Transcriptional regulation of congocidine (netropsin) biosynthesis and resistance.

The production of specialized metabolites by Streptomyces bacteria is usually temporally regulated. This regulation is complex and frequently involves both global and pathway-specific mechanisms. Streptomyces ambofaciens ATCC23877 produces several specialized metabolites, including spiramycins, stambomycins, kinamycins and congocidine. The production of the first three molecules has been shown to be controlled by one or several cluster-situated transcriptional regulators. However, nothing is known regarding the regulation of congocidine biosynthesis. Congocidine (netropsin) belongs to the family of pyrrolamide metabolites, which also includes distamycin and anthelvencins. Most pyrrolamides bind into the minor groove of DNA, specifically in A/T-rich regions, which gives them numerous biological activities, such as antimicrobial and antitumoral activities. We previously reported the characterization of the pyrrolamide biosynthetic gene clusters of congocidine ( cgc ) in S. ambofaciens ATCC23877, distamycin ( dst ) in Streptomyces netropsis DSM40846 and anthelvencins ( ant ) in Streptomyces venezuelae ATCC14583. The three gene clusters contain a gene encoding a putative transcriptional regulator, cgc1 , dst1 and ant1 respectively. Cgc1, Dst1 and Ant1 present a high percentage of amino acid sequence similarity. We demonstrate here that Cgc1, an atypical orphan response regulator, activates the transcription of all cgc genes in the stationary phase of S. ambofaciens growth. We also show that the cgc cluster is constituted of eight main transcriptional units. Finally, we show that congocidine induces the expression of the transcriptional regulator Cgc1 and of the operon containing the resistance genes ( cgc20 and cgc21 , coding for an ABC transporter), and propose a model for the transcriptional regulation of the cgc gene cluster. Importance Understanding the mechanisms of regulation of specialized metabolite production can have important implications both at the level of specialized metabolism study (expression of silent gene clusters) and the biotechnological level (increase of the production of a metabolite of interest). We report here a study on the regulation of the biosynthesis of a metabolite from the pyrrolamide family, congocidine. We show that congocidine biosynthesis and resistance is controlled by Cgc1, a cluster-situated regulator. As the gene clusters directing the biosynthesis of the pyrrolamides distamycin and anthelvencin encode a homolog of Cgc1, our findings may be relevant for the biosynthesis of other pyrrolamides. In addition, our results reveal a new type of feed-forward induction mechanism, in which congocidine induces its own biosynthesis through the induction of the transcription of cgc1 .

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Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6

Applied and Environmental Microbiology ◽

10.1128/aem.72.3.1825-1832.2006 ◽

2006 ◽

Vol 72 (3) ◽

pp. 1825-1832 ◽

Cited By ~ 46

Author(s):

Mikio Sasoh ◽

Eiji Masai ◽

Satoko Ishibashi ◽

Hirofumi Hara ◽

Naofumi Kamimura ◽

...

Keyword(s):

Sequence Similarity ◽

Large Subunit ◽

Transcriptional Regulator ◽

Gene Clusters ◽

Small Subunit ◽

Amino Acid Sequence Similarity ◽

Comamonas Testosteroni ◽

Cell Extracts ◽

Two Component

ABSTRACT We isolated Comamonas sp. strain E6, which utilizes terephthalate (TPA) as the sole carbon and energy source via the protocatechuate (PCA) 4,5-cleavage pathway. Two almost identical TPA degradation gene clusters, tphR I C I A2 I A3 I B I A1 I and tphR II C II A2 II A3 II B II A1 II, were isolated from this strain. Based on amino acid sequence similarity, the genes tphR, tphC, tphA2, tphA3, tphB, and tphA1 were predicted to code, respectively, for an IclR-type transcriptional regulator, a periplasmic TPA binding receptor, the large subunit of the oxygenase component of TPA 1,2-dioxygenase (TPADO), the small subunit of the oxygenase component of TPADO, a 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD) dehydrogenase, and a reductase component of TPADO. The growth of E6 on TPA was not affected by disruption of either tphA2 I or tphA2 II singly; however, the tphA2 I tphA2 II double mutant no longer grew on TPA, suggesting that both TPADO genes are involved in TPA degradation. Introduction of a plasmid carrying tphR II C II A2 II A3 II B II A1 II conferred the TPA utilization phenotype on Comamonas testosteroni IAM 1152, which is able to grow on PCA but not on TPA. Disruption of either tphR II or tphC II on this plasmid resulted in the loss of the growth of IAM 1152 on TPA, suggesting that these genes are essential to convert TPA to PCA in E6. The genes tphA1 II, tphA2 II, tphA3 II, and tphB II were expressed in Escherichia coli, and the resultant cell extracts containing TphA1II, TphA2II, and TphA3II converted TPA in the presence of NADPH into a product which was transformed to PCA by TphBII. On the basis of these results, TPADO was strongly suggested to be a two-component dioxygenase which consists of the terminal oxygenase component (TphA2 and TphA3) and the reductase (TphA1), and tphB codes for the DCD dehydrogenase.

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Genome mining and UHPLC-MS/MS illuminate the specificity of secondary metabolite synthetic gene clusters in Bacillus subtilis NCD-2

10.21203/rs.3.rs-33091/v1 ◽

2020 ◽

Author(s):

Zhenhe Su ◽

Xiuye Chen ◽

Xiaomeng Liu ◽

Qinggang Guo ◽

Shezeng Li ◽

...

Keyword(s):

Bacillus Subtilis ◽

Secondary Metabolites ◽

Secondary Metabolite ◽

Biological Activities ◽

Genome Mining ◽

Gene Clusters ◽

Synthetic Gene ◽

Integrative Approach ◽

Amino Acid Sequence Similarity ◽

Genetic Characteristics

Abstract Background Bacillus subtilisstrain NCD-2 is anexcellent biocontrol agent against plant soil-borne diseases and shows broad-spectrum antifungal activities. This study aimed to explore all the secondary metabolite synthetic gene clusters and related bioactive compounds in NCD-2. An integrative approach, which coupled genome mining with structural identification technologies using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-MS/MS), was conducted to interpret the chemical origins of the significant biological activities in NCD-2. Results Genome mining revealed that NCD-2 contained nine gene clustershaving predicted functionsinvolving secondary metabolites with bioactive abilities. They encoded six known products-fengycin, surfactin, bacillaene, subtilosin, bacillibactin, and bacilysin-as well as three unknown products.Interestingly, the synthetic gene clusters for surfactin and fengycin showed 83% and 92% amino acid sequence similarity levels with the corresponding productsin Bacillus velezensisstrain FZB42. A further comparison of gene clusters encoding fengycin and surfactinrevealed that strain NCD-2 had lost thefenC and fenDgenes in the fengycinbiosynthetic operon, and that the surfactin synthetic enzyme-related gene srfAB was divided into two parts.Abioinformatics analysis showed that fenEAmay function as fenCD in synthesizing fengycinand that the structure of thisfengycin synthetic gene clusteris likely unique to NCD-2.Five kinds of fengycin,with 26 homologs, and surfactin,with 4 homologs,were detectedfrom strain NCD-2, which indicated the non-typical and unique nature of this fengycin biosynthetic gene cluster.To the best of our knowledge, this is the first report of a non-typical gene cluster related to fengycin synthesis. Conclusions The data provide the genetic characteristics of secondary metabolite synthetic gene clusters for fengycinand surfactin in B. subtilis NCD-2, including the unique synthetic mechanism for fengycin, and suggest that bioactive secondary metabolites explain the biological activities of NCD-2.

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Dynamics of the compartmentalized Streptomyces chromosome during metabolic differentiation

Nature Communications ◽

10.1038/s41467-021-25462-1 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Virginia S. Lioy ◽

Jean-Noël Lorenzi ◽

Soumaya Najah ◽

Thibault Poinsignon ◽

Hervé Leh ◽

...

Keyword(s):

Chromosome Structure ◽

Gene Clusters ◽

Structural Features ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Linear Chromosome ◽

Chromosome Architecture ◽

Specialized Metabolites ◽

Streptomyces Ambofaciens ◽

Core Genes

AbstractBacteria of the genus Streptomyces are prolific producers of specialized metabolites, including antibiotics. The linear chromosome includes a central region harboring core genes, as well as extremities enriched in specialized metabolite biosynthetic gene clusters. Here, we show that chromosome structure in Streptomyces ambofaciens correlates with genetic compartmentalization during exponential phase. Conserved, large and highly transcribed genes form boundaries that segment the central part of the chromosome into domains, whereas the terminal ends tend to be transcriptionally quiescent compartments with different structural features. The onset of metabolic differentiation is accompanied by a rearrangement of chromosome architecture, from a rather ‘open’ to a ‘closed’ conformation, in which highly expressed specialized metabolite biosynthetic genes form new boundaries. Thus, our results indicate that the linear chromosome of S. ambofaciens is partitioned into structurally distinct entities, suggesting a link between chromosome folding, gene expression and genome evolution.

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Desulfovibrio africanus subsp. uniflagellum subsp. nov., a sulfate-reducing bacterium from a uranium-contaminated subsurface aquifer

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.006668-0 ◽

2010 ◽

Vol 60 (4) ◽

pp. 880-886 ◽

Cited By ~ 14

Author(s):

I. Nydia Castañeda-Carrión ◽

Cody S. Sheik ◽

Lee R. Krumholz

Keyword(s):

Dna Sequence ◽

Dna Sequences ◽

Type Strain ◽

Sequence Similarity ◽

Gene Clusters ◽

Contaminated Site ◽

Amino Acid Sequence Similarity ◽

Trna Genes ◽

Rrna Gene ◽

Small Plasmid

The bacterial strain SR-1T was isolated from subsurface sediments of a uranium-contaminated site in Shiprock, New Mexico, USA. Cells are vibrioid and motile by means of a single polar flagellum. Strain SR-1T grows on sulfate, oxidizing formate, lactate and H2, but not malate, and ferments pyruvate. The DNA sequences of the 16S rRNA gene and the 16S–23S internal transcribed spacer of strain SR-1T showed 99.9 and 99.4 % similarity, respectively, to those of the type strain Desulfovibrio africanus DSM 2603T. The DNA sequence of the ITS region is 300 bases in length and contains two tRNA genes (tRNAIle, tRNAAla). The partial DNA sequence of the dsrAB gene showed 94.6 % amino acid sequence similarity to that of D. africanus. The DNA G+C content of strain SR-1T was 62.4 mol% and it showed 72 % DNA–DNA similarity to D. africanus. DNA typing methods that target gene clusters and whole genomes revealed characteristic genomic fingerprints for strain SR-1T. A small plasmid was detected by gel electrophoresis. On the basis of distinct phenotypic and genotypic characteristics, strain SR-1T represents a novel subspecies of D. africanus, for which the name Desulfovibrio africanus subsp. uniflagellum subsp. nov. is proposed. The type strain is SR-1T (=JCM 15510T =LS KCTC 5649T).

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Staphylococcus aureus AgrA Binding to the RNAIII-agr Regulatory Region

Journal of Bacteriology ◽

10.1128/jb.186.22.7549-7555.2004 ◽

2004 ◽

Vol 186 (22) ◽

pp. 7549-7555 ◽

Cited By ~ 130

Author(s):

Robbin L. Koenig ◽

Jessica L. Ray ◽

Soheila J. Maleki ◽

Mark S. Smeltzer ◽

Barry K. Hurlburt

Keyword(s):

Staphylococcus Aureus ◽

Response Regulator ◽

Osmotic Shock ◽

Sequence Similarity ◽

Regulatory Region ◽

Amino Acid Sequence Similarity ◽

Direct Repeats ◽

Promoter Regions ◽

Mobility Shift ◽

Partial Control

ABSTRACT The control of virulence gene expression in the human pathogen Staphylococcus aureus is under the partial control of the two-component quorum-sensing system encoded by genes of the agr locus. The product of the agrA gene has been shown by amino acid sequence similarity to be the putative response regulator; however, binding of AgrA to promoters under its control has not yet been demonstrated. In this study, we isolated and purified soluble AgrA by expression under osmotic shock conditions and ion-exchange chromatography. Purified AgrA showed high-affinity binding to the RNAIII-agr intergenic region by electrophoretic mobility shift assays. Binding was localized by DNase I protection assays to a pair of direct repeats in the P2 and P3 promoter regions of the agr locus. We found that this binding was enhanced by the addition of the small phosphoryl donor, acetyl phosphate. The difference in binding affinity between these two promoters was found to result from a 2-bp difference between the downstream direct repeats of the P2 and P3 sites. Mutation of these base pairs in the P3 site to match those found in the P2 site increased the affinity of AgrA for the P3 site relative to that for the P2 site. These results are consistent with the function of AgrA as a response regulator with recognition sites in the promoter regions of RNAIII and the agr locus.

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Copy Number of Pilus Gene Clusters inHaemophilus influenzae and Variation in the hifEPilin Gene

Infection and Immunity ◽

10.1128/iai.66.4.1622-1631.1998 ◽

1998 ◽

Vol 66 (4) ◽

pp. 1622-1631 ◽

Cited By ~ 11

Author(s):

Timothy D. Read ◽

Sarah W. Satola ◽

Jason A. Opdyke ◽

Monica M. Farley

Keyword(s):

Sequence Similarity ◽

Gene Clusters ◽

Amino Acid Sequence Similarity ◽

Nucleotide Sequencing ◽

Gene Probe ◽

Type B ◽

Putative Receptor ◽

Brazilian Purpuric Fever ◽

Chaperone Binding ◽

Serotype B

ABSTRACT Brazilian purpuric fever (BPF)-associated Haemophilus influenzae biogroup aegyptius strain F3031 contains two identical copies of a five gene cluster (hifA to hifE) encoding pili similar to well-characterized Hif fimbriae of H. influenzae type b. HifE, the putative pilus tip adhesin of F3031, shares only 40% amino acid sequence similarity with the same molecule from type b strains, whereas the other four proteins have 75 to 95% identity. To determine whether pilus cluster duplication and thehifE F3031 allele were special features of BPF-associated bacteria, we analyzed a collection of H. influenzae strains by PCR with hifA- andhifE-specific oligonucleotides, by Southern hybridization with a hifC gene probe, and by nucleotide sequencing. The presence of two pilus clusters was limited to some H. influenzae biogroup aegyptius strains. ThehifE F3031 allele was limited to H. influenzae biogroup aegyptius. Two strains contained one copy ofhifE F3031 and one copy of a varianthifE allele. We determined the nucleotide sequences of fourhifE genes from H. influenzae biogroup aegyptius and H. influenzae capsule serotypes a and c. The predicted proteins produced by these genes demonstrated only 35 to 70% identity to the three published HifE proteins from nontypeable H. influenzae, serotype b, and BPF strains. The C-terminal third of the molecules implicated in chaperone binding was the most highly conserved region. Three conserved domains in the otherwise highly variable N-terminal putative receptor-binding region of HifE were similar to conserved portions in the N terminus ofNeisseria pilus adhesin PilC. We concluded that two pilus clusters and hifE F3031 were not specific for BPF-causing H. influenzae, and we also identified portions of HifE possibly involved in binding mammalian cell receptors.

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Novel Aeromonas hydrophila PPD134/91 Genes Involved in O-Antigen and Capsule Biosynthesis

Infection and Immunity ◽

10.1128/iai.70.5.2326-2335.2002 ◽

2002 ◽

Vol 70 (5) ◽

pp. 2326-2335 ◽

Cited By ~ 31

Author(s):

Y. L. Zhang ◽

E. Arakawa ◽

K. Y. Leung

Keyword(s):

Amino Acid ◽

Gene Cluster ◽

Aeromonas Hydrophila ◽

Sequence Similarity ◽

Gene Clusters ◽

Inhibitory Effect ◽

Amino Acid Sequence Similarity ◽

O Antigen ◽

Serotype O ◽

Surface Polysaccharides

ABSTRACT The sequences of the O-antigen and capsule gene clusters of the virulent Aeromonas hydrophila strain PPD134/91 were determined. The O-antigen gene cluster is 17,296 bp long and comprises 17 genes. Seven pathway genes for the synthesis of rhamnose and mannose, six transferase genes, one O unit flippase gene, and one O-antigen chain length determinant gene were identified by amino acid sequence similarity. PCR and Southern blot analysis were performed to survey the distribution of these 17 genes among 11 A. hydrophila strains of different serotypes. A. hydrophila PPD134/91 might belong to serotype O:18, as represented by JCM3980; it contained all the same O-antigen genes as JCM3980 (97 to 100% similarity at the DNA and amino acid levels). The capsule gene cluster of A. hydrophila PPD134/91 is 17,562 bp long and includes 13 genes, which were assembled into three distinct regions similar to those of the group II capsule gene cluster of Escherichia coli and other bacteria. Regions I and III contained four and two capsule transport genes, respectively. Region II had five genes which were highly similar to capsule synthesis pathway genes found in other bacteria. Both the purified O-antigen and capsular polysaccharides increased the ability of the avirulent A. hydrophila strain PPD35/85 to survive in naïve tilapia serum. However, the purified surface polysaccharides had no inhibitory effect on the adhesion of A. hydrophila PPD134/91 to carp epithelial cells.

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Production of Plant-Associated Volatiles by Select Model and Industrially Important Streptomyces spp.

Microorganisms ◽

10.3390/microorganisms8111767 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1767 ◽

Cited By ~ 1

Author(s):

Zhenlong Cheng ◽

Sean McCann ◽

Nicoletta Faraone ◽

Jody-Ann Clarke ◽

E. Abbie Hudson ◽

...

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Biological Activities ◽

Gene Clusters ◽

Gas Chromatography Mass Spectrometry ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Specialized Metabolites ◽

Streptomyces Spp ◽

Volatile Organic

The Streptomyces produce a great diversity of specialized metabolites, including highly volatile compounds with potential biological activities. Volatile organic compounds (VOCs) produced by nine Streptomyces spp., some of which are of industrial importance, were collected and identified using gas chromatography–mass spectrometry (GC-MS). Biosynthetic gene clusters (BGCs) present in the genomes of the respective Streptomyces spp. were also predicted to match them with the VOCs detected. Overall, 33 specific VOCs were identified, of which the production of 16 has not been previously reported in the Streptomyces. Among chemical classes, the most abundant VOCs were terpenes, which is consistent with predicted biosynthetic capabilities. In addition, 27 of the identified VOCs were plant-associated, demonstrating that some Streptomyces spp. can also produce such molecules. It is possible that some of the VOCs detected in the current study have roles in the interaction of Streptomyces with plants and other higher organisms, which might provide opportunities for their application in agriculture or industry.

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Characterization of the Isophthalate Degradation Genes of Comamonas sp. Strain E6

Applied and Environmental Microbiology ◽

10.1128/aem.01270-09 ◽

2009 ◽

Vol 76 (2) ◽

pp. 519-527 ◽

Cited By ~ 27

Author(s):

Yuki Fukuhara ◽

Keisuke Inakazu ◽

Norimichi Kodama ◽

Naofumi Kamimura ◽

Daisuke Kasai ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Sequence Similarity ◽

Transcriptional Regulator ◽

Complete Loss ◽

Transcriptional Unit ◽

Energy Sources ◽

Amino Acid Sequence Similarity ◽

Two Component

ABSTRACT The isophthalate (IPA) degradation gene cluster (iphACBDR) responsible for the conversion of IPA into protocatechuate (PCA) was isolated from Comamonas sp. strain E6, which utilizes phthalate isomers as sole carbon and energy sources via the PCA 4,5-cleavage pathway. Based on amino acid sequence similarity, the iphA, iphC, iphB, iphD, and iphR genes were predicted to code for an oxygenase component of IPA dioxygenase (IPADO), a periplasmic IPA binding receptor, a 1,2-dihydroxy-3,5-cyclohexadiene-1,5-dicarboxylate (1,5-DCD) dehydrogenase, a reductase component of IPADO, and an IclR-type transcriptional regulator, respectively. The iphACBDR genes constitute a single transcriptional unit, and transcription of the iph catabolic operon was induced during growth of E6 on IPA. The iphA, iphD, and iphB genes were expressed in Escherichia coli. Crude IphA and IphD converted IPA in the presence of NADPH into a product which was transformed to PCA by IphB. These results suggested that IPADO is a two-component dioxygenase that consists of a terminal oxygenase component (IphA) and a reductase component (IphD) and that iphB encodes the 1,5-DCD dehydrogenase. Disruption of iphA and iphB resulted in complete loss of growth of E6 on IPA. Inactivation of iphD significantly affected growth on IPA, and the iphC mutant did not grow on IPA at neutral pH. These results indicated that the iphACBD genes are essential for the catabolism of IPA in E6. Disruption of iphR resulted in faster growth of E6 on IPA, suggesting that iphR encodes a repressor for the iph catabolic operon. Promoter analysis of the operon supported this notion.

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Three New NifA-Regulated Genes in the Bradyrhizobium japonicum Symbiotic Gene Region Discovered by Competitive DNA-RNA Hybridization

Journal of Bacteriology ◽

10.1128/jb.182.6.1472-1480.2000 ◽

2000 ◽

Vol 182 (6) ◽

pp. 1472-1480 ◽

Cited By ~ 29

Author(s):

Andrea Nienaber ◽

Alexander Huber ◽

Michael Göttfert ◽

Hauke Hennecke ◽

Hans-Martin Fischer

Keyword(s):

Nitrogen Fixation ◽

Bradyrhizobium Japonicum ◽

Sequence Similarity ◽

Regulatory Protein ◽

Gene Clusters ◽

Amino Acid Sequence Similarity ◽

Wild Type ◽

Significant Similarity ◽

Symbiotic Gene

ABSTRACT The so-called symbiotic region of the Bradyrhizobium japonicum chromosome (C. Kündig, H. Hennecke, and M. Göttfert, J. Bacteriol. 175:613–622, 1993) was screened for the presence of genes controlled by the nitrogen fixation regulatory protein NifA. Southern blots of restriction enzyme-digested cosmids that represent an ordered, overlapping library of the symbiotic region were competitively hybridized with in vitro-labeled RNA from anaerobically grown wild-type cells and an excess of RNA isolated either from anaerobically grown nifA and rpoNmutant cells or from aerobically grown wild-type cells. In addition to the previously characterized nif and fixgene clusters, we identified three new NifA-regulated genes that were named nrgA, nrgB, and nrgC(nrg stands for NifA-regulated gene). The latter two probably form an operon, nrgBC. The proteins encoded bynrgC and nrgA exhibited amino acid sequence similarity to bacterial hydroxylases andN-acetyltransferases, respectively. The product ofnrgB showed no significant similarity to any protein with a database entry. Primer extension experiments and expression studies with translational lacZ fusions revealed the presence of a functional −24/−12-type promoter upstream ofnrgA and nrgBC and proved the NifA- and RpoN (ς54)-dependent transcription of the respective genes. Null mutations introduced into nrgA and nrgBCresulted in mutant strains that exhibited wild-type-like symbiotic properties, including nitrogen fixation, when tested on soybean, cowpea, or mung bean host plants. Thus, the discovery ofnrgA and nrgBC further emphasizes the previously suggested role of NifA as an activator of anaerobically induced genes other than the classical nitrogen fixation genes.

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