The LysR-Type Transcriptional Regulator CrgA Negatively Regulates the Flagellar Master Regulator flhDC in Ralstonia solanacearum GMI1000

ABSTRACT The invasion and colonization of host plants by the destructive pathogen Ralstonia solanacearum rely on its cell motility, which is controlled by multiple factors. Here, we report that the LysR-type transcriptional regulator CrgA (RS_RS16695) represses cell motility in R. solanacearum GMI1000. CrgA possesses common features of a LysR-type transcriptional regulator and contains an N-terminal helix-turn-helix motif as well as a C-terminal LysR substrate-binding domain. Deletion of crgA results in an enhanced swim ring and increased transcription of flhDC. In addition, the ΔcrgA mutant possesses more polar flagella than wild-type GMI1000 and exhibits higher expression of the flagellin gene fliC. Despite these alterations, the ΔcrgA mutant did not have a detectable growth defect in culture. Yeast one-hybrid and electrophoretic mobility shift assays revealed that CrgA interacts directly with the flhDC promoter. Expressing the β-glucuronidase (GUS) reporter under the control of the crgA promoter showed that crgA transcription is dependent on cell density. Soil-soaking inoculation with the crgA mutant caused wilt symptoms on tomato (Solanum lycopersicum L. cv. Hong yangli) plants earlier than inoculation with the wild-type GMI1000 but resulted in lower disease severity. We conclude that the R. solanacearum regulator CrgA represses flhDC expression and consequently affects the expression of fliC to modulate cell motility, thereby conditioning disease development in host plants. IMPORTANCE Ralstonia solanacearum is a widely distributed soilborne plant pathogen that causes bacterial wilt disease on diverse plant species. Motility is a critical virulence attribute of R. solanacearum because it allows this pathogen to efficiently invade and colonize host plants. In R. solanacearum, motility-defective strains are markedly affected in pathogenicity, which is coregulated with multiple virulence factors. In this study, we identified a new LysR-type transcriptional regulator (LTTR), CrgA, that negatively regulates motility. The mutation of the corresponding gene leads to the precocious appearance of wilt symptoms on tomato plants when the pathogen is introduced using soil-soaking inoculation. This study indicates that the regulation of R. solanacearum motility is more complex than previously thought and enhances our understanding of flagellum regulation in R. solanacearum.

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Transcriptional Activation of Multiple Operons Involved inpara-Nitrophenol Degradation by Pseudomonas sp. Strain WBC-3

Applied and Environmental Microbiology ◽

10.1128/aem.02720-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 220-230 ◽

Cited By ~ 22

Author(s):

Wen-Mao Zhang ◽

Jun-Jie Zhang ◽

Xuan Jiang ◽

Hongjun Chao ◽

Ning-Yi Zhou

Keyword(s):

Transcriptional Activation ◽

Transcriptional Regulator ◽

Pseudomonas Sp ◽

Mobility Shift ◽

Content Type ◽

High Affinity ◽

Dnase I Footprinting ◽

Nitrophenol Degradation ◽

Transcriptional Start Sites ◽

Electrophoretic Mobility Shift Assays

ABSTRACTPseudomonassp. strain WBC-3 utilizespara-nitrophenol (PNP) as a sole carbon and energy source. The genes involved in PNP degradation are organized in the following three operons:pnpA,pnpB, andpnpCDEFG. How the expression of the genes is regulated is unknown. In this study, an LysR-type transcriptional regulator (LTTR) is identified to activate the expression of the genes in response to the specific inducer PNP. While the LTTR coding genepnpRwas found to be not physically linked to any of the three catabolic operons, it was shown to be essential for the growth of strain WBC-3 on PNP. Furthermore, PnpR positively regulated its own expression, which is different from the function of classical LTTRs. A regulatory binding site (RBS) with a 17-bp imperfect palindromic sequence (GTT-N11-AAC) was identified in allpnpA,pnpB,pnpC, andpnpRpromoters. Through electrophoretic mobility shift assays and mutagenic analyses, this motif was proven to be necessary for PnpR binding. This consensus motif is centered at positions approximately −55 bp relative to the four transcriptional start sites (TSSs). RBS integrity was required for both high-affinity PnpR binding and transcriptional activation ofpnpA,pnpB, andpnpR. However, this integrity was essential only for high-affinity PnpR binding to the promoter ofpnpCDEFGand not for its activation. Intriguingly, unlike other LTTRs studied, no changes in lengths of the PnpR binding regions of thepnpAandpnpBpromoters were observed after the addition of the inducer PNP in DNase I footprinting.

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Diverse Genetic Regulon of the Virulence-Associated Transcriptional Regulator MucR in Brucella abortus 2308

Infection and Immunity ◽

10.1128/iai.01097-12 ◽

2013 ◽

Vol 81 (4) ◽

pp. 1040-1051 ◽

Cited By ~ 23

Author(s):

Clayton C. Caswell ◽

Ahmed E. M. Elhassanny ◽

Emilie E. Planchin ◽

Christelle M. Roux ◽

Jenni N. Weeks-Gorospe ◽

...

Keyword(s):

Brucella Abortus ◽

Iron Homeostasis ◽

Brucella Melitensis ◽

Cell Envelope ◽

Transcriptional Regulator ◽

Growth Defect ◽

Mobility Shift ◽

Content Type ◽

Polysaccharide Biosynthesis

ABSTRACTThe Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence inBrucella. Here, we show that aBrucella abortusin-framemucRdeletion strain exhibits a pronounced growth defect duringin vitrocultivation and, more importantly, that themucRmutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation ofBrucella mucRmutants has not been defined previously, but in the present study the genes regulated by MucR inB. abortushave been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). InB. abortus2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified isarsR6(nolR), which encodes a transcriptional regulator previously linked to virulence inBrucella melitensis16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (includingarsR6[nolR]), and inBrucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR inBrucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect ofBrucella mucRmutants.

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A Novel CO-Responsive Transcriptional Regulator and Enhanced H2Production by an Engineered Thermococcus onnurineus NA1 Strain

Applied and Environmental Microbiology ◽

10.1128/aem.03019-14 ◽

2014 ◽

Vol 81 (5) ◽

pp. 1708-1714 ◽

Cited By ~ 25

Author(s):

Min-Sik Kim ◽

Ae Ran Choi ◽

Seong Hyuk Lee ◽

Hae-Chang Jung ◽

Seung Seob Bae ◽

...

Keyword(s):

Production Rate ◽

Gene Cluster ◽

Type Strain ◽

Wild Type Strain ◽

Regulatory System ◽

Transcriptional Regulator ◽

Bioreactor Culture ◽

Wild Type ◽

Content Type ◽

Transcriptional Regulatory

ABSTRACTGenome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism inThermococcus onnurineusNA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes ofThermococcusspecies and “CandidatusKorarchaeum cryptofilum” OPF8. In-frame deletion of eithercorQorcorRcaused a severe impairment in CO-dependent growth and H2production. WhencorQandcorRdeletion mutants were complemented by introducing thecorQRgenes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integratedcorQR(ΔCorR/corQR↑) compared with those in the wild-type strain. In addition, the ΔCorR/corQR↑strain exhibited a much higher H2production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2production rate (191.9 mmol liter−1h−1) and the specific H2production rate (249.6 mmol g−1h−1) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that thecorQRgenes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2production.

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Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti

Microbiology ◽

10.1099/mic.0.000909 ◽

2020 ◽

Vol 166 (5) ◽

pp. 484-497 ◽

Cited By ~ 1

Author(s):

Alejandra Arteaga Ide ◽

Victor M. Hernández ◽

Liliana Medina-Aparicio ◽

Edson Carcamo-Noriega ◽

Lourdes Girard ◽

...

Keyword(s):

Type Species ◽

Sinorhizobium Meliloti ◽

Arginase Activity ◽

Wild Type ◽

Mobility Shift ◽

Content Type ◽

Link Type ◽

Arginine Catabolism

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti , that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

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Presence of atrs-Like Motif Promotes Rep-Mediated Wild-Type Adeno-Associated Virus Type 2 Integration

Journal of Virology ◽

10.1128/jvi.00426-15 ◽

2015 ◽

Vol 89 (14) ◽

pp. 7428-7432 ◽

Cited By ~ 9

Author(s):

Karl Petri ◽

Richard Gabriel ◽

Leticia Agundez ◽

Raffaele Fronza ◽

Saira Afzal ◽

...

Keyword(s):

Virus Type ◽

Integration Site ◽

Dermal Fibroblasts ◽

Wild Type ◽

Mobility Shift ◽

Adeno Associated Virus ◽

Rep Protein ◽

Electrophoretic Mobility Shift Assays ◽

First Time

High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.

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The Novel Streptococcal Transcriptional Regulator XtgS Negatively Regulates Bacterial Virulence and Directly Represses PseP Transcription

Infection and Immunity ◽

10.1128/iai.00035-20 ◽

2020 ◽

Vol 88 (10) ◽

Author(s):

Guangjin Liu ◽

Tingting Gao ◽

Xiaojun Zhong ◽

Jiale Ma ◽

Yumin Zhang ◽

...

Keyword(s):

Strong Association ◽

Challenge Test ◽

Transcriptional Regulator ◽

Transcriptional Regulators ◽

Negative Regulator ◽

Broad Host Range ◽

Bacterial Survival ◽

Mobility Shift ◽

Upstream Gene ◽

Content Type

ABSTRACT Streptococcus agalactiae (group B streptococcus [GBS]) has received continuous attention for its involvement in invasive infections and its broad host range. Transcriptional regulators have an important impact on bacterial adaptation to various environments. Research on transcriptional regulators will shed new light on GBS pathogenesis. In this study, we identified a novel XRE-family transcriptional regulator encoded on the GBS genome, designated XtgS. Our data demonstrate that XtgS inactivation significantly increases bacterial survival in host blood and animal challenge test, suggesting that it is a negative regulator of GBS pathogenicity. Further transcriptomic analysis and quantitative reverse transcription-PCR (qRT-PCR) mainly indicated that XtgS significantly repressed transcription of its upstream gene pseP. Based on electrophoretic mobility shift and lacZ fusion assays, we found that an XtgS homodimer directly binds a palindromic sequence in the pseP promoter region. Meanwhile, the PseP and XtgS combination naturally coexists in diverse Streptococcus genomes and has a strong association with sequence type, serotype diversification and host adaptation of GBS. Therefore, this study reveals that XtgS functions as a transcriptional regulator that negatively affects GBS virulence and directly represses PseP expression, and it provides new insights into the relationships between transcriptional regulator and genome evolution.

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NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

Journal of Bacteriology ◽

10.1128/jb.00349-15 ◽

2015 ◽

Vol 197 (20) ◽

pp. 3339-3353 ◽

Cited By ~ 14

Author(s):

Jihong Li ◽

John C. Freedman ◽

Bruce A. McClane

Keyword(s):

Sialic Acid ◽

Clostridium Perfringens ◽

Host Cells ◽

Start Codon ◽

Null Mutant ◽

Wild Type ◽

Mobility Shift ◽

Content Type ◽

Type D ◽

Epsilon Toxin

ABSTRACTClostridium perfringenstype D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011,http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, ananInull mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due tonanIgene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions incodYandccpAgene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A doublecodYccpAnull mutant produced even less ETX than acodYorccpAsingle null mutant. CcpA bound directly to sequences upstream of theetxorcodYstart codon, and bioinformatics identified putative CcpA-bindingcresites immediately upstream of both thecodYandetxstart codons, suggesting possible direct CcpA regulatory effects. AccpAmutation also decreasedcodYtranscription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects oncodYtranscription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease.IMPORTANCEClostridium perfringensNanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators. Results obtained using singleccpAorcodYnull mutants and accpAcodYdouble null mutant showed thatcodYandccpAregulate ETX production independently of one another but thatccpAalso affectscodYtranscription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulateetxtranscription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producingC. perfringensstrains, via CcpA and CodY, to upregulate ETX production and cause disease.

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SREBP-Dependent Triazole Susceptibility in Aspergillus fumigatus Is Mediated through Direct Transcriptional Regulation oferg11A(cyp51A)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05027-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 248-257 ◽

Cited By ~ 41

Author(s):

Sara J. Blosser ◽

Robert A. Cramer

Keyword(s):

Aspergillus Fumigatus ◽

Regulatory Element ◽

Critical Role ◽

Clinical Importance ◽

Growth Defect ◽

Ergosterol Biosynthesis ◽

Wild Type ◽

Transcript Levels ◽

Content Type ◽

Conditional Expression

ABSTRACTAs triazole antifungal drug resistance during invasiveAspergillus fumigatusinfection has become more prevalent, the need to understand mechanisms of resistance inA. fumigatushas increased. The presence of twoerg11(cyp51) genes inAspergillusspp. is hypothesized to account for the inherent resistance of this mold to the triazole fluconazole (FLC). Recently, anA. fumigatusnull mutant of a transcriptional regulator in the sterol regulatory element binding protein (SREBP) family, the ΔsrbAstrain, was found to have increased susceptibility to FLC and voriconazole (VCZ). In this study, we examined the mechanism engendering the observed increase inA. fumigatustriazole susceptibility in the absence of SrbA. We observed a significant reduction in theerg11Atranscript in the ΔsrbAstrain in response to FLC and VCZ. Transcript levels oferg11Bwere also reduced but not to the extent oferg11A. Interestingly,erg11Atranscript levels increased upon extended VCZ, but not FLC, exposure. Construction of anerg11Aconditional expression strain in the ΔsrbAstrain was able to restoreerg11Atranscript levels and, consequently, wild-type MICs to the triazole FLC. The VCZ MIC was also partially restored upon increasederg11Atranscript levels; however, total ergosterol levels remained significantly reduced compared to those of the wild type. Induction of theerg11Aconditional strain did not restore the hypoxia growth defect of the ΔsrbAstrain. Taken together, our results demonstrate a critical role for SrbA-mediated regulation of ergosterol biosynthesis and triazole drug interactions inA. fumigatusthat may have clinical importance.

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The Streptococcus iniae Transcriptional Regulator CpsY Is Required for Protection from Neutrophil-Mediated Killing and Proper GrowthInVitro

Infection and Immunity ◽

10.1128/iai.05567-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4638-4648 ◽

Cited By ~ 16

Author(s):

Jonathan P. Allen ◽

Melody N. Neely

Keyword(s):

Whole Blood ◽

Local Environment ◽

Growth Defect ◽

Infection Model ◽

Streptococcus Iniae ◽

Virulence Determinants ◽

Wild Type ◽

Content Type ◽

Proteose Peptone ◽

Lysr Family

ABSTRACTThe ability of a pathogen to metabolically adapt to the local environment for optimal expression of virulence determinants is a continued area of research. Orthologs of theStreptococcus iniaeLysR family regulator CpsY have been shown to regulate methionine biosynthesis and uptake pathways but appear to influence expression of several virulence genes as well. AnS. iniaemutant with an in-frame deletion ofcpsY(ΔcpsYmutant) is highly attenuated in a zebrafish infection model. The ΔcpsYmutant displays a methionine-independent growth defect in serum, which differs from the methionine-dependent defect observed for orthologous mutants ofStreptococcus mutansandStreptococcus agalactiae. On the contrary, the ΔcpsYmutant can grow in excess of the wild type (WT) when supplemented with proteose peptone, suggesting an inability to properly regulate growth. CpsY is critical for protection ofS. iniaefrom clearance by neutrophils in whole blood but is dispensable for intracellular survival in macrophages. Susceptibility of the ΔcpsYmutant to killing in whole blood is not due to a growth defect, because inhibition of neutrophil phagocytosis rescues the mutant to WT levels. Thus, CpsY appears to have a pleiotropic regulatory role forS. iniae, integrating metabolism and virulence. Furthermore,S. iniaeprovides a unique model to investigate the paradigm of CpsY-dependent regulation during systemic streptococcal infection.

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Ca14-3-3 Interacts With CaWRKY58 to Positively Modulate Pepper Response to Low-Phosphorus Starvation

Frontiers in Plant Science ◽

10.3389/fpls.2020.607878 ◽

2021 ◽

Vol 11 ◽

Author(s):

Jinsen Cai ◽

Weiwei Cai ◽

Xueying Huang ◽

Sheng Yang ◽

Jiayu Wen ◽

...

Keyword(s):

Ralstonia Solanacearum ◽

Phosphorus Deficiency ◽

Negative Regulator ◽

Bimolecular Fluorescence Complementation ◽

Virus Induced Gene Silencing ◽

Mobility Shift ◽

Natural Habitats ◽

Low Phosphorus ◽

Low Phosphorus Stress ◽

Electrophoretic Mobility Shift Assays

Low-phosphorus stress (LPS) and pathogen attack are two important stresses frequently experienced by plants in their natural habitats, but how plant respond to them coordinately remains under-investigated. Here, we demonstrate that CaWRKY58, a known negative regulator of the pepper (Capsicum annuum) response to attack by Ralstonia solanacearum, is upregulated by LPS. Virus-induced gene silencing (VIGS) and overexpression of CaWRKY58 in Nicotiana benthamiana plants in combination with chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) demonstrated that CaWRKY58 positively regulates the response of pepper to LPS by directly targeting and regulating genes related to phosphorus-deficiency tolerance, including PHOSPHATE STARVATION RESPONSE1 (PHR1). Yeast two-hybrid assays revealed that CaWRKY58 interacts with a 14-3-3 protein (Ca14-3-3); this interaction was confirmed by pull-down, bimolecular fluorescence complementation (BiFC), and microscale thermophoresis (MST) assays. The interaction between Ca14-3-3 and CaWRKY58 enhanced the activation of PHR1 expression by CaWRKY58, but did not affect the expression of the immunity-related genes CaNPR1 and CaDEF1, which are negatively regulated by CaWRKY58 in pepper upon Ralstonia solanacearum inoculation. Collectively, our data indicate that CaWRKY58 negatively regulates immunity against Ralstonia solanacearum, but positively regulates tolerance to LPS and that Ca14-3-3 transcriptionally activates CaWRKY58 in response to LPS.

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