scholarly journals Cloning and Characterization of a Gene Cluster for Hatomarubigin Biosynthesis in Streptomyces sp. Strain 2238-SVT4

2010 ◽  
Vol 76 (13) ◽  
pp. 4201-4206 ◽  
Author(s):  
Takashi Kawasaki ◽  
Reiko Hirashima ◽  
Tomoka Maruta ◽  
Haruka Sato ◽  
Ayumi Maeda ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Streptomyces Lividans ◽  
Open Reading Frames ◽  
Specific Primers ◽  
Streptomyces Sp ◽  
Dimeric Structure ◽  
Genes Expression ◽  
Reading Frames

ABSTRACT Streptomyces sp. strain 2238-SVT4 produces hatomarubigins A, B, C, and D, which belong to the angucycline family. Among them, hatomarubigin D has a unique dimeric structure with a methylene linkage. PCR using aromatase and cyclase gene-specific primers identified the hrb gene cluster for angucycline biosynthesis in Streptomyces sp. 2238-SVT4. The cluster consisted of 30 open reading frames, including those for the minimal polyketide synthase, ketoreductase, aromatase, cyclase, O-methyltransferase, oxidoreductase, and oxygenase genes. Expression of a part of the gene cluster containing hrbR1 to hrbX in Streptomyces lividans TK23 resulted in the production of hatomarubigins A, B, and C. Hatomarubigin D was obtained from the conversion of hatomarubigin C by a purified enzyme encoded by hrbY, among the remaining genes.

scholarly journals Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis inStreptomyces griseus DSM40695

2000 ◽  
Vol 44 (7) ◽  
pp. 1809-1817 ◽  
Author(s):  
Wyatt C. Smith ◽  
Longkuan Xiang ◽  
Ben Shen
Keyword(s):  
Polyketide Synthase ◽  
Isotope Labeling ◽  
Streptomyces Griseus ◽  
Open Reading Frames ◽  
A Cell ◽  
Cyclic Polyethers ◽  
The Difference ◽  
Reading Frames

ABSTRACT The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2E-enoic acid can be converted into NA by a cell-free preparation from Streptomyces lividans that expresses nonS. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from Streptomyces griseus DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported nonR andnonS genes. Inactivation of nonS in vivo completely abolished macrotetrolide production. Complementation of thenonS mutant by the expression of nonS intrans fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of thenonS mutant in the presence of exogenous (±)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of nonS in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the nonS mutant by the plasmid-borne nonSgene and fermentation of the nonS mutant in the presence of exogenously added (±)-NA suggests that NonS catalyzes the formation of (−)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis inS. griseus. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis.

scholarly journals Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in “Streptomyces carzinostaticus”

2004 ◽  
Vol 48 (9) ◽  
pp. 3468-3476 ◽  
Author(s):  
Miyuki Otsuka ◽  
Koji Ichinose ◽  
Isao Fujii ◽  
Yutaka Ebizuka
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Streptomyces Coelicolor ◽  
Open Reading Frames ◽  
Modular Organization ◽  
Type I ◽  
Orf3 Protein ◽  
Polyketide Synthase Gene ◽  
Polyketide Chain ◽  
Reading Frames

ABSTRACT Neocarzilins (NCZs) are antitumor chlorinated polyenones produced by “Streptomyces carzinostaticus” var. F-41. The gene cluster responsible for the biosynthesis of NCZs was cloned and characterized. DNA sequence analysis of a 33-kb region revealed a cluster of 14 open reading frames (ORFs), three of which (ORF4, ORF5, and ORF6) encode type I polyketide synthase (PKS), which consists of four modules. Unusual features of the modular organization is the lack of an obvious acyltransferase domain on modules 2 and 4 and the presence of longer interdomain regions more than 200 amino acids in length on each module. Involvement of the PKS genes in NCZ biosynthesis was demonstrated by heterologous expression of the cluster in Streptomyces coelicolor CH999, which produced the apparent NCZ biosynthetic intermediates dechloroneocarzillin A and dechloroneocarzilin B. Disruption of ORF5 resulted in a failure of NCZ production, providing further evidence that the cluster is essential for NCZ biosynthesis. Mechanistic consideration of NCZ formation indicates the iterative use of at least one module of the PKS, which subsequently releases its product by decarboxylation to generate an NCZ skeleton, possibly catalyzed by a type II thioesterase encoded by ORF7. This is a novel type I PKS system of bacterial origin for the biosynthesis of a reduced polyketide chain. Additionally, the protein encoded by ORF3, located upstream of the PKS genes, closely resembles the FADH2-dependent halogenases involved in the formation of halometabolites. The ORF3 protein could be responsible for the halogenation of NCZs, presenting a unique example of a halogenase involved in the biosynthesis of an aliphatic halometabolite.

scholarly journals Identification and Characterization of a Gene Cluster for Synthesis of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol from Pseudomonas fluorescens Q2-87

1999 ◽  
Vol 181 (10) ◽  
pp. 3155-3163 ◽  
Author(s):  
M. Gita Bangera ◽  
Linda S. Thomashow
Keyword(s):  
Genomic Dna ◽  
Plant Pathogens ◽  
Polyketide Synthase ◽  
Open Reading Frames ◽  
Fungal Plant Pathogens ◽  
Repressor Proteins ◽  
Flanking Regions ◽  
Identification And Characterization ◽  
Reading Frames

The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) is produced by many strains of fluorescent Pseudomonas spp. with biocontrol activity against soilborne fungal plant pathogens. Genes required for 2,4-DAPG synthesis by P. fluorescensQ2-87 are encoded by a 6.5-kb fragment of genomic DNA that can transfer production of 2,4-DAPG to 2,4-DAPG-nonproducing recipientPseudomonas strains. In this study the nucleotide sequence was determined for the 6.5-kb fragment and flanking regions of genomic DNA from strain Q2-87. Six open reading frames were identified, four of which (phlACBD) comprise an operon that includes a set of three genes (phlACB) conserved between eubacteria and archaebacteria and a gene (phlD) encoding a polyketide synthase with homology to chalcone and stilbene synthases from plants. The biosynthetic operon is flanked on either side by phlEand phlF, which code respectively for putative efflux and regulatory (repressor) proteins. Expression in Escherichia coli of phlA, phlC, phlB, andphlD, individually or in combination, identified a novel polyketide biosynthetic pathway in which PhlD is responsible for the production of monoacetylphloroglucinol (MAPG). PhlA, PhlC, and PhlB are necessary to convert MAPG to 2,4-DAPG, and they also may function in the synthesis of MAPG.

scholarly journals Characterization of the Type 8 Capsular Gene Cluster of Streptococcus pneumoniae

1999 ◽  
Vol 181 (19) ◽  
pp. 6214-6219 ◽  
Author(s):  
Rosario Muñoz ◽  
Marta Mollerach ◽  
Rubens López ◽  
Ernesto García
Keyword(s):  
Streptococcus Pneumoniae ◽  
Nucleotide Sequence ◽  
Gene Cluster ◽  
Complete Nucleotide Sequence ◽  
Capsular Polysaccharide ◽  
Open Reading Frames ◽  
Dna Fragment ◽  
Type 3 ◽  
Reading Frames

ABSTRACT The complete nucleotide sequence of the capsular gene cluster (cap8) responsible for the biosynthesis of the capsular polysaccharide of Streptococcus pneumoniae type 8 has been determined. The cap8 gene cluster, located between the genes dexB and aliA, is composed of 12 open reading frames. A 14.7-kb DNA fragment embracing the cap8genes was sufficient to transform an unencapsulated type 3 S. pneumoniae strain to a strain with the type 8 capsule. A possible scenario for the evolution of pneumococcal types 2 and 8 is outlined.

scholarly journals Gene Cluster on pAO1 of Arthrobacter nicotinovorans Involved in Degradation of the Plant Alkaloid Nicotine: Cloning, Purification, and Characterization of 2,6-Dihydroxypyridine 3-Hydroxylase

2001 ◽  
Vol 183 (18) ◽  
pp. 5262-5267 ◽  
Author(s):  
Daniel Baitsch ◽  
Cristinel Sandu ◽  
Roderich Brandsch ◽  
Gabor L. Igloi
Keyword(s):  
Gene Cluster ◽  
Flavin Adenine Dinucleotide ◽  
Open Reading Frames ◽  
Hypothetical Proteins ◽  
Purification And Characterization ◽  
Irreversible Inhibitors ◽  
Nicotine Degradation ◽  
Arthrobacter Nicotinovorans ◽  
Reading Frames

ABSTRACT A 27,690-bp gene cluster involved in the degradation of the plant alkaloid nicotine was characterized from the plasmid pAO1 ofArthrobacter nicotinovorans. The genes of the heterotrimeric, molybdopterin cofactor (MoCo)-, flavin adenine dinucleotide (FAD)-, and [Fe-S] cluster-dependent 6-hydroxypseudooxynicotine (ketone) dehydrogenase (KDH) were identified within this cluster. The gene of the large MoCo subunit of KDH was located 4,266 bp from the FAD and [Fe-S] cluster subunit genes. Deduced functions of proteins encoded by open reading frames (ORFs) of the cluster were correlated to individual steps in nicotine degradation. The gene for 2,6-dihydroxypyridine 3-hydroxylase was cloned and expressed in Escherichia coli. The purified homodimeric enzyme of 90 kDa contained 2 mol of tightly bound FAD per mol of dimer. Enzyme activity was strictly NADH-dependent and specific for 2,6-dihydroxypyridine. 2,3-Dihydroxypyridine and 2,6-dimethoxypyridine acted as irreversible inhibitors. Additional ORFs were shown to encode hypothetical proteins presumably required for holoenzyme assembly, interaction with the cell membrane, and transcriptional regulation, including a MobA homologue predicted to be specific for the synthesis of the molybdopterin cytidine dinucleotide cofactor.

scholarly journals Identification of the Novobiocin Biosynthetic Gene Cluster of Streptomyces spheroides NCIB 11891

2000 ◽  
Vol 44 (5) ◽  
pp. 1214-1222 ◽  
Author(s):  
Marion Steffensky ◽  
Agnes Mühlenweg ◽  
Zhao-Xin Wang ◽  
Shu-Ming Li ◽  
Lutz Heide
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Streptomyces Lividans ◽  
Open Reading Frames ◽  
Biosynthetic Gene ◽  
Insertional Inactivation ◽  
Key Enzyme ◽  
Novobiocin Resistance ◽  
Reading Frames

ABSTRACT The novobiocin biosynthetic gene cluster from Streptomyces spheroides NCIB 11891 was cloned by using homologous deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratase gene fragments as probes. Double-stranded sequencing of 25.6 kb revealed the presence of 23 putative open reading frames (ORFs), including the gene for novobiocin resistance, gyrB r, and at least 11 further ORFs to which a possible role in novobiocin biosynthesis could be assigned. An insertional inactivation experiment with a dNDP-glucose 4,6-dehydratase fragment resulted in abolishment of novobiocin production, since biosynthesis of the deoxysugar moiety of novobiocin was blocked. Heterologous expression of a key enzyme of novobiocin biosynthesis, i.e., novobiocic acid synthetase, inStreptomyces lividans TK24 further confirmed the involvement of the analyzed genes in the biosynthesis of the antibiotic.

scholarly journals Biosynthetic Gene Cluster for the Polyenoyltetramic Acid α-Lipomycin

2006 ◽  
Vol 50 (6) ◽  
pp. 2113-2121 ◽  
Author(s):  
C. Bihlmaier ◽  
E. Welle ◽  
C. Hofmann ◽  
K. Welzel ◽  
A. Vente ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Biosynthetic Gene ◽  
Peptide Synthetases ◽  
Insight Into ◽  
Reading Frames ◽  
Module System

ABSTRACT The gram-positive bacterium Streptomyces aureofaciens Tü117 produces the acyclic polyene antibiotic α-lipomycin. The entire biosynthetic gene cluster (lip gene cluster) was cloned and characterized. DNA sequence analysis of a 74-kb region revealed the presence of 28 complete open reading frames (ORFs), 22 of them belonging to the biosynthetic gene cluster. Central to the cluster is a polyketide synthase locus that encodes an eight-module system comprised of four multifunctional proteins. In addition, one ORF shows homology to those for nonribosomal peptide synthetases, indicating that α-lipomycin belongs to the classification of hybrid peptide-polyketide natural products. Furthermore, the lip cluster includes genes responsible for the formation and attachment of d-digitoxose as well as ORFs that resemble those for putative regulatory and export functions. We generated biosynthetic mutants by insertional gene inactivation. By analysis of culture extracts of these mutants, we could prove that, indeed, the genes involved in the biosynthesis of lipomycin had been cloned, and additionally we gained insight into an unusual biosynthesis pathway.

scholarly journals Expression of the Clostridium botulinum A2 Neurotoxin Gene Cluster Proteins and Characterization of the A2 Complex

2009 ◽  
Vol 76 (1) ◽  
pp. 40-47 ◽  
Author(s):  
Guangyun Lin ◽  
William H. Tepp ◽  
Christina L. Pier ◽  
Mark J. Jacobson ◽  
Eric A. Johnson
Keyword(s):  
Gene Cluster ◽  
Clostridium Botulinum ◽  
Polyclonal Antibodies ◽  
Expression System ◽  
Middle Part ◽  
Open Reading Frames ◽  
Protein Sequencing ◽  
Medium Size ◽  
Reading Frames

ABSTRACT Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Polyclonal antibodies against individual orfX cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2 orfX cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that C. botulinum A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.

scholarly journals Identification and Characterization of Catabolic para-Nitrophenol 4-Monooxygenase and para-Benzoquinone Reductase from Pseudomonas sp. Strain WBC-3

2009 ◽  
Vol 191 (8) ◽  
pp. 2703-2710 ◽  
Author(s):  
Jun-Jie Zhang ◽  
Hong Liu ◽  
Yi Xiao ◽  
Xian-En Zhang ◽  
Ning-Yi Zhou
Keyword(s):  
Gene Cluster ◽  
Flavin Adenine Dinucleotide ◽  
Gel Filtration ◽  
Sole Source ◽  
Open Reading Frames ◽  
Flavin Mononucleotide ◽  
Large Component ◽  
Identification And Characterization ◽  
Reading Frames

ABSTRACT Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P)H:quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxyphenylacetate hydroxylase from Pseudomonas putida U and PnpB is 58% identical to an NAD(P)H:quinone oxidoreductase from Escherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP to para-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependent p-benzoquinone reductase that catalyzes the reduction of p-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that both pnpA and pnpB play essential roles in PNP mineralization in strain WBC-3. Furthermore, the pnpCDEF gene cluster next to pnpAB shares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) in Pseudomonas fluorescens ACB (M. J. Moonen, N. M. Kamerbeek, A. H. Westphal, S. A. Boeren, D. B. Janssen, M. W. Fraaije, and W. J. van Berkel, J. Bacteriol. 190:5190-5198, 2008), suggesting that the genes involved in PNP degradation are physically linked.

scholarly journals Identification and Characterization of Two Novel Methyltransferase Genes That Determine the Serotype 12-Specific Structure of Glycopeptidolipids of Mycobacterium intracellulare

2007 ◽  
Vol 190 (3) ◽  
pp. 1064-1071 ◽  
Author(s):  
Noboru Nakata ◽  
Nagatoshi Fujiwara ◽  
Takashi Naka ◽  
Ikuya Yano ◽  
Kazuo Kobayashi ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Structural Difference ◽  
Open Reading Frames ◽  
Specific Gene ◽  
Bacterial Physiology ◽  
Mycobacterium Intracellulare ◽  
Functional Analyses ◽  
Identification And Characterization ◽  
Reading Frames

ABSTRACT The Mycobacterium avium complex is distributed ubiquitously in the environment. It is an important cause of pulmonary and extrapulmonary diseases in humans and animals. The species in this complex produce polar glycopeptidolipids (GPLs); of particular interest is their serotype-specific antigenicity. Several reports have described that GPL structure may play an important role in bacterial physiology and pathogenesis and in the host immune response. Recently, we determined the complete structure of the GPL derived from Mycobacterium intracellulare serotype 7 and characterized the serotype 7 GPL-specific gene cluster. The structure of serotype 7 GPL closely resembles that of serotype 12 GPL, except for O methylation. In the present study, we isolated and characterized the serotype 12-specific gene cluster involved in glycosylation of the GPL. Ten open reading frames (ORFs) and one pseudogene were observed in the cluster. The genetic organization of the serotype 12-specific gene cluster resembles that of the serotype 7-specific gene cluster, but two novel ORFs (orfA and orfB) encoding putative methyltransferases are present in the cluster. Functional analyses revealed that orfA and orfB encode methyltransferases that synthesize O-methyl groups at the C-4 position in the rhamnose residue next to the terminal hexose and at the C-3 position in the terminal hexose, respectively. Our results show that these two methyltransferase genes determine the structural difference of serotype 12-specific GPL from serotype 7-specific GPL.

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