Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding byStreptomycesAntibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators inStreptomyces natalensis

ABSTRACTThe biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. AStreptomycesantibiotic regulatory protein–large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene,pimM. A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimRSARPappears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding.IMPORTANCEHere, we have shown that a modulation of the production of the antifungal pimaricin inStreptomyces natalensiscan be accomplished via promoter engineering of the PAS-LuxR transcriptional activatorpimM. The expression of this gene is controlled by theStreptomycesantibiotic regulatory protein–large ATP-binding regulator of the LuxR family (SARP-LAL) regulator PimR, which binds a series of heptameric direct repeats in its promoter region. The structure and importance of such repeats in protein binding, transcriptional activation, and polyene production have been investigated. These findings should provide important clues to understand the regulatory machinery that modulates antibiotic biosynthesis inStreptomycesand open new possibilities for the manipulation of metabolite production. The presence of PimR orthologues encoded by gene clusters for different secondary metabolites and the conservation of their operators suggest that the improvements observed in the activation of pimaricin biosynthesis byStreptomyces natalensiscould be extrapolated to the production of different compounds by other species.

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RbsR Activates Capsule but Represses therbsUDKOperon in Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00640-15 ◽

2015 ◽

Vol 197 (23) ◽

pp. 3666-3675 ◽

Cited By ~ 13

Author(s):

Mei G. Lei ◽

Chia Y. Lee

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factor ◽

Transcriptional Activation ◽

Promoter Region ◽

Mobility Shift ◽

Content Type ◽

Virulence Regulation ◽

Important Virulence Factor ◽

Dna Fragment ◽

Mobility Shift Assay

ABSTRACTStaphylococcus aureuscapsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of thecapoperon. A 10-bp inverted repeat (IR) located 13 bp upstream of the −35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of thecappromoter. To search for potential proteins which directly interact with thecappromoter region (Pcap), we directly analyzed the proteins interacting with the PcapDNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulatecapgene expression by specifically binding to thecappromoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed thatrbsRwas directly controlled by SigB and that RbsR was a repressor of therbsUDKoperon, involved in ribose uptake and phosphorylation. The repression ofrbsUDKby RbsR could be derepressed byd-ribose. However,d-ribose did not affect RbsR activation of capsule.IMPORTANCEStaphylococcus aureusis an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression ofrbsR. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits therbsoperon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence inS. aureus. Thus, this study further advances our understanding of staphylococcal virulence regulation.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00617-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5083-5092 ◽

Cited By ~ 7

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.

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

Journal of Bacteriology ◽

10.1128/jb.00262-15 ◽

2015 ◽

Vol 197 (15) ◽

pp. 2536-2544 ◽

Cited By ~ 24

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.

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Discovery of 16-Demethylrifamycins by Removing the Predominant Polyketide Biosynthesis Pathway in Micromonospora sp. Strain TP-A0468

Applied and Environmental Microbiology ◽

10.1128/aem.02597-18 ◽

2018 ◽

Vol 85 (4) ◽

Cited By ~ 1

Author(s):

Qiang Zhou ◽

Guang-Cai Luo ◽

Huizhan Zhang ◽

Gong-Li Tang

Keyword(s):

Natural Products ◽

Transcriptional Activation ◽

Metabolic Flux ◽

Genome Mining ◽

Gene Clusters ◽

Bioinformatic Analysis ◽

Transcriptional Analysis ◽

Content Type ◽

In The Wild ◽

New Compounds

ABSTRACT A number of strategies have been developed to mine novel natural products based on biosynthetic gene clusters and there have been dozens of successful cases facilitated by the development of genomic sequencing. During our study on biosynthesis of the antitumor polyketide kosinostatin (KST), we found that the genome of Micromonospora sp. strain TP-A0468, the producer of KST, contains other potential polyketide gene clusters, with no encoded products detected. Deletion of kst cluster led to abolishment of KST and the enrichment of several new compounds, which were isolated and characterized as 16-demethylrifamycins (referred to here as compounds 3 to 6). Transcriptional analysis demonstrated that the expression of the essential genes related to the biosynthesis of compounds 3 to 6 was comparable to the level in the wild-type and in the kst cluster deletion strain. This indicates that the accumulation of these compounds was due to the redirection of metabolic flux rather than transcriptional activation. Genetic disruption, chemical complementation, and bioinformatic analysis revealed that the production of compounds 3 to 6 was accomplished by cross talk between the two distantly placed polyketide gene clusters pks3 and M-rif. This finding not only enriches the analogue pool and the biosynthetic diversity of rifamycins but also provides an auxiliary strategy for natural product discovery through genome mining in polyketide-producing microorganisms. IMPORTANCE Natural products are essential in the development of novel clinically used drugs. Discovering new natural products and modifying known compounds are still the two main ways to generate new candidates. Here, we have discovered several rifamycins with varied skeleton structures by redirecting the metabolic flux from the predominant polyketide biosynthetic pathway to the rifamycin pathway in the marine actinomycetes species Micromonospora sp. strain TP-A0468. Rifamycins are indispensable chemotherapeutics in the treatment of various diseases such as tuberculosis, leprosy, and AIDS-related mycobacterial infections. This study exemplifies a useful method for the discovery of cryptic natural products in genome-sequenced microbes. Moreover, the 16-demethylrifamycins and their genetically manipulable producer provide a new opportunity in the construction of novel rifamycin derivates to aid in the defense against the ever-growing drug resistance of Mycobacterium tuberculosis.

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The Leucine-Responsive Regulatory Protein Lrp Participates in Virulence Regulation Downstream of Small RNA ArcZ inErwinia amylovora

mBio ◽

10.1128/mbio.00757-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 6

Author(s):

Jeffrey K. Schachterle ◽

George W. Sundin

Keyword(s):

Small Rna ◽

Transcriptional Activation ◽

Fire Blight ◽

Erwinia Amylovora ◽

Regulatory Protein ◽

Regulatory Control ◽

Transcriptional Level ◽

Content Type ◽

Virulence Regulation ◽

Blight Disease

ABSTRACTErwinia amylovoracauses the devastating fire blight disease of apple and pear trees. During systemic infection of host trees, pathogen cells must rapidly respond to changes in their environment as they move through different host tissues that present distinct challenges and sources of nutrition. Growing evidence indicates that small RNAs (sRNAs) play an important role in disease progression as posttranscriptional regulators. The sRNA ArcZ positively regulates the motility phenotype and transcription of flagellar genes inE. amylovoraEa1189 yet is a direct repressor of translation of the flagellar master regulator, FlhD. We utilized transposon mutagenesis to conduct a forward genetic screen and identified suppressor mutations that increase motility in the Ea1189ΔarcZmutant background. This enabled us to determine that the mechanism of transcriptional activation of theflhDCmRNA by ArcZ is mediated by the leucine-responsive regulatory protein, Lrp. We show that Lrp contributes to expression of virulence and several virulence-associated traits, including production of the exopolysaccharide amylovoran, levansucrase activity, and biofilm formation. We further show that Lrp is regulated posttranscriptionally by ArcZ through destabilization oflrpmRNA. Thus, ArcZ regulation of FlhDC directly and indirectly through Lrp forms an incoherent feed-forward loop that regulates levansucrase activity and motility as outputs. This work identifies Lrp as a novel participant in virulence regulation inE. amylovoraand places it in the context of a virulence-associated regulatory network.IMPORTANCEFire blight disease continues to plague the commercial production of apples and pears despite more than a century of research into disease epidemiology and disease control. The causative agent of fire blight,Erwinia amylovoracoordinates turning on or off specific virulence-associated traits at the appropriate time during disease development. The development of novel control strategies requires an in-depth understanding ofE. amylovoraregulatory mechanisms, including regulatory control of virulence-associated traits. This study investigates how the small RNA ArcZ regulates motility at the transcriptional level and identifies the transcription factor Lrp as a novel participant in the regulation of several virulence-associated traits. We report that ArcZ and Lrp together affect key virulence-associated traits through integration of transcriptional and posttranscriptional mechanisms. Further understanding of the topology of virulence regulatory networks can uncover weak points that can subsequently be exploited to controlE. amylovora.

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Role of the nifB1 and nifB2 Promoters in Cell-Type-Specific Expression of Two Mo Nitrogenases in the Cyanobacterium Anabaena variabilis ATCC 29413

Journal of Bacteriology ◽

10.1128/jb.00674-16 ◽

2016 ◽

Vol 199 (4) ◽

Cited By ~ 6

Author(s):

Susan A. Vernon ◽

Brenda S. Pratte ◽

Teresa Thiel

Keyword(s):

Promoter Region ◽

Expression Patterns ◽

Gene Clusters ◽

Anabaena Variabilis ◽

Cell Type ◽

Specific Expression ◽

Vegetative Cells ◽

Content Type ◽

Cell Type Specific Expression ◽

Cell Type Specific

ABSTRACT Anabaena variabilis ATCC 29413 has one Mo nitrogenase that is made under oxic growth conditions in specialized cells called heterocysts and a second Mo nitrogenase that is made only under anoxic conditions in vegetative cells. The two large nif gene clusters responsible for these two nitrogenases are under the control of the promoter of the first gene in the operon, nifB1 or nifB2. Despite differences in the expression patterns of nifB1 and nifB2, related to oxygen and cell type, the regions upstream of their transcription start sites (tss) show striking homology, including three highly conserved sequences (CS). CS1, CS2, and the region just upstream from the tss were required for optimal expression from the nifB1 promoter, but CS3 and the 5′ untranslated region (UTR) were not. Hybrid fusions of the nifB1 and nifB2 upstream regions revealed that the region including CS1, CS2, and CS3 of nifB2 could substitute for the similar region of nifB1; however, the converse was not true. Expression from the nifB2 promoter region required the CS1, CS2, and CS3 regions of nifB2 and also required the nifB2 5′ UTR. A hybrid promoter that was mostly nifB2 but that had the region from about position −40 to the tss of nifB1 was expressed in heterocysts and in anoxic vegetative cells. Thus, addition of the nifB1 promoter region (from about position −40 to the tss of nifB1) in the nifB hybrid promoter supported expression in heterocysts but did not prevent the mostly nifB2 promoter from also functioning in anoxic vegetative cells. IMPORTANCE In the filamentous cyanobacterium Anabaena variabilis, two Mo nitrogenase gene clusters, nif1 and nif2, function under different environmental conditions in different cell types. Little is known about the regulation of transcription from the promoter upstream of the first gene of the cluster, which drives transcription of each of these two large operons. The similarity in the sequences upstream of the primary promoters for the two nif gene clusters belies the differences in their expression patterns. Analysis of these nif promoters in strains with mutations in the conserved sequences and in strains with hybrid promoters, comprising parts from nif1 and nif2, provides strong evidence that each promoter has key elements required for cell-type-specific expression of the nif1 and nif2 gene clusters.

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In Salmonella enterica, Ethanolamine Utilization Is Repressed by 1,2-Propanediol To Prevent Detrimental Mixing of Components of Two Different Bacterial Microcompartments

Journal of Bacteriology ◽

10.1128/jb.00215-15 ◽

2015 ◽

Vol 197 (14) ◽

pp. 2412-2421 ◽

Cited By ~ 18

Author(s):

Ryan Sturms ◽

Nicholas A. Streauslin ◽

Shouqiang Cheng ◽

Thomas A. Bobik

Keyword(s):

Protein Interactions ◽

Regulatory Protein ◽

Gene Clusters ◽

Content Type ◽

Bacterial Microcompartments ◽

Shell Protein ◽

Bacterial Microcompartment ◽

Genes Encoding ◽

Shell Proteins ◽

High Conservation

ABSTRACTBacterial microcompartments (MCPs) are a diverse family of protein-based organelles composed of metabolic enzymes encapsulated within a protein shell. The function of bacterial MCPs is to optimize metabolic pathways by confining toxic and/or volatile metabolic intermediates. About 20% of bacteria produce MCPs, and there are at least seven different types. Different MCPs vary in their encapsulated enzymes, but all have outer shells composed of highly conserved proteins containing bacterial microcompartment domains. Many organisms have genes encoding more than one type of MCP, but given the high homology among shell proteins, it is uncertain whether multiple MCPs can be functionally expressed in the same cell at the same time. In these studies, we examine the regulation of the 1,2-propanediol (1,2-PD) utilization (Pdu) and ethanolamine utilization (Eut) MCPs inSalmonella. Studies showed that 1,2-PD (shown to induce the Pdu MCP) represses transcription of the Eut MCP and that the PocR regulatory protein is required. The results indicate that repression of the Eut MCP by 1,2-PD is needed to prevent detrimental mixing of shell proteins from the Eut and Pdu MCPs. Coexpression of both MCPs impaired the function of the Pdu MCP and resulted in the formation of hybrid MCPs composed of Eut and Pdu MCP components. We also show that plasmid-based expression of individual shell proteins from the Eut MCP or the β-carboxysome impaired the function of Pdu MCP. Thus, the high conservation among bacterial microcompartment (BMC) domain shell proteins is problematic for coexpression of the Eut and Pdu MCPs and perhaps other MCPs as well.IMPORTANCEBacterial MCPs are encoded by nearly 20% of bacterial genomes, and almost 40% of those genomes contain multiple MCP gene clusters. In this study, we examine how the regulation of two different MCP systems (Eut and Pdu) is integrated inSalmonella. Our findings indicate that 1,2-PD (shown to induce the Pdu MCP) represses the Eut MCP to prevent detrimental mixing of Eut and Pdu shell proteins. These findings suggest that numerous organisms which produce more than one type of MCP likely need some mechanism to prevent aberrant shell protein interactions.

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VASP is upregulated by WDR5-MYC nexus and promotes cell migration in breast cancer

10.21203/rs.3.rs-40146/v1 ◽

2020 ◽

Author(s):

Xiaolong Xu ◽

Yihao Tian ◽

Fangfang Chen ◽

Yang Gao ◽

Jingjing Xu ◽

...

Keyword(s):

Breast Cancer ◽

Cell Migration ◽

Cancer Cells ◽

Transcriptional Activation ◽

Promoter Region ◽

Breast Cancer Cells ◽

Regulatory Protein ◽

Site Directed Mutagenesis ◽

Regulatory Function ◽

Normal Tissues

Abstract Backguroud: Breast cancer is one of the most threatening diseases for women, whose metastasis and recurrence are important causes of death in breast cancer patients. Vasodilator-stimulated phosphoprotein (VASP) is a cytoskeletal regulatory protein that promotes invasion and metastasis of tumor cells by regulating cell migration. Bioinformatics data indicated that H3K4me3, WDR5 and MYC co-enriched in the VASP promoter region. Aims: The purpose of this study is to demonstrate the regulatory function and mechanism of WDR5-MYC nexus complex on VASP in breast cancer.Method: In this present study, the expression of VASP in breast cancer and adjacent normal tissues was detected by RT-qPCR. The enrichment of H3K4me3, WDR5 and MYC on the VASP promoter was analyzed by ChIPseeker R package and verified by ChIP-PCR. The interaction of H3K4me3, WDR5 and MYC in breast cancer cells was detected by immunoprecipitation and immunofluorescence. Transcriptional activation function of MYC on VASP was detected by site-directed mutagenesis and dual fluorescence reporter system. The regulatory effect of WDR5 on breast cancer cell migration was tested by wund healing and transwell.Result: VASP is up-regulated in breast cancer tissues as compared with adjacent normal tissues. There is interaction between H3K4me3, WDR5 and MYC and co-enrichment on the VASP promoter. MYC can activate VASP transcription by binding to the VASP promoter-842 binding site. WDR5 and MYC can enhance the migration ability of breast cancer cells by up-regulating VASPConclusion: Our results suggest that WDR5-MYC nexus can activate the transcription of VASP by binding to the VASP promoter region, and promote the migration of breast cancer by up-regulating the expression level of VASP.

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SanG, a transcriptional activator, controls nikkomycin biosynthesis through binding to the sanN–sanO intergenic region in Streptomyces ansochromogenes

Microbiology ◽

10.1099/mic.0.033605-0 ◽

2010 ◽

Vol 156 (3) ◽

pp. 828-837 ◽

Cited By ~ 20

Author(s):

Xihong He ◽

Rui Li ◽

Yuanyuan Pan ◽

Gang Liu ◽

Huarong Tan

Keyword(s):

Transcriptional Activation ◽

Promoter Region ◽

Target Genes ◽

Regulatory Protein ◽

Transcriptional Level ◽

Gtpase Activity ◽

Mobility Shift ◽

Target Dna ◽

Streptomyces Ansochromogenes

Streptomyces ansochromogenes SanG is a pathway-specific regulator that mainly controls the transcription of two transcriptional units involved in nikkomycin biosynthesis. SanG consists of three major functional domains: an N-terminal Streptomyces antibiotic regulatory protein (SARP) domain, a central ATPase domain, and a C-terminal half homologous to guanylate cyclases belonging to the LuxR family. SanG was expressed in Escherichia coli as a C-terminally His6-tagged protein. The purified SanG-His6 was shown to be a dimer in solution by dynamic light scattering. An electrophoretic mobility-shift assay showed that the purified SanG protein could bind to the DNA fragment containing the bidirectional sanN–sanO promoter region. The SanG-binding sites within the bidirectional sanN–sanO promoter region were determined by footprinting analysis and identified a consensus-directed repeat sequence 5′-CGGCAAG-3′. SanG showed significant ATPase/GTPase activity in vitro, and addition of ATP/GTP enhanced the affinity of SanG for target DNA, but ATP/GTP hydrolysis was not essential for SanG binding to the target DNA. However, real-time reverse transcription PCR showed that mutation of the ATPase/GTPase domain of SanG significantly decreased the transcriptional level of sanN–I and sanO–V. These results indicated that the ATPase/GTPase activity of SanG modulated the transcriptional activation of SanG target genes during nikkomycin biosynthesis.

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Bacillus subtilis Fur Is a Transcriptional Activator for the PerR-Repressed pfeT Gene, Encoding an Iron Efflux Pump

Journal of Bacteriology ◽

10.1128/jb.00697-19 ◽

2020 ◽

Vol 202 (8) ◽

Cited By ~ 3

Author(s):

Azul Pinochet-Barros ◽

John D. Helmann

Keyword(s):

Bacillus Subtilis ◽

Listeria Monocytogenes ◽

Transcriptional Activation ◽

Promoter Region ◽

Iron Homeostasis ◽

Efflux Pump ◽

Regulatory Element ◽

Efflux Pumps ◽

Iron Starvation ◽

Content Type

ABSTRACT The physiological relevance of bacterial iron efflux has only recently been appreciated. The Bacillus subtilis P1B4-type ATPase PfeT (peroxide-induced ferrous efflux transporter) was one of the first iron efflux pumps to be characterized, and cells lacking pfeT accumulate high levels of intracellular iron. The pfeT promoter region has binding sites for both PerR, a peroxide-sensing Fur-family metalloregulator, and the ferric uptake repressor Fur. Both Fur and PerR bind DNA with Fe(II) as a cofactor. While reaction of PerR-Fe(II) with peroxide can account for the induction of pfeT under oxidative stress, binding of Fur-Fe(II) would be expected to lead to repression, which is inconsistent with the known role of PfeT as an iron efflux protein. Here, we show that expression of pfeT is repressed by PerR, as anticipated, and induced by Fur in response to Fe(II). Activation by Fur is mediated both by antagonism of the PerR repressor and by direct transcriptional activation, as confirmed using in vitro transcription assays. A similar mechanism of regulation can explain the iron induction of the Listeria monocytogenes PfeT ortholog and virulence factor, FrvA. Mutational studies support a model in which Fur activation involves regions both upstream and downstream of the pfeT promoter, and Fur and PerR have overlapping recognition of a shared regulatory element in this complex promoter region. This work demonstrates that B. subtilis Fur can function as an iron-dependent activator of transcription. IMPORTANCE Iron homeostasis plays a key role at the host-pathogen interface during the process of infection. Bacterial growth restriction resulting from host-imposed iron starvation (nutritional immunity) highlights the importance of iron import during pathogenesis. Conversely, bacterial iron efflux pumps function as virulence factors in several systems. The requirement for iron efflux in pathogens such as Listeria monocytogenes, Streptococcus pyogenes, and Mycobacterium tuberculosis suggests that both import and efflux are needed for cells to successfully navigate rapidly changing levels of iron availability in the host. Here, we provide insight into how iron efflux genes are controlled, an aspect of bacterial iron homeostasis relevant to infectious disease processes.

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