAaAbAcAdB operon is positively regulated by CbsR, whereas the clcABCDE operon is activated by ClcR. Primer extension analysis was used to locate the transcription start sites of the cbsFAaAbAcAdB and cbsR operons. Electrophoretic mobility shift assay analyses showed that CbsR is bound to the sites in the promoter regions of cbsFAaAbAcAdB and cbsR operons. The XylR/NtrC-type regulator CbsR positively regulates the transcription of the cbsFAaAbAcAdB operon encoding the upstream pathway of chlorobenzene catabolism, while the LysR-type regulator ClcR activates the clcABCDE operon encoding the downstream pathway.

BysR, a LysR-Type Pleiotropic Regulator, Controls Production of Occidiofungin by Activating the LuxR-Type Transcriptional Regulator AmbR1 in Burkholderia sp. Strain JP2-270.

Occidiofungin is a highly effective antifungal glycopeptide produced by certain Burkholderia strains. The ocf gene cluster, responsible for occidiofungin biosynthesis, is regulated by the cluster-specific regulators encoded by an ambR homolog(s) within the same gene cluster, while the extent to which occidiofungin biosynthesis is connected with the core regulation network remains unknown. Here, we report that the LysR-type regulator BysR acts as a pleiotropic regulator and is essential for occidiofungin biosynthesis. Magnaporthe oryzae was used as an antifungal target in this study, and deletion of bysR and ocfE abolished the antagonistic activity against M. oryzae in Burkholderia sp. strain JP2-270. The ΔbysR defect can be recovered by constitutively expressing bysR or ambR1, but not ambR2. Electrophoretic mobility shift assays (EMSAs) collectively showed that BysR regulates ambR1 by directly binding to its promoter region. In addition, transcriptomic analysis revealed altered expression of 350 genes in response to bysR deletion, and the genes engaged in flagellar assembly and bacterial chemotaxis constitute the most enriched pathways. Also, 400 putative BysR-targeted loci were identified by DNA affinity purification sequencing (DAP-seq) in JP2-270. These loci include not only genes engaged in key metabolic pathways but also those involved in secondary metabolic pathways. To conclude, the occidiofungin produced by JP2-270 is the main substance inhibiting M. oryzae, and BysR controls occidiofungin production by directly targeting ambR1, an intracluster transcriptional regulatory gene that further activates the transcription of the ocf gene cluster. IMPORTANCE We report for the first time that occidiofungin production is regulated by the global transcriptional factor BysR, by directly targeting the specific regulator ambR1, which further promotes the transcription of ocf genes. BysR also acts as a pleiotropic regulator that controls various cellular processes in Burkholderia sp. strain JP2-270. This study provides insight into the regulatory mechanism of occidiofungin synthesis and enhances our understanding of the regulatory patterns of the LysR-type regulator.

Identification of a Novel LysR Family Transcriptional Regulator Controlling Acquisition of Sulfur Sources in Acinetobacter baumannii

<sc>l</sc>-cysteine biosynthesis from inorganic sulfur represents a major mechanism by which reduced sulfur is incorporated into organic compounds. Cysteine biosynthesis and regulation is well characterized in Escherichia coli. However, the regulation of sulfur metabolism in Acinetobacter baumannii is only partly understood, with the LysR-type regulator, GigC known to control some aspects of sulfur reduction. In this study, we have used transcriptomics and bioinformatic analyses to characterize a novel LysR-type transcriptional regulator encoded by ABUW_1016 (cbl), in a highly multidrug resistant and virulent isolate of A. baumannii. We have shown that Cbl is involved in controlling expression of the genes required for uptake and reduction of various sulfur sources in A. baumannii. Collectively, we have identified the global regulon of Cbl and proposed a model of cysteine biosynthesis and its regulation by Cbl and GigC in A. baumannii.

A LysR Transcriptional Regulator Manipulates Macrophage Autophagy Flux During Brucella Infection.

Brucella, the intracellular bacteria, have evolved subtle strategies to efficiently survive and replicate in macrophages. However, the virulence effector proteins involved are still unclear. LysR-type transcriptional regulators (lttrs) are the largest regulator family with diverse function in prokaryotes. However, very little is known about the role of LysR regulators in the Brucella spp. Here, a BSS2_II0858 gene, encoded as one of the LysR-type regulators, was studied. We successfully constructed a BSS2_II0858 deletion mutant, Δ0858, and complementation strain CΔ0858 in Brucella suis S2. The cell apoptosis induced by B. suis S2 and its derivatives were detected by flow cytometry. The autophagy was then assessed by immunoblot analysis using the IL3I/II and p62 makers. In addition, the autophagy flux was evaluated by double fluorescent labeling method for autophagy marker protein LC3. Our studies demonstrated that B. suis S2 and its derivatives inhibited the programmed cell death in early stage and promoted apoptosis in the later stage during infection in RAW264.7 cells. The BSS2_II0858 gene was found to play no role during apoptosis according to these results. Compared with the wild-type strain, Δ0858 mutant can stimulate the conversion of LC3-I to LC3-II and markedly inhibited the autophagy flux at early stage leading to obvious autophagosome accumulation. This study explored the function of BSS2_II0858 gene and may provide new insights for understanding the mechanisms involved in the survival of Brucella in macrophages.

The Lon protease negatively regulates pyoluteorin biosynthesis through the Gac/Rsm-RsmE cascade and directly degrades the transcriptional activator PltR in Pseudomonas protegens H78.

Pyoluteorin (Plt) is a broad-spectrum antibiotic with antibacterial and antifungal activities. In Pseudomonas protegens H78, the Plt biosynthetic operon pltLABCDEFG is transcriptionally activated by the LysR-type regulator PltR and is positively regulated by the Gac/Rsm signal transduction cascade (GacS/A-RsmXYZ-RsmE-pltR/pltAB). Additionally, Plt biosynthesis has been shown to be significantly enhanced by mutation of the Lon protease-encoding gene. This study aims to understand the negative regulation pathway and molecular mechanism by which Lon functions in Plt biosynthesis. lon deletion was first found to improve the antimicrobial ability of strain H78 due to its increased Plt production, while partially inhibiting the growth of H78 strain. Lon protease decreases the abundance and stability of the two-component system response regulator GacA and thus participates in the abovementioned Gac/Rsm cascade and negatively regulates Plt biosynthesis. Similarly, Lon protease also decreases the abundance and stability of transcriptional activator PltR. PltR protein can be directly degraded by the Lon protease but not by a mutated form of Lon protease with an amino acid replacement of S674 -A. In summary, Lon protease negatively regulates Plt biosynthesis via both the Gac/Rsm-mediated global regulatory pathway and the direct degradation of the transcriptional activator PltR in P. protegens H78.

Pervasive RNA Regulation of Metabolism Enhances the Root Colonization Ability of Nitrogen-Fixing Symbiotic α-Rhizobia.

ABSTRACTThe rhizosphere and rhizoplane are nutrient-rich but selective environments for the root microbiome. Here, we deciphered a posttranscriptional network regulated by the homologous trans-small RNAs (sRNAs) AbcR1 and AbcR2, which rewire the metabolism of the nitrogen-fixing α-rhizobium Sinorhizobium meliloti during preinfection stages of symbiosis with its legume host alfalfa. The LysR-type regulator LsrB, which transduces the cell redox state, is indispensable for AbcR1 expression in actively dividing bacteria, whereas the stress-induced transcription of AbcR2 depends on the alternative σ factor RpoH1. MS2 affinity purification coupled with RNA sequencing unveiled exceptionally large and overlapping AbcR1/2 mRNA interactomes, jointly representing ⁓6% of the S. meliloti protein-coding genes. Most mRNAs encode transport/metabolic proteins whose translation is silenced by base pairing to two distinct anti-Shine Dalgarno motifs that function independently in both sRNAs. A metabolic model-aided analysis of the targetomes predicted changes in AbcR1/2 expression driven by shifts in carbon/nitrogen sources, which were confirmed experimentally. Low AbcR1/2 levels in some defined media anticipated overexpression growth phenotypes linked to the silencing of specific mRNAs. As a proof of principle, we confirmed AbcR1/2-mediated downregulation of the l-amino acid AapQ permease. AbcR1/2 interactomes are well represented in rhizosphere-related S. meliloti transcriptomic signatures. Remarkably, a lack of AbcR1 specifically compromised the ability of S. meliloti to colonize the root rhizoplane. The AbcR1 regulon likely ranks the utilization of available substrates to optimize metabolism, thus conferring on S. meliloti an advantage for efficient rhizosphere/rhizoplane colonization. AbcR1 regulation is predicted to be conserved in related α-rhizobia, which opens unprecedented possibilities for engineering highly competitive biofertilizers.

Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube.

The current study aims at the functional and kinetic characterization of protocatechuate (PCA) 4,5-dioxygenase (PcaA) from Pseudarthrobacter phenanthrenivorans Sphe3. This is the first single subunit Type II dioxygenase characterized in Actinobacteria. RT-PCR analysis demonstrated that pcaA and the adjacent putative genes implicated in the PCA meta-cleavage pathway comprise a single transcriptional unit. The recombinant PcaA is highly specific for PCA and exhibits Michaelis–Menten kinetics with Km and Vmax values of 21 ± 1.6 μM and 44.8 ± 4.0 U × mg−1, respectively, in pH 9.5 and at 20 °C. PcaA also converted gallate from a broad range of substrates tested. The enzymatic reaction products were identified and characterized, for the first time, through in situ biotransformation monitoring inside an NMR tube. The PCA reaction product demonstrated a keto-enol tautomerization, whereas the gallate reaction product was present only in the keto form. Moreover, the transcriptional levels of pcaA and pcaR (gene encoding a LysR-type regulator of the pathway) were also determined, showing an induction when cells were grown on PCA and phenanthrene. Studying key enzymes in biodegradation pathways is significant for bioremediation and for efficient biocatalysts development.

Crystal structure of the full‐length LysR‐type transcription regulator CbnR in complex with promoter DNA

LysR-type transcription regulators (LTTRs) comprise one of the largest families of transcriptional regulators in bacteria. They are typically homo-tetrameric proteins and interact with promoter DNA of approximately 50-60 bp. Earlier biochemical studies have suggested that LTTR binding to promoter DNA bends the DNA and, upon inducer binding, the bend angle of the DNA is reduced through a quaternary structure change of the tetrameric LTTR, leading to the activation of transcription. To date, crystal structures of full-length LTTRs, DNA-binding domains with their target DNAs, and the regulatory domains with and without inducer molecules have been reported. However, these crystal structures have not provided direct evidence of the quaternary structure changes of LTTRs or of the molecular mechanism underlying these changes. Here, we report the first crystal structure of a full-length LTTR, CbnR, in complex with its promoter DNA. The crystal structure showed that, in the absence of bound inducer molecules, the four DNA-binding domains of the tetrameric CbnR interact with the promoter DNA, bending the DNA by approximately 70°. Structural comparison between the DNA-free and -bound forms demonstrates that the quaternary structure change of the tetrameric CbnR required for promoter region-binding arises from relative orientation changes of the three domains in each subunit. The mechanism of the quaternary structure change caused by inducer binding is also discussed based on the present crystal structure, affinity analysis between CbnR and the promoter DNA, and earlier mutational studies on CbnR.

A LysR-type transcriptional regulator controls the expression of numerous small RNAs in Agrobacterium tumefaciens.

Small RNAs (sRNAs) are universal posttranscriptional regulators of gene expression and hundreds of sRNAs are frequently found in each and every bacterium. In order to coordinate cellular processes in response to ambient conditions, many sRNAs are differentially expressed. Here, we asked how these small regulators are regulated using Agrobacterium tumefaciens as a model system. Among the best-studied sRNAs in this plant pathogen are AbcR1 regulating numerous ABC transporters and PmaR, a regulator of peptidoglycan biosynthesis, motility, and ampicillin resistance. We report that the LysR-type regulator VtlR (also known as LsrB) controls expression of AbcR1 and PmaR. A vtlR/lsrB deletion strain showed growth defects, was sensitive to antibiotics and severely compromised in plant tumor formation. Transcriptome profiling by RNA-sequencing revealed more than 1,200 genes with altered expression in the mutant. Consistent with the function of VtlR/LsrB as regulator of AbcR1, many ABC transporter genes were affected. Interestingly, the transcription factor did not only control the expression of AbcR1 and PmaR. In the mutant, 102 sRNA genes were significantly up- or downregulated. Thus, our study uncovered VtlR/LsrB as the master regulator of numerous sRNAs. Thereby, the transcriptional regulator harnesses the regulatory power of sRNAs to orchestrate the expression of distinct sub-regulons.

RovC - a novel type of hexameric transcriptional activator promoting type VI secretion gene expression.

Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment.

OnfD, an AraC-Type Transcriptional Regulator Encoded by Rhizobium tropici CIAT 899 and Involved in Nod Factor Synthesis and Symbiosis.

Rhizobium tropici CIAT 899 is a broad-host-range rhizobial strain that establishes symbiotic interactions with legumes and tolerates different environmental stresses such as heat, acidity, or salinity. This rhizobial strain produces a wide variety of symbiotically active nodulation factors (NF) induced not only by the presence of plant-released flavonoids but also under osmotic stress conditions through the LysR-type transcriptional regulators NodD1 (flavonoids) and NodD2 (osmotic stress). However, the activation of NodD2 under high-osmotic-stress conditions remains elusive. Here, we have studied the role of a new AraC-type regulator (named as OnfD) in the symbiotic interaction of R. tropici CIAT 899 with Phaseolus vulgaris and Lotus plants. We determined that OnfD is required under salt stress conditions for the transcriptional activation of the nodulation genes and therefore the synthesis and export of NF, which are required for a successful symbiosis with P. vulgaris Moreover, using bacterial two-hybrid analysis, we demonstrated that the OnfD and NodD2 proteins form homodimers and OnfD/NodD2 form heterodimers, which could be involved in the production of NF in the presence of osmotic stress conditions since both regulators are required for NF synthesis in the presence of salt. A structural model of OnfD is presented and discussed.IMPORTANCE The synthesis and export of rhizobial NF are mediated by a conserved group of LysR-type regulators, the NodD proteins. Here, we have demonstrated that a non-LysR-type regulator, an AraC-type protein, is required for the transcriptional activation of symbiotic genes and for the synthesis of symbiotically active NF under salt stress conditions.

Rhizobium leguminosarum bv. trifolii NodD2 Enhances Competitive Nodule Colonization in the Clover-Rhizobium Symbiosis.

Establishment of the symbiotic relationship that develops between rhizobia and their legume hosts is contingent upon an interkingdom signal exchange. In response to host legume flavonoids, NodD proteins from compatible rhizobia activate expression of nodulation genes that produce lipochitin oligosaccharide signaling molecules known as Nod factors. Root nodule formation commences upon legume recognition of compatible Nod factor. Rhizobium leguminosarum was previously considered to contain one copy of nodD; here, we show that some strains of the Trifolium (clover) microsymbiont R. leguminosarum bv. trifolii contain a second copy designated nodD2. nodD2 genes were present in 8 out of 13 strains of R. leguminosarum bv. trifolii, but were absent from the genomes of 16 R. leguminosarum bv. viciae strains. Analysis of single and double nodD1 and nodD2 mutants in R. leguminosarum bv. trifolii strain TA1 revealed that NodD2 was functional and enhanced nodule colonization competitiveness. However, NodD1 showed significantly greater capacity to induce nod gene expression and infection thread formation. Clover species are either annual or perennial and this phenological distinction is rarely crossed by individual R. leguminosarum bv. trifolii microsymbionts for effective symbiosis. Of 13 strains with genome sequences available, 7 of the 8 effective microsymbionts of perennial hosts contained nodD2, whereas the 3 microsymbionts of annual hosts did not. We hypothesize that NodD2 inducer recognition differs from NodD1, and NodD2 functions to enhance competition and effective symbiosis, which may discriminate in favor of perennial hosts.IMPORTANCE Establishment of the rhizobium-legume symbiosis requires a highly specific and complex signal exchange between both participants. Rhizobia perceive legume flavonoid compounds through LysR-type NodD regulators. Often, rhizobia encode multiple copies of nodD, which is one determinant of host specificity. In some species of rhizobia, the presence of multiple copies of NodD extends their symbiotic host-range. Here, we identified and characterized a second copy of nodD present in some strains of the clover microsymbiont Rhizobium leguminosarum bv. trifolii. The second nodD gene contributed to the competitive ability of the strain on white clover, an important forage legume. A screen for strains containing nodD2 could be utilized as one criterion to select strains with enhanced competitive ability for use as inoculants for pasture production.

Transcriptional regulation of drug resistance mechanisms in Salmonella: where we stand and what we need to know.

Salmonellae have evolved a wide range of molecular mechanisms to neutralize the effect of antibiotics and evade the host immune system response. These mechanisms are exquisitely controlled by global and local regulators and enable the pathogens to use its energy as per need and hence allow the pathogen to economize the consumption of energy by its cellular machinery. Several families that regulate the expression of different drug resistance genes are known; some of these are: the TetR family (which affects tetracycline resistance genes), the AraC/XylS family (regulators that can act as both transcriptional activators and repressors), two-component signal transduction systems (e.g. PhoPQ, a key regulator for virulence), mercury resistance Mer-R and multiple antibiotic resistance Mar-R regulators, LysR-type global regulators (e.g. LeuO) and histone-like protein regulators (involved in the repression of newly transferred resistance genes). This minireview focuses on the role of different regulators harbored by the Salmonella genome and characterized for mediating the drug resistance mechanisms particularly via efflux and influx systems. Understanding of such transcriptional regulation mechanisms is imperative to address drug resistance issues in Salmonella and other bacterial pathogens.