scholarly journals GouR, a TetR Family Transcriptional Regulator, Coordinates the Biosynthesis and Export of Gougerotin in Streptomyces graminearus

2013 ◽  
Vol 80 (2) ◽  
pp. 714-722 ◽  
Author(s):  
Junhong Wei ◽  
Yuqing Tian ◽  
Guoqing Niu ◽  
Huarong Tan
Keyword(s):  
Biological Activities ◽  
Regulatory Protein ◽  
Specific Inhibitor ◽  
Biosynthetic Gene Cluster ◽  
Binding Activity ◽  
Major Facilitator Superfamily ◽  
Transcriptional Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Inhibitor Of Protein Synthesis

ABSTRACTGougerotin is a peptidyl nucleoside antibiotic. It functions as a specific inhibitor of protein synthesis by binding ribosomal peptidyl transferase and exhibits a broad spectrum of biological activities.gouR, situated in the gougerotin biosynthetic gene cluster, encodes a TetR family transcriptional regulatory protein. Gene disruption and genetic complementation revealed thatgouRplays an important role in the biosynthesis of gougerotin. Transcriptional analysis suggested that GouR represses the transcription of thegouL-to-gouBoperon consisting of 11 structural genes and activates the transcription of the major facilitator superfamily (MFS) transporter gene (gouM). Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting experiments showed that GouR has specific DNA-binding activity for the promoter regions ofgouL,gouM, andgouR. Our data suggested that GouR modulates gougerotin production by coordinating its biosynthesis and export inStreptomyces graminearus.

scholarly journals Involvement of PatE, a Prophage-Encoded AraC-Like Regulator, in the Transcriptional Activation of Acid Resistance Pathways of Enterohemorrhagic Escherichia coli Strain EDL933

2012 ◽  
Vol 78 (15) ◽  
pp. 5083-5092 ◽  
Author(s):  
Jennifer K. Bender ◽  
Judyta Praszkier ◽  
Matthew J. Wakefield ◽  
Kathryn Holt ◽  
Marija Tauschek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Activation ◽  
Regulatory Protein ◽  
Acid Resistance ◽  
Enterohemorrhagic Escherichia Coli ◽  
Transcriptional Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Infectious Dose ◽  
Genes Encoding

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) O157:H7 is a lethal human intestinal pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. EHEC is transmitted by the fecal-oral route and has a lower infectious dose than most other enteric bacterial pathogens in that fewer than 100 CFU are able to cause disease. This low infectious dose has been attributed to the ability of EHEC to survive in the acidic environment of the human stomach.In silicoanalysis of the genome of EHEC O157:H7 strain EDL933 revealed a gene,patE, for a putative AraC-like regulatory protein within the prophage island, CP-933H. Transcriptional analysis inE. colishowed that the expression ofpatEis induced during stationary phase. Data from microarray assays demonstrated that PatE activates the transcription of genes encoding proteins of acid resistance pathways. In addition, PatE downregulated the expression of a number of genes encoding heat shock proteins and the type III secretion pathway of EDL933. Transcriptional analysis and electrophoretic mobility shift assays suggested that PatE also activates the transcription of the gene for the acid stress chaperonehdeAby binding to its promoter region. Finally, assays of acid tolerance showed that increasing the expression of PatE in EHEC greatly enhanced the ability of the bacteria to survive in different acidic environments. Together, these findings indicate that EHEC strain EDL933 carries a prophage-encoded regulatory system that contributes to acid resistance.

scholarly journals PapR6, a Putative Atypical Response Regulator, Functions as a Pathway-Specific Activator of Pristinamycin II Biosynthesis in Streptomyces pristinaespiralis

2014 ◽  
Vol 197 (3) ◽  
pp. 441-450 ◽  
Author(s):  
Junling Dun ◽  
Yawei Zhao ◽  
Guosong Zheng ◽  
Hong Zhu ◽  
Lijun Ruan ◽  
...  
Keyword(s):  
Response Regulator ◽  
Biosynthetic Gene Cluster ◽  
Transcriptional Analysis ◽  
Upstream Region ◽  
Binding Motif ◽  
Regulation Mechanism ◽  
Mobility Shift ◽  
Content Type ◽  
Streptomyces Pristinaespiralis ◽  
Specific Regulation

There are up to seven regulatory genes in the pristinamycin biosynthetic gene cluster ofStreptomyces pristinaespiralis, which infers a complicated regulation mechanism for pristinamycin production. In this study, we revealed that PapR6, a putative atypical response regulator, acts as a pathway-specific activator of pristinamycin II (PII) biosynthesis. Deletion of thepapR6gene resulted in significantly reduced PII production, and its overexpression led to increased PII formation, compared to that of the parental strain HCCB 10218. However, eitherpapR6deletion or overexpression had very little effect on pristinamycin I (PI) biosynthesis. Electrophoretic mobility shift assays (EMSAs) demonstrated that PapR6 bound specifically to the upstream region ofsnaF, the first gene of thesnaFE1E2GHIJKoperon, which is likely responsible for providing the precursor isobutyryl-coenzyme A (isobutyryl-CoA) and the intermediate C11αβ-unsaturated thioester for PII biosynthesis. A signature PapR6-binding motif comprising two 4-nucleotide (nt) inverted repeat sequences (5′-GAGG-4 nt-CCTC-3′) was identified. Transcriptional analysis showed that inactivation of thepapR6gene led to markedly decreased expression ofsnaFE1E2GHIJK. Furthermore, we found that a mutant (snaFmu) with base substitutions in the identified PapR6-binding sequence in the genome exhibited the same phenotype as that of the ΔpapR6strain. Therefore, it may be concluded that pathway-specific regulation of PapR6 in PII biosynthesis is possibly exerted via controlling the provision of isobutyryl-CoA as well as the intermediate C11αβ-unsaturated thioester.

An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element

Blood ◽  
2001 ◽  
Vol 98 (8) ◽  
pp. 2555-2562 ◽  
Author(s):  
Mark Loyevsky ◽  
Timothy LaVaute ◽  
Charles R. Allerson ◽  
Robert Stearman ◽  
Olakunle O. Kassim ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Regulatory Protein ◽  
Protein S ◽  
Fold Increase ◽  
Binding Activity ◽  
Mobility Shift ◽  
Iron Responsive Element ◽  
Element Binding Protein ◽  
Responsive Element ◽  
Electrophoretic Mobility Shift Assays

Abstract This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)–like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex fromP falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.

scholarly journals WblAch, a Pivotal Activator of Natamycin Biosynthesis and Morphological Differentiation in Streptomyces chattanoogensis L10, Is Positively Regulated by AdpAch

2014 ◽  
Vol 80 (22) ◽  
pp. 6879-6887 ◽  
Author(s):  
Pin Yu ◽  
Shui-Ping Liu ◽  
Qing-Ting Bu ◽  
Zhen-Xing Zhou ◽  
Zhen-Hong Zhu ◽  
...  
Keyword(s):  
Morphological Differentiation ◽  
Binding Activity ◽  
Transcriptional Analysis ◽  
Antibiotic Biosynthesis ◽  
Content Type ◽  
Real Time Quantitative Pcr ◽  
Dna Binding Activity ◽  
In The Wild ◽  
Natamycin Production ◽  
Streptomyces Chattanoogensis

ABSTRACTDetailed mechanisms ofWhiB-like (Wbl) proteins involved in antibiotic biosynthesis and morphological differentiation are poorly understood. Here, we characterize the role of WblAch, aStreptomyces chattanoogensisL10 protein belonging to this superfamily. Based on DNA microarray data and verified by real-time quantitative PCR (qRT-PCR), the expression ofwblAchwas shown to be positively regulated by AdpAch. Gel retardation assays and DNase I footprinting experiments showed that AdpAchhas specific DNA-binding activity for the promoter region ofwblAch. Gene disruption and genetic complementation revealed that WblAchacts in a positive manner to regulate natamycin production. WhenwblAchwas overexpressed in the wild-type strain, the natamycin yield was increased by ∼30%. This provides a strategy to generate improved strains for natamycin production. Moreover, transcriptional analysis showed that the expression levels ofwhigenes (includingwhiA,whiB,whiH, andwhiI) were severely depressed in the ΔwblAchmutant, suggesting that WblAchplays a part in morphological differentiation by influencing the expression of thewhigenes.

scholarly journals Implication of Antigenic Conversion of Helicobacter pylori Lipopolysaccharides That Involve Interaction with Surfactant Protein D

2012 ◽  
Vol 80 (8) ◽  
pp. 2956-2962 ◽  
Author(s):  
Shin-ichi Yokota ◽  
Ken-ichi Amano ◽  
Chiaki Nishitani ◽  
Shigeru Ariki ◽  
Yoshio Kuroki ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Biological Activities ◽  
Surfactant Protein ◽  
Binding Activity ◽  
Surfactant Protein D ◽  
Dependent Manner ◽  
Antigenic Epitope ◽  
Binding Assays ◽  
Content Type ◽  
Gastric Cancer Patients

ABSTRACTWe propose two antigenic types ofHelicobacter pylorilipopolysaccharides (LPS): highly antigenic epitope-carrying LPS (HA-LPS) and weakly antigenic epitope-carrying LPS (WA-LPS) based on human serum reactivity. Strains carrying WA-LPS are highly prevalent in isolates from gastric cancer patients. WA-LPS exhibits more potent biological activities compared to HA-LPS, namely, upregulation of Toll-like receptor 4 (TLR4) expression and induction of enhanced epithelial cell proliferation. The results of competitive binding assays using monosaccharides and methylglycosides, as well as binding assays using glycosidase-treated LPS, suggested that β-linkedN-acetyl-d-glucosamine and β-linkedd-galactose residues largely contributed to the highly antigenic epitope and the weakly antigenic epitope, respectively. WA-LPS exhibited greater binding activity to surfactant protein D (SP-D) in a Ca2+-dependent manner, and this interaction was inhibited by methyl-β-d-galactoside. The biological activities of WA-LPS were markedly enhanced by the addition of SP-D. Lines of evidence suggested that removal of β-N-acetyl-d-glucosamine residue, which comprises the highly antigenic epitope, results in exposure of the weakly antigenic epitope. The weakly antigenic epitope interacted preferentially with SP-D, and SP-D enhanced the biological activity of WA-LPS.

scholarly journals Regulation of the Cobalt/Nickel Efflux OperondmeRFin Agrobacterium tumefaciens and a Link between the Iron-Sensing Regulator RirA and Cobalt/Nickel Resistance

2016 ◽  
Vol 82 (15) ◽  
pp. 4732-4742 ◽  
Author(s):  
Thanittra Dokpikul ◽  
Paweena Chaoprasid ◽  
Kritsakorn Saninjuk ◽  
Sirin Sirirakphaisarn ◽  
Jaruwan Johnrod ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Iron Homeostasis ◽  
Regulatory Protein ◽  
Nickel Resistance ◽  
Mobility Shift ◽  
Potent Inducer ◽  
Content Type ◽  
Cobalt Nickel ◽  
Metal Efflux ◽  
Cobalt And Nickel

ABSTRACTTheAgrobacterium tumefaciensC58 genome harbors an operon containing thedmeR(Atu0890) anddmeF(Atu0891) genes, which encode a transcriptional regulatory protein belonging to the RcnR/CsoR family and a metal efflux protein belonging to the cation diffusion facilitator (CDF) family, respectively. ThedmeRFoperon is specifically induced by cobalt and nickel, with cobalt being the more potent inducer. Promoter-lacZtranscriptional fusion, an electrophoretic mobility shift assay, and DNase I footprinting assays revealed that DmeR repressesdmeRFtranscription through direct binding to the promoter region upstream ofdmeR. A strain lackingdmeFshowed increased accumulation of intracellular cobalt and nickel and exhibited hypersensitivity to these metals; however, this strain displayed full virulence, comparable to that of the wild-type strain, when infecting aNicotiana benthamianaplant model under the tested conditions. Cobalt, but not nickel, increased the expression of many iron-responsive genes and reduced the induction of the SoxR-regulated genesodBII. Furthermore, control of iron homeostasis via RirA is important for the ability ofA. tumefaciensto cope with cobalt and nickel toxicity.IMPORTANCEThe molecular mechanism of the regulation ofdmeRFtranscription by DmeR was demonstrated. This work provides evidence of a direct interaction of apo-DmeR with the corresponding DNA operator siteinvitro. The recognition site for apo-DmeR consists of 10-bp AT-rich inverted repeats separated by six C bases (5′-ATATAGTATACCCCCCTATAGTATAT-3′). Cobalt and nickel cause DmeR to dissociate from thedmeRFpromoter, which leads to expression of the metal efflux genedmeF. This work also revealed a connection between iron homeostasis and cobalt/nickel resistance inA. tumefaciens.

scholarly journals Two Master Switch Regulators Trigger A40926 Biosynthesis in Nonomuraea sp. Strain ATCC 39727

2015 ◽  
Vol 197 (15) ◽  
pp. 2536-2544 ◽  
Author(s):  
Letizia Lo Grasso ◽  
Sonia Maffioli ◽  
Margherita Sosio ◽  
Mervyn Bibb ◽  
Anna Maria Puglia ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Antibiotic Production ◽  
Regulatory Protein ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Regulatory Mechanisms ◽  
Antibiotic Biosynthesis ◽  
Strain Atcc ◽  
Protein Coding ◽  
Content Type

ABSTRACTThe actinomyceteNonomuraeasp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by thedbvgene cluster, which contains 37 protein-coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926-biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and liquid chromatography-mass spectrometry (LC-MS) analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation ofdbv6had no effect. In addition, overexpression ofdbv3led to higher levels of A40926 production. Transcriptional and quantitative reverse transcription (RT)-PCR analyses showed that Dbv4 is essential for the transcription of two operons,dbv14-dbv8anddbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4,dbv29,dbv36, anddbv37) and of six operons (dbv2-dbv1,dbv14-dbv8,dbv17-dbv15,dbv21-dbv20,dbv24-dbv28, anddbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription ofdbv4and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export, and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 directly regulates biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions, including the four cross-links, halogenation, glycosylation, and acylation.IMPORTANCEThis report expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomyceteNonomuraeasp. strain ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of skin infections by Gram-positive bacteria. Therefore, understanding the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control two other similar glycopeptides (balhimycin and teicoplanin) have been elucidated, and beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.

scholarly journals Hierarchical control of virginiamycin production in Streptomyces virginiae by three pathway-specific regulators: VmsS, VmsT and VmsR

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1250-1259 ◽  
Author(s):  
Nattika Pulsawat ◽  
Shigeru Kitani ◽  
Eriko Fukushima ◽  
Takuya Nihira
Keyword(s):  
Gene Cluster ◽  
Response Regulator ◽  
Expression Profiles ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Biosynthetic Gene Cluster ◽  
Transcriptional Analysis ◽  
Biosynthetic Gene ◽  
Biosynthetic Genes ◽  
Streptomyces Virginiae

Two regulatory genes encoding a Streptomyces antibiotic regulatory protein (vmsS) and a response regulator (vmsT) of a bacterial two-component signal transduction system are present in the left-hand region of the biosynthetic gene cluster of the antibiotic virginiamycin, which is composed of virginiamycin M (VM) and virginiamycin S (VS), in Streptomyces virginiae. Disruption of vmsS abolished both VM and VS biosynthesis, with drastic alteration of the transcriptional profile for virginiamycin biosynthetic genes, whereas disruption of vmsT resulted in only a loss of VM biosynthesis, suggesting that vmsS is a pathway-specific regulator for both VM and VS biosynthesis, and that vmsT is a pathway-specific regulator for VM biosynthesis alone. Gene expression profiles determined by semiquantitative RT-PCR on the virginiamycin biosynthetic gene cluster demonstrated that vmsS controls the biosynthetic genes for VM and VS, and vmsT controls unidentified gene(s) of VM biosynthesis located outside the biosynthetic gene cluster. In addition, transcriptional analysis of a deletion mutant of vmsR located in the clustered regulatory region in the virginiamycin cluster (and which also acts as a SARP-family activator for both VM and VS biosynthesis) indicated that the expression of vmsS and vmsT is under the control of vmsR, and vmsR also contributes to the expression of VM and VS biosynthetic genes, independent of vmsS and vmsT. Therefore, coordinated virginiamycin biosynthesis is controlled by three pathway-specific regulators which hierarchically control the expression of the biosynthetic gene cluster.

scholarly journals Negative Regulation of Ectoine Uptake and Catabolism in Sinorhizobium meliloti: Characterization of the EhuR Gene

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Qinli Yu ◽  
Hanlin Cai ◽  
Yanfeng Zhang ◽  
Yongzhi He ◽  
Lincai Chen ◽  
...  
Keyword(s):  
Dna Binding ◽  
Sinorhizobium Meliloti ◽  
Binding Activity ◽  
Mobility Shift ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Dna Binding Activity ◽  
Dnase I Footprinting ◽  
Gntr Family

ABSTRACT Ectoine has osmoprotective effects on Sinorhizobium meliloti that differ from its effects in other bacteria. Ectoine does not accumulate in S. meliloti cells; instead, it is degraded. The products of the ehuABCD-eutABCDE operon were previously discovered to be responsible for the uptake and catabolism of ectoine in S. meliloti. However, the mechanism by which ectoine is involved in the regulation of the ehuABCD-eutABCDE operon remains unclear. The ehuR gene, which is upstream of and oriented in the same direction as the ehuABCD-eutABCDE operon, encodes a member of the MocR/GntR family of transcriptional regulators. Quantitative reverse transcription-PCR and promoter-lacZ reporter fusion experiments revealed that EhuR represses transcription of the ehuABCD-eutABCDE operon, but this repression is inhibited in the presence of ectoine. Electrophoretic mobility shift assays and DNase I footprinting assays revealed that EhuR bound specifically to the DNA regions overlapping the −35 region of the ehuA promoter and the +1 region of the ehuR promoter. Surface plasmon resonance assays further demonstrated direct interactions between EhuR and the two promoters, although EhuR was found to have higher affinity for the ehuA promoter than for the ehuR promoter. In vitro, DNA binding by EhuR could be directly inhibited by a degradation product of ectoine. Our work demonstrates that EhuR is an important negative transcriptional regulator involved in the regulation of ectoine uptake and catabolism and is likely regulated by one or more end products of ectoine catabolism. IMPORTANCE Sinorhizobium meliloti is an important soil bacterium that displays symbiotic interactions with legume hosts. Ectoine serves as a key osmoprotectant for S. meliloti. However, ectoine does not accumulate in the cells; rather, it is degraded. In this study, we characterized the transcriptional regulation of the operon responsible for ectoine uptake and catabolism in S. meliloti. We identified and characterized the transcription repressor EhuR, which is the first MocR/GntR family member found to be involved in the regulation of compatible solute uptake and catabolism. More importantly, we demonstrated for the first time that an ectoine catabolic end product could modulate EhuR DNA-binding activity. Therefore, this work provides new insights into the unique mechanism of ectoine-induced osmoprotection in S. meliloti.

scholarly journals Transcriptome-Based Analysis of the Pantoea stewartii Quorum-Sensing Regulon and Identification of EsaR Direct Targets

2014 ◽  
Vol 80 (18) ◽  
pp. 5790-5800 ◽  
Author(s):  
Revathy Ramachandran ◽  
Alison Kernell Burke ◽  
Guy Cormier ◽  
Roderick V. Jensen ◽  
Ann M. Stevens
Keyword(s):  
Quorum Sensing ◽  
Cell Envelope ◽  
Regulatory Protein ◽  
Homoserine Lactone ◽  
Sequencing Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Proteomic Approach ◽  
Stewart's Wilt ◽  
Cell Densities

ABSTRACTPantoeastewartiisubsp.stewartiiis a proteobacterium that causes Stewart's wilt disease in corn plants. The bacteria form a biofilm in the xylem of infected plants and produce capsule that blocks water transport, eventually causing wilt. At low cell densities, the quorum-sensing (QS) regulatory protein EsaR is known to directly repress expression ofesaRitself as well as the genes for the capsular synthesis operon transcription regulator,rcsA, and a 2,5-diketogluconate reductase,dkgA. It simultaneously directly activates expression of genes for a putative small RNA,esaS, the glycerol utilization operon,glpFKX, and another transcriptional regulator,lrhA. At high bacterial cell densities, all of this regulation is relieved when EsaR binds an acylated homoserine lactone signal, which is synthesized constitutively over growth. QS-dependent gene expression is critical for the establishment of disease in the plant. However, the identity of the full set of genes controlled by EsaR/QS is unknown. A proteomic approach previously identified around 30 proteins in the QS regulon. In this study, a whole-transcriptome, next-generation sequencing analysis of rRNA-depleted RNA from QS-proficient and -deficientP. stewartiistrains was performed to identify additional targets of EsaR. EsaR-dependent transcriptional regulation of a subset of differentially expressed genes was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Electrophoretic mobility shift assays demonstrated that EsaR directly bound 10 newly identified target promoters. Overall, the QS regulon ofP. stewartiiorchestrates three major physiological responses: capsule and cell envelope biosynthesis, surface motility and adhesion, and stress response.

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