Two-component Response Regulator Research Articles

Enhancement of phenol biodegradation: Metabolic division of labor in co-culture of Stenotrophomonas sp. N5 and Advenella sp. B9

The aim of this work is to study the synergistic effect of Stenotrophomonas sp. N5 and Advenella sp. B9 co-culture (COC) on enhancement of phenol biodegradation. These two strains utilizing phenol as sole carbon and energy source were isolated from phenol-containing coking wastewater. The results of biodegradation experiment showed the COC of N5 and B9 has stronger capability to degrade phenol than either of mono-culture (MOC). Growth kinetics studies indicated inhibitory effect of phenol on COC was reduced by the interaction of N5 and B9 in COC. The RNA-Seq results demonstrated that phenol biodegradation was enhanced by metabolic division of labor (DOL) in COC based on the expression of key genes for phenol degradation. GO enrichment analysis of differentially expressed genes (DEGs) indicated DEGs between COC and MOC degradation systems are mainly concentrated in the synthesis of cell components, microbial growth and metabolism, and catalytic activity. The expression of 3 transcriptional factors (LysR, Two-component system response regulator, and TetR families) which can regulate degradation of aromatic compounds, was identified beneficial to phenol degradation.

New Roles for Two-Component System Response Regulators of Salmonella enterica Serovar Typhi during Host Cell Interactions.

In order to survive external stresses, bacteria need to adapt quickly to changes in their environment. One adaptive mechanism is to coordinate and alter their gene expression by using two-component systems (TCS). TCS are composed of a sensor kinase that activates a transcriptional response regulator by phosphorylation. TCS are involved in motility, virulence, nutrient acquisition, and envelope stress in many bacteria. The pathogenic bacteria Salmonella enterica serovar Typhi (S. Typhi) possess 30 TCSs, is specific to humans, and causes typhoid fever. Here, we have individually deleted each of the 30 response regulators. We have determined their role during interaction with host cells (epithelial cells and macrophages). Deletion of most of the systems (24 out of 30) resulted in a significant change during infection. We have identified 32 new phenotypes associated with TCS of S. Typhi. Some previously known phenotypes associated with TCSs in Salmonella were also confirmed. We have also uncovered phenotypic divergence between Salmonella serovars, as distinct phenotypes between S. Typhi and S. Typhimurium were identified for cpxR. This finding highlights the importance of specifically studying S. Typhi to understand its pathogenesis mechanisms and to develop strategies to potentially reduce typhoid infections.

In silico characterization, and expression analysis of rice golden 2-like (OsGLK) members in response to low phosphorous.

The availability of phosphorus (P) affects productivity of rice. Under acidic soil conditions (pH < 5.5), P is rapidly immobilized in the soil. Several transcription factors play an important role in low Pi tolerance response, including MYB family members but their role in acidic soil is yet unknown. In this study, genome wide identification and characterization of golden 2-like (GLK) members belonging to GARP superfamily from rice (OsGLK) led to identification of 46 members distributed over 12 chromosomes. We assigned gene nomenclature, analyzed gene structure and identified mutant orthologs and phenotypes in maize and rice, respectively. On the basis of biological functions three categories viz., (a) two-component response regulator (five members), (b) putative transcription factor (21 members) and (c) phosphate starvation response (8 members) were identified. Phylogenetic analysis revealed a total of nine subgroups with MYB homeodomain-like and MYB CC-type domains conserved across members. Expression profiling of OsGLKs in response to 24 and 48 h of low Pi in four contrasting rice genotypes, revealed significantly higher expression of OsGLK10, OsGLK15, OsGLK22 and OsGLK30 in tolerant genotypes as compared to susceptible genotypes, suggesting their role in Pi starvation tolerance. Meta analyses and cis-regulatory elements (CREs) profiling of OsGLK showed diverse expression pattern in various tissues and organs and also modulation in response to various abiotic and biotic stresses. Our results highlight the versatile role of this diverse and complex GLK family, in particular to abiotic stress. These genes will form the basis of future studies on low Pi tolerance in acidic soils.

Overexpression of the response regulator rpaA causes an impaired cell division in the Cyanobacterium Synechocystis sp. PCC 6803.

In photosynthetic microorganisms, cell cycle progression depends on day and night cycles; however, how cell division is regulated in response to these environmental changes is poorly understood. RpaA has been implicated in the signal output from both circadian clocks and light/dark conditions in the unicellular spherical-celled cyanobacterium Synechocystis sp. PCC 6803. In the present study, we investigated the involvement of a two-component response regulator RpaA in cell division regulation. Firstly, we examined the effects of rpaA overexpression on cell morphology and the expression levels of cell division genes. We observed an increase in the volume of non-dividing cells and a high proportion of dividing cells in rpaA-overexpressing strains by light microscopy. The expression levels of selected cell division-related genes were higher in the rpaA-overexpressing strain than in the wild type, including minD of the Min system; cdv3 and zipN, which encode two divisome components; and murB, murC, and pbp2, which are involved in peptidoglycan (PG) synthesis. Moreover, in the rpaA-overexpressing strain, the outer membrane and cell wall PG layer were not smooth, and the outer membrane was not clearly visible by transmission electron microscopy. These results demonstrated that rpaA overexpression causes an impaired cell division, which is accompanied by transcriptional activation of cell division genes and morphological changes in the PG layer and outer membrane.

RstA, a two-component response regulator, plays important roles in multiple virulence-associated processes in enterohemorrhagic Escherichia coli O157:H7

BackgroundEnterohemorrhagic Escherichia coli O157:H7 (EHEC O157) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC O157 encounters varied microenvironments during infection, and can efficiently adapt to these using the two-component system (TCS). Recently, a functional TCS, RstAB, has been implicated in the regulation of virulence of several bacterial pathogens. However, the regulatory function of RstAB in EHEC O157 is poorly understood. This study aimed at providing insights into the global effects of RstA on gene expression in EHEC O157.ResultsIn the present study, we analyzed gene expression differences between the EHEC O157 wild-type strain and a ΔrstA mutant using RNA-seq technology. Genes with differential expression in the ΔrstA mutant compared to that in the wild-type strain were identified and grouped into clusters of orthologous categories. RstA promoted EHEC O157 LEE gene expression, adhesion in vitro, and colonization in vivo by indirect regulation. We also found that RstA could bind directly to the promoter region of hdeA and yeaI to enhance acid tolerance and decrease biofilm formation by modulating the concentration of c-di-GMP.ConclusionsIn summary, the RstAB TCS in EHEC O157 plays a major role in the regulation of virulence, acid tolerance, and biofilm formation. We clarified the regulatory function of RstA, providing an insight into mechanisms that may be potential drug targets for treatment of EHEC O157-related infections.

ITRAQ-based proteomics reveals key role of γ-aminobutyric acid (GABA) in regulating drought tolerance in perennial creeping bentgrass (Agrostis stolonifera)

γ-Aminobutyric acid (GABA), a non-proteinaceous amino acid, modulates plant growth and stress tolerance. However, the potential role of GABA in regulating key metabolic pathways and stress-defensive proteins against drought in plants has never been explored. Creeping bentgrass (Agrostis stolonifera) plants were pretreated with or without GABA and then subjected to water stress for 8 days in controlled growth chambers (23/19 °C, day/night). Physiological analysis showed that elevated endogenous GABA level via exogenous GABA application significantly mitigated water stress damage to creeping bentgrass, as manifested by increased leaf relative water content, water use efficiency, osmotic adjustment (OA), photochemical efficiency (Fv/Fm), net photosynthetic rate, and reduced oxidative damage. iTRAQ-based proteomics found that enhanced chaperones accumulation, carbohydrates, amino acids, and energy metabolism played important roles in protein protection, OA, energy maintenance, and metabolic balance, which is important adaptive response to drought stress in creeping bentgrass. The GABA further promoted energy production and conversion, antioxidant defense, and DHN3 accumulation that were essential for energy requirement, ROS-scavenging, and the prevention of cell dehydration in leaf during drought stress. In addition, GABA-treated plants maintained significantly higher abundance of dicarboxylate transporter 2.1, ATP-dependent zinc metalloprotease, receptor-like protein kinase HERK1, o-acyltransferase WSD1, omega-6 fatty acid desaturase, and two-component response regulator ORR21 than untreated plants under drought stress. The result provides new evidences that GABA-induced drought tolerance is possibly involved in the improvement of nitrogen recycling, protection of photosystem II, mitigation of drought-depressed cell elongation, wax biosynthesis, fatty acid desaturase, and delaying leaf senescence in creeping bentgrass.

Identification of genes regulated by the two-component system response regulator NarP of Actinobacillus pleuropneumoniae via DNA-affinity-purified sequencing

Identifying the direct target genes of response regulators (RRs) of a bacterial two-component system (TCS) is critical to understand the roles of TCS in bacterial environmental adaption and pathogenesis. Actinobacillus pleuropneumoniae is an important respiratory bacterial pathogen that causes considerable economic losses to swine industry worldwide. The targets of A. pleuropneumoniae NarP (nitrate/nitrite RR), which is the cognate RR of the nitrate/nitrite sensor histidine kinase NarQ, are still unknown. In the present study, a DNA-affinity-purified sequencing (DAP-Seq) approach was established. The upstream regions of a total of 131 candidate genes from the genome of A. pleuropneumoniae were co-purified with the activated NarP protein. Electrophoretic mobility shift assay (EMSA) results confirmed the interactions of NarP with the promoter regions of five selected target genes, including dmsA, pgaA, ftpA, cstA and ushA. The EMSA-confirmed target genes were significantly up-regulated in the narP-deleted mutant in the presence of additional nitrate, whilst the transcriptional changes were restored in the complemented strain. The NarP binding motif in the upstream regions of the target genes dmsA and ftpA were further identified and confirmed by EMSA using the truncated binding motif. The NarP binding sites were present in a total of 25.2% of the DNA fragments captured by DAP-Seq. These results demonstrated that the established DAP-Seq method is effective for exploring the direct targets of RRs of bacterial TCSs and that the A. pleuropneumoniae NarP could be a repressor in response to nitrate.

Surveillance and Genomics of Toxigenic Vibrio cholerae O1 From Fish, Phytoplankton and Water in Lake Victoria, Tanzania.

The occurrence of toxigenic Vibrio cholerae O1 during a non- outbreak period in Lake Victoria was studied and genetic characteristics for environmental persistence and relatedness to pandemic strains were assessed. We analyzed 360 samples of carps, phytoplankton and water collected in 2017 during dry and rainy seasons in the Tanzanian basin of Lake Victoria. Samples were tested using PCR (ompW and ctxA) with DNA extracted from bacterial isolates and samples enriched in alkaline peptone water. Isolates were screened with polyvalent antiserum O1 followed by antimicrobial susceptibility testing. Whole genome sequencing and bioinformatics tools were employed to investigate the genomic characteristics of the isolates. More V. cholerae positive samples were recovered by PCR when DNA was obtained from enriched samples than from isolates (69.0% vs. 21.3%, p < 0.05), irrespectively of season. We identified ten V. cholerae O1 among 22 ctxA-positive isolates. Further studies are needed to serotype the remaining ctxA-positive non-O1 strains. Sequenced strains belonged to El Tor atypical biotype of V. cholerae O1 of MLST ST69 harboring the seventh pandemic gene. Major virulence genes, ctxA, ctxB, zot, ace, tcpA, hlyA, rtxA, ompU, toxR, T6SS, alsD, makA and pathogenicity islands VPI-1, VPI-2, VSP-1, and VSP-2 were found in all strains. The strains contained Vibrio polysaccharide biosynthesis enzymes, the mshA gene and two-component response regulator proteins involved in stress response and autoinducers for quorum sensing and biofilm formation. They carried the SXT integrative conjugative element with phenotypic and genotypic resistance to aminoglycoside, sulfamethoxazole, trimethoprim, phenicol, and quinolones. Strains contained a multidrug efflux pump component and were resistant to toxic compounds with copper homeostasis and cobalt-zinc-cadmium resistance proteins. The environmental strains belonged to the third wave of the seventh pandemic and most are genetically closely related to recent outbreak strains from Tanzania, Kenya, and Uganda with as low as three SNPs difference. Some strains have persisted longer in the environment and were more related to older outbreak strains in the region. V. cholerae O1 of outbreak potential seem to persist in Lake Victoria through interactions with fish and phytoplankton supported by the optimum water parameters and intrinsic genetic features enhancing survival in the aquatic environment.

Highly multiplexed AmpliSeq technology identifies novel variation of flowering time-related genes in soybean (Glycine max).

Whole-genome re-sequencing is a powerful approach to detect gene variants, but it is expensive to analyse only the target genes. To circumvent this problem, we attempted to detect novel variants of flowering time-related genes and their homologues in soybean mini-core collection by target re-sequencing using AmpliSeq technology. The average depth of 382 amplicons targeting 29 genes was 1,237 with 99.85% of the sequence data mapped to the reference genome. Totally, 461 variants were detected, of which 150 sites were novel and not registered in dbSNP. Known and novel variants were detected in the classical maturity loci—E1, E2, E3, and E4. Additionally, large indel alleles, E1-nl and E3-tr, were successfully identified. Novel loss-of-function and missense variants were found in FT2a, MADS-box, WDR61, phytochromes, and two-component response regulators. The multiple regression analysis showed that four genes—E2, E3, Dt1, and two-component response regulator—can explain 51.1–52.3% of the variation in flowering time of the mini-core collection. Among them, the two-component response regulator with a premature stop codon is a novel gene that has not been reported as a soybean flowering time-related gene. These data suggest that the AmpliSeq technology is a powerful tool to identify novel alleles.

LurR is a regulator of the central lactate oxidation pathway in sulfate-reducing Desulfovibrio species.

The central carbon/lactate utilization pathway in the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, is encoded by the highly conserved operon DVU3025-3033. Our earlier in vitro genome-wide study had suggested a network of four two-component system regulators that target this large operon; however, how these four regulators control this operon was not known. Here, we probe the regulation of the lactate utilization operon with mutant strains and DNA-protein binding assays. We show that the LurR response regulator is required for optimal growth and complete lactate utilization, and that it activates the DVU3025-3033 lactate oxidation operon as well as DVU2451, a lactate permease gene, in the presence of lactate. We show by electrophoretic mobility shift assays that LurR binds to three sites in the upstream region of DVU3025, the first gene of the operon. NrfR, a response regulator that is activated under nitrite stress, and LurR share similar binding site motifs and bind the same sites upstream of DVU3025. The DVU3025 promoter also has a binding site motif (Pho box) that is bound by PhoB, a two-component response regulator activated under phosphate limitation. The lactate utilization operon, the regulator LurR, and LurR binding sites are conserved across the order Desulfovibrionales whereas possible modulation of the lactate utilization genes by additional regulators such as NrfR and PhoB appears to be limited to D. vulgaris.

Response of Lactobacillus plantarum WCFS1 to the Gram-Negative Pathogen-Associated Quorum Sensing Molecule N-3-Oxododecanoyl Homoserine Lactone.

The bacterial quorum sensing phenomenon has been well studied since its discovery and has traditionally been considered to include signaling pathways recognized exclusively within either Gram-positive or Gram-negative bacteria. These groups of bacteria synthesize structurally distinct signaling molecules to mediate quorum sensing, where Gram-positive bacteria traditionally utilize small autoinducing peptides (AIPs) and Gram-negatives use small molecules such as acyl-homoserine lactones (AHLs). The structural differences between the types of signaling molecules have historically implied a lack of cross-talk among Gram-positive and Gram-negative quorum sensing systems. Recent investigations, however, have demonstrated the ability for AIPs and AHLs to be produced by non-canonical organisms, implying quorum sensing systems may be more universally recognized than previously hypothesized. With that in mind, our interests were piqued by the organisms Lactobacillus plantarum, a Gram-positive commensal probiotic known to participate in AIP-mediated quorum sensing, and Pseudomonas aeruginosa, a characterized Gram-negative pathogen whose virulence is in part controlled by AHL-mediated quorum sensing. Both health-related organisms are known to inhabit the human gut in various instances, both are characterized to elicit distinct effects on host immunity, and some studies hint at the putative ability of L. plantarum to degrade AHLs produced by P. aeruginosa. We therefore wanted to determine if L. plantarum cultures would respond to the addition of N-(3-oxododecanoyl)-L-homoserine lactone (3OC12) from P. aeruginosa by analyzing changes on both the transcriptome and proteome over time. Based on the observed upregulation of various two-component systems, response regulators, and native quorum sensing related genes, the resulting data provide evidence of an AHL recognition and response by L. plantarum.

Deep-Sea Biofilms, Historic Shipwreck Preservation and the Deepwater Horizon Spill

Exposure to oil from the Deepwater Horizon spill may have lasting impacts on preservation of historic shipwrecks in the Gulf of Mexico. Submerged steel structures, including shipwrecks, serve as artificial reefs and become hotspots of biodiversity in the deep-sea. Marine biofilms on submerged structures support settlement of micro- and macrobiota and may enhance and protect against corrosion. Disruptions in the local environment, including oil spills, may impact the role that biofilms play in reef preservation. To determine how the Deepwater Horizon spill potentially impacted shipwreck biofilms and the functional roles of the biofilm microbiome, experiments containing carbon steels disks (CSDs) were placed at five historic shipwreck sites located within, and external to the benthic footprint of the Deepwater Horizon spill. The CSDs were incubated for 16 weeks to enable colonization by biofilm-forming microorganisms and to provide time for in situ corrosion to occur. Biofilms from the CSDs, as well as sediment and water microbiomes, were collected and analyzed by 16S rRNA amplicon gene sequencing to describe community composition and determine the source of taxa colonizing biofilms. Biofilm metagenomes were sequenced to compare differential gene abundances at spill-impacted and reference sites. Biofilms were dominated by Zeta-, Alpha-, Epsilon and Gammaproteobacteria. Sequences affiliated with the Mariprofundus and Sulfurimonas genera were prolific, and Roseobacter, and Colwellia genera were also abundant. Analysis of 16S rRNA sequences from sediment, water, and biofilms revealed sediment to be the main known source of taxa to biofilms at impacted sites. Differential gene abundance analysis revealed the two-component response regulator CreC, a gene involved in environmental stress response, to be elevated at reference sites compared to impacted sites within the spill plume fallout area on the seafloor. Genes for chemotaxis, motility, and alcohol dehydrogenases were differentially abundant at reference vs. impacted sites. Metal loss on CSDs was elevated at sites within the spill fallout plume. Time series images reveal that metal loss at a heavily impacted site, the German Submarine U-166, has accelerated since the spill in 2010. This study provides evidence that spill residues on the seafloor may impact biofilm communities and the preservation of historic steel shipwrecks.

Role of Two-Component System Response Regulator bceR in the Antimicrobial Resistance, Virulence, Biofilm Formation, and Stress Response of Group B Streptococcus.

Group B Streptococcus (GBS; Streptococcus agalactiae) is a leading cause of sepsis in neonates and pregnant mothers worldwide. Whereas the hyper-virulent serogroup III clonal cluster 17 has been associated with neonatal disease and meningitis, serogroup III ST283 was recently implicated in invasive disease among non-pregnant adults in Asia. Here, through comparative genome analyses of invasive and non-invasive ST283 strains, we identified a truncated DNA-binding regulator of a two-component system in a non-invasive strain that was homologous to Bacillus subtilis bceR, encoding the bceRSAB response regulator, which was conserved among GBS strains. Using isogenic knockout and complementation mutants of the ST283 strain, we demonstrated that resistance to bacitracin and the human antimicrobial peptide cathelicidin LL-37 was reduced in the ΔbceR strain with MICs changing from 64 and 256 μg/ml to 0.25 and 64 μg/ml, respectively. Further, the ATP-binding cassette transporter was upregulated by sub-inhibitory concentrations of bacitracin in the wild-type strain. Upregulation of dltA in the wild-type strain was also observed and thought to explain the increased resistance to antimicrobial peptides. DltA, an enzyme involved in D-alanylation during the synthesis of wall teichoic acids, which mediates reduced antimicrobial susceptibility, was previously shown to be regulated by the bceR-type regulator in Staphylococcus aureus. In a murine infection model, we found that the ΔbceR mutation significantly reduced the mortality rate compared to that with the wild-type strain (p < 0.01). Moreover, this mutant was more susceptible to oxidative stress compared to the wild-type strain (p < 0.001) and was associated with reduced biofilm formation (p < 0.0001). Based on 2-DGE and mass spectrometry, we showed that downregulation of alkyl hydroperoxide reductase (AhpC), a Gls24 family stress protein, and alcohol dehydrogenase (Adh) in the ΔbceR strain might explain the attenuated virulence and compromised stress response. Together, we showed for the first time that the bceR regulator in GBS plays an important role in bacitracin and antimicrobial peptide resistance, virulence, survival under oxidative stress, and biofilm formation.

Proteomic analysis of the rice (Oryza officinalis) provides clues on molecular tagging of proteins for brown planthopper resistance

BackgroundAmong various pests, the brown planthopper (BPH) that damages rice is the major destructive pests. Understanding resistance mechanisms is a critical step toward effective control of BPH. This study investigates the proteomics of BPH interactions with three rice cultivars: the first resistant (PR) to BPH, the second susceptible (PS), and the third hybrid (HR) between the two, in order to understand mechanisms of BPH resistance in rice.ResultsOver 4900 proteins were identified from these three rice cultivars using iTRAQ proteomics study. A total of 414, 425 and 470 differentially expressed proteins (DEPs) were detected from PR, PS and HR, respectively, after BPH infestation. Identified DEPs are mainly enriched in categories related with biosynthesis of secondary metabolites, carbon metabolism, and glyoxylate and dicarboxylate metabolism. A two-component response regulator protein (ORR22) may participate in the early signal transduction after BPH infestation. In the case of the resistant rice cultivar (PR), 6 DEPs, i.e. two lipoxygenases (LOXs), a lipase, two dirigent proteins (DIRs) and an Ent-cassa-12,15-diene synthase (OsDTC1) are related to inheritable BPH resistance. A heat shock protein (HSP20) may take part in the physiological response to BPH infestation, making it a potential target for marker-assisted selection (MAS) of rice. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed eight genes encoding various metabolic proteins involved in BPH resistance. During grain development the expressions of these genes varied at the transcriptional and translational levels.ConclusionsThis study provides comprehensive details of key proteins under compatible and incompatible interactions during BPH infestation, which will be useful for further investigation of the molecular basis of rice resistance to BPH and for breeding BPH-resistant rice cultivars.

The ArlR-MgrA regulatory cascade regulates PIA-dependent and protein-mediated biofilm formation in Rbf-dependent and Rbf-independent pathways

The two-component system response regulator ArlR and the global regulator MgrA in Staphylococcus aureus participated in numerous biological processes including biofilm formation inhibition. Previous studies have shown that these two regulators could function as a regulatory cascade. Rbf is a positive regulator of biofilm formation enhancing the production of PIA (polysaccharide intercellular adhesin). Here we have demonstrated that both ArlR and MgrA can directly bind to the promoter of rbf and repress its expression. ArlR and MgrA can also directly bind to the promoter of ica operon and enhance the expression of icaA and PIA production, revealing that the ArlR-MgrA regulatory cascade controls PIA-dependent biofilm formation. In addition, we have found that Rbf can directly bind to the aur promoter and repress the expression of aur, which encodes a protease initiating a protease cascade to inhibit protein-mediated biofilm formation. Moreover, our data indicate that the ArlR-MgrA regulatory cascade can promote the expression of aur by directly binding to its promoter and inhibit protein-mediated biofilm formation. These findings shed light on the molecular mechanisms of both PIA-dependent and protein-mediated biofilm formation modulated by the ArlR-MgrA regulatory cascade and the new role of Rbf in protein-mediated biofilm formation, and broaden our understanding of the biofilm formation regulation in S. aureus.

The spermidine acetyltransferase SpeG regulates transcription of the small RNA rprA.

Spermidine N-acetyltransferase (SpeG) acetylates and thus neutralizes toxic polyamines. Studies indicate that SpeG plays an important role in virulence and pathogenicity of many bacteria, which have evolved SpeG-dependent strategies to control polyamine concentrations and survive in their hosts. In Escherichia coli, the two-component response regulator RcsB is reported to be subject to Nε-acetylation on several lysine residues, resulting in reduced DNA binding affinity and reduced transcription of the small RNA rprA; however, the physiological acetylation mechanism responsible for this behavior has not been fully determined. Here, we performed an acetyltransferase screen and found that SpeG inhibits rprA promoter activity in an acetylation-independent manner. Surface plasmon resonance analysis revealed that SpeG can physically interact with the DNA-binding carboxyl domain of RcsB. We hypothesize that SpeG interacts with the DNA-binding domain of RcsB and that this interaction might be responsible for SpeG-dependent inhibition of RcsB-dependent rprA transcription. This work provides a model for SpeG as a modulator of E. coli transcription through its ability to interact with the transcription factor RcsB. This is the first study to provide evidence that an enzyme involved in polyamine metabolism can influence the function of the global regulator RcsB, which integrates information concerning envelope stresses and central metabolic status to regulate diverse behaviors.

Regulatory Targets of the Response Regulator RR_1586 from Clostridioides difficile Identified Using a Bacterial One-Hybrid Screen.

The Clostridioides difficile R20291 genome encodes 57 response regulator proteins that, as part of two-component signaling pathways, regulate adaptation to environmental conditions. Genomic and transcriptomic studies in C. difficile have been limited, due to technical challenges, to the analysis of either high-throughput screens or high-priority targets, such as primary regulators of toxins or spore biology. We present the use of several technically accessible and generally applicable techniques to elucidate the putative regulatory targets of a response regulator, RR_1586, involved in sporulation of the hypervirulent C. difficile strain R20291. A DNA-binding specificity motif for RR_1586 was determined using a bacterial one-hybrid assay originally developed for Drosophila transcription factors. Comparative bioinformatics approaches identified and in vitro experiments confirmed RR_1586 binding sites upstream of putative target genes, including those that encode phosphate ion transporters, spermidine/putrescine biosynthesis and transport pathways, ABC type transport systems, known regulators of sporulation, and genes encoding spore structural proteins. Representative examples of these regulatory interactions have been tested and confirmed in Escherichia coli-based reporter assays. Finally, evidence of possible regulatory mechanisms is also presented. A working model includes self-regulation by RR_1586 and phosphorylation-dependent and -independent DNA binding at low- and high-fidelity binding sites, respectively. Broad application of this and similar approaches is anticipated to be an important catalyst for the study of gene regulation by two-component systems from pathogenic or technically challenging bacteria.IMPORTANCEClostridioides difficile spores survive under harsh conditions and can germinate into actively dividing cells capable of causing disease. An understanding of the regulatory networks controlling sporulation and germination in C. difficile could be exploited for therapeutic advantage. However, such studies are hindered by the challenges of working with an anaerobic pathogen recalcitrant to genetic manipulation. Although two-component response regulators can be identified from genetic sequences, identification of their downstream regulatory networks requires further development. This work integrates experimental and bioinformatic approaches, which provide practical advantages over traditional transcriptomic analyses, to identify the putative regulon of the C. difficile response regulator RR_1586 by first screening for protein-DNA interactions in E. coli and then predicting regulatory outputs in C. difficile.

Desiccation tolerance in Acinetobacter baumannii is mediated by the two-component response regulator BfmR.

For the opportunistic pathogen Acinetobacter baumannii, desiccation tolerance is thought to contribute significantly to the persistence of these bacteria in the healthcare environment. We investigated the ability of A. baumannii to survive rapid drying, and found that some strains exhibited a profoundly desiccation-resistant phenotype, characterized by the ability of a large proportion of cells to survive on a dry surface for an extended period of time. However, this phenotype was only displayed during the stationary phase of growth. Most interestingly, we found that drying resistance could be lost after extended cultivation in liquid medium. Genome sequencing of isolates that became drying-sensitive identified mutations in bfmR, which encodes a two-component response regulator that is important for A. baumannii virulence. Additionally, BfmR was necessary for the expression of stress-related proteins during stationary phase, and one of these, KatE, was important for long-term drying survival. These results suggested that BfmR may control stress responses, and we demonstrated that the ΔbfmR mutant was more sensitive to hydrogen peroxide, nutrient starvation, and increased osmolarity. We also found that cross-protection against drying could be stimulated by either starvation, which required BfmR, or increased osmolarity. These results imply that BfmR plays a role in controlling stress responses in A. baumannii which help protect cells during desiccation, and they provide a regulatory link between this organism’s ability to persist in the environment and pathogenicity.

An in silico comparative genomic report on transcription factors in three Arabidopsis species

Transcription factors (TF) are the elements, which regulate gene expression. Regulatory function of TFs play an important role in plant biological processes and mechanisms. They may interconnect with other transcription factors or functional genes to modulate their expression in response to an internal/external factor like life cycle stage, growth, development and stress. Arabidopsis is the well-known and the most used model organism. Transcription factors of three Arabidopsis species including A. halleri, A. lyrata and A. thaliana, were compared. basic/helix-loop-helix (bHLH) with 220 TFs was the most abundant family among three Arabidopsis species while MYB and MYB related families considering as a whole group were more than bHLH with 308 TFs. No STERILE APETALA (SAP) TF homolog was found for A.halleri. The common transcription factors among three species were 4,172 grouped in 1,212 clusters. The species-specific clustered TFs were 12, 30 and 58 for A. halleri, A. lyrata and A. thaliana respectively. Eight hundred ninety two single-copy gene clusters those have one gene copy from each species, i.e. 2,676 genes, were listed. Four hundred forty five TF singletons were not clustered and are unique among three species. For clustered TF belonging to each species, GO terms and SwissProt hits showed that A. halleri has two species-specific TFs involved in heavy metal response including Zinc finger protein AZF2 and two-component response regulator ARR11 while for A. lyrata specific TFs are involved in stress response and plant development. A. thaliana specific clustered TFs work on plant flower development and acclimation.

An in silico comparative genomic report on transcription factors in three Arabidopsis species

Transcription factors (TF) are the elements, which regulate gene expression. Regulatory function of TFs play an important role in plant biological processes and mechanisms. They may interconnect with other transcription factors or functional genes to modulate their expression in response to an internal/external factor like life cycle stage, growth, development and stress. Arabidopsis is the well-known and the most used model organism. Transcription factors of three Arabidopsis species including A. halleri, A. lyrata and A. thaliana, were compared. basic/helix-loop-helix (bHLH) with 220 TFs was the most abundant family among three Arabidopsis species while MYB and MYB related families considering as a whole group were more than bHLH with 308 TFs. No STERILE APETALA (SAP) TF homolog was found for A.halleri. The common transcription factors among three species were 4,172 grouped in 1,212 clusters. The species-specific clustered TFs were 12, 30 and 58 for A. halleri, A. lyrata and A. thaliana respectively. Eight hundred ninety two single-copy gene clusters those have one gene copy from each species, i.e. 2,676 genes, were listed. Four hundred forty five TF singletons were not clustered and are unique among three species. For clustered TF belonging to each species, GO terms and SwissProt hits showed that A. halleri has two species-specific TFs involved in heavy metal response including Zinc finger protein AZF2 and two-component response regulator ARR11 while for A. lyrata specific TFs are involved in stress response and plant development. A. thaliana specific clustered TFs work on plant flower development and acclimation.