Deciphering Tuberactinomycin Biosynthesis: Isolation, Sequencing, and Annotation of the Viomycin Biosynthetic Gene Cluster

ABSTRACT The tuberactinomycin antibiotics are essential components in the drug arsenal against Mycobacterium tuberculosis infections and are specifically used for the treatment of multidrug-resistant tuberculosis. These antibiotics are also being investigated for their targeting of the catalytic RNAs involved in viral replication and for the treatment of bacterial infections caused by methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant enterococci. We report on the isolation, sequencing, and annotation of the biosynthetic gene cluster for one member of this antibiotic family, viomycin, from Streptomyces sp. strain ATCC 11861. This is the first gene cluster for a member of the tuberactinomycin family of antibiotics sequenced, and the information gained can be extrapolated to all members of this family. The gene cluster covers 36.3 kb of DNA and encodes 20 open reading frames that we propose are involved in the biosynthesis, regulation, export, and activation of viomycin, in addition to self-resistance to the antibiotic. These results enable us to predict the metabolic logic of tuberactinomycin production and begin steps toward the combinatorial biosynthesis of these antibiotics to complement existing chemical modification techniques to produce novel tuberactinomycin derivatives.

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Identification of the Biosynthetic Gene Cluster and an Additional Gene for Resistance to the Antituberculosis Drug Capreomycin

Applied and Environmental Microbiology ◽

10.1128/aem.00485-07 ◽

2007 ◽

Vol 73 (13) ◽

pp. 4162-4170 ◽

Cited By ~ 58

Author(s):

Elizabeth A. Felnagle ◽

Michelle R. Rondon ◽

Andrew D. Berti ◽

Heidi A. Crosby ◽

Michael G. Thomas

Keyword(s):

Gene Cluster ◽

Resistance Gene ◽

Aminoglycoside Antibiotic ◽

Biosynthetic Gene Cluster ◽

Multidrug Resistant ◽

23S Rrna ◽

Peptide Antibiotics ◽

Biosynthetic Gene ◽

Essential Components ◽

Initial Work

ABSTRACT Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.

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Biosynthetic Investigations of Lactonamycin and Lactonamycin Z: Cloning of the Biosynthetic Gene Clusters and Discovery of an Unusual Starter Unit

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00717-07 ◽

2007 ◽

Vol 52 (2) ◽

pp. 574-585 ◽

Cited By ~ 27

Author(s):

Xiujun Zhang ◽

Lawrence B. Alemany ◽

Hans-Peter Fiedler ◽

Michael Goodfellow ◽

Ronald J. Parry

Keyword(s):

Gene Cluster ◽

Genetic Locus ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Open Reading Frames ◽

Antibacterial Drug ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Starter Unit ◽

High Degree

ABSTRACT The antibiotics lactonamycin and lactonamycin Z provide attractive leads for antibacterial drug development. Both antibiotics contain a novel aglycone core called lactonamycinone. To gain insight into lactonamycinone biosynthesis, cloning and precursor incorporation experiments were undertaken. The lactonamycin gene cluster was initially cloned from Streptomyces rishiriensis. Sequencing of ca. 61 kb of S. rishiriensis DNA revealed the presence of 57 open reading frames. These included genes coding for the biosynthesis of l-rhodinose, the sugar found in lactonamycin, and genes similar to those in the tetracenomycin biosynthetic gene cluster. Since lactonamycin production by S. rishiriensis could not be sustained, additional proof for the identity of the S. rishiriensis cluster was obtained by cloning the lactonamycin Z gene cluster from Streptomyces sanglieri. Partial sequencing of the S. sanglieri cluster revealed 15 genes that exhibited a very high degree of similarity to genes within the lactonamycin cluster, as well as an identical organization. Double-crossover disruption of one gene in the S. sanglieri cluster abolished lactonamycin Z production, and production was restored by complementation. These results confirm the identity of the genetic locus cloned from S. sanglieri and indicate that the highly similar locus in S. rishiriensis encodes lactonamycin biosynthetic genes. Precursor incorporation experiments with S. sanglieri revealed that lactonamycinone is biosynthesized in an unusual manner whereby glycine or a glycine derivative serves as a starter unit that is extended by nine acetate units. Analysis of the gene clusters and of the precursor incorporation data suggested a hypothetical scheme for lactonamycinone biosynthesis.

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Biosynthetic engineering of the antifungal, anti-MRSA auroramycin

10.1101/829812 ◽

2019 ◽

Author(s):

Wan Lin Yeo ◽

Elena Heng ◽

Lee Ling Tan ◽

Yi Wee Lim ◽

Kuan Chieh Ching ◽

...

Keyword(s):

Antifungal Activity ◽

Genome Editing ◽

Gene Cluster ◽

Biosynthetic Pathway ◽

Biosynthetic Gene Cluster ◽

Combinatorial Biosynthesis ◽

Biosynthetic Gene ◽

Antifungal Activities ◽

Biosynthetic Engineering

AbstractUsing an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseoporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, it also displays antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Six auroramycin analogs with variations in C-, N-methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.

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Biosynthetic Pathway for Mannopeptimycins, Lipoglycopeptide Antibiotics Active against Drug-Resistant Gram-Positive Pathogens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01545-05 ◽

2006 ◽

Vol 50 (6) ◽

pp. 2167-2177 ◽

Cited By ~ 63

Author(s):

Nathan A. Magarvey ◽

Brad Haltli ◽

Min He ◽

Michael Greenstein ◽

John A. Hucul

Keyword(s):

Amino Acid ◽

Gene Cluster ◽

Biosynthetic Pathway ◽

Domain Analysis ◽

Biosynthetic Gene Cluster ◽

Multidrug Resistant ◽

Drug Resistant ◽

Biosynthetic Gene ◽

Peptide Synthetases ◽

Multidrug Resistant Pathogens

ABSTRACT The mannopeptimycins are a novel class of lipoglycopeptide antibiotics active against multidrug-resistant pathogens with potential as clinically useful antibacterials. This report is the first to describe the biosynthesis of this novel class of mannosylated lipoglycopeptides. Included here are the cloning, sequencing, annotation, and manipulation of the mannopeptimycin biosynthetic gene cluster from Streptomyces hygroscopicus NRRL 30439. Encoded by genes within the mannopeptimycin biosynthetic gene cluster are enzymes responsible for the generation of the hexapeptide core (nonribosomal peptide synthetases [NRPS]) and tailoring reactions (mannosylation, isovalerylation, hydroxylation, and methylation). The NRPS system is noncanonical in that it has six modules utilizing only five amino acid-specific adenylation domains and it lacks a prototypical NRPS macrocyclizing thioesterase domain. Analysis of the mannopeptimycin gene cluster and its engineering has elucidated the mannopeptimycin biosynthetic pathway and provides the framework to make new and improved mannopeptimycins biosynthetically.

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GIP2, a Putative Transcription Factor That Regulates the Aurofusarin Biosynthetic Gene Cluster in Gibberella zeae

Applied and Environmental Microbiology ◽

10.1128/aem.72.2.1645-1652.2006 ◽

2006 ◽

Vol 72 (2) ◽

pp. 1645-1652 ◽

Cited By ~ 55

Author(s):

Jung-Eun Kim ◽

Jianming Jin ◽

Hun Kim ◽

Jin-Cheol Kim ◽

Sung-Hwan Yun ◽

...

Keyword(s):

Gene Cluster ◽

Biosynthetic Gene Cluster ◽

Gibberella Zeae ◽

Open Reading Frames ◽

Biosynthetic Gene ◽

Targeted Gene Deletion ◽

Binuclear Cluster ◽

Putative Transcription Factor ◽

Type Strains ◽

Reading Frames

ABSTRACT Gibberella zeae (anamorph: Fusarium graminearum) is an important pathogen of maize, wheat, and rice. Colonies of G. zeae produce yellow-to-tan mycelia with the white-to-carmine red margins. In this study, we focused on nine putative open reading frames (ORFs) closely linked to PKS12 and GIP1, which are required for aurofusarin biosynthesis in G. zeae. Among them is an ORF designated GIP2 (for Gibberella zeae pigment gene 2), which encodes a putative protein of 398 amino acids that carries a Zn(II)2Cys6 binuclear cluster DNA-binding domain commonly found in transcription factors of yeasts and filamentous fungi. Targeted gene deletion and complementation analyses confirmed that GIP2 is required for aurofusarin biosynthesis. Expression of GIP2 in carrot medium correlated with aurofusarin production by G. zeae and was restricted to vegetative mycelia. Inactivation of the 10 contiguous genes in the ΔGIP2 strain delineates an aurofusarin biosynthetic gene cluster. Overexpression of GIP2 in both the ΔGIP2 and the wild-type strains increases aurofusarin production and reduces mycelial growth. Thus, GIP2 is a putative positive regulator of the aurofusarin biosynthetic gene cluster, and aurofusarin production is negatively correlated with vegetative growth by G. zeae.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.5.1214-1222.2000 ◽

2000 ◽

Vol 44 (5) ◽

pp. 1214-1222 ◽

Cited By ~ 133

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.

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Analysis of Genes Involved in Biosynthesis of Coronafacic Acid, the Polyketide Component of the Phytotoxin Coronatine

Journal of Bacteriology ◽

10.1128/jb.180.13.3330-3338.1998 ◽

1998 ◽

Vol 180 (13) ◽

pp. 3330-3338 ◽

Cited By ~ 24

Author(s):

Vidhya Rangaswamy ◽

Robin Mitchell ◽

Matthias Ullrich ◽

Carol Bender

Keyword(s):

Gene Cluster ◽

Pseudomonas Syringae ◽

Biosynthetic Gene Cluster ◽

Open Reading Frames ◽

Plant Pathogenic Bacterium ◽

Biosynthetic Gene ◽

Future Studies ◽

Coronafacic Acid ◽

Single Transcript ◽

Reading Frames

ABSTRACT Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant-pathogenic bacteriumPseudomonas syringae. The genes involved in CFA biosynthesis are encoded by a single transcript which encompasses 19 kb of the COR gene cluster. In the present study, the nucleotide sequence was determined for a 4-kb region located at the 3′ end of the CFA biosynthetic gene cluster. Three open reading frames were identified and designated cfa8, cfa9, andtnp1; the predicted translation products of these genes showed relatedness to oxidoreductases, thioesterases, and transposases, respectively. The translational products of cfa8 andcfa9 were overproduced in Escherichia coliBL21; however, tnp1 was not translated in these experiments. Mutagenesis and complementation analysis indicated thatcfa8 is required for the production of CFA and COR. Analysis of a cfa9 mutant indicated that this gene is dispensable for CFA and COR production but may increase the release of enzyme-bound products from the COR pathway; tnp1, however, had no obvious function in CFA or COR biosynthesis. A genetic strategy was used to produce CFA in a P. syringae strain which lacks the COR gene cluster; this approach will be useful in future studies designed to investigate biosynthetic products of the CFA gene cluster.

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Identification and Analysis of the Biosynthetic Gene Cluster Encoding the Thiopeptide Antibiotic Cyclothiazomycin in Streptomyces hygroscopicus 10-22

Applied and Environmental Microbiology ◽

10.1128/aem.01790-09 ◽

2010 ◽

Vol 76 (7) ◽

pp. 2335-2344 ◽

Cited By ~ 43

Author(s):

Jiang Wang ◽

Yi Yu ◽

Kexuan Tang ◽

Wen Liu ◽

Xinyi He ◽

...

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Posttranslational Modifications ◽

Biosynthetic Gene Cluster ◽

Open Reading Frames ◽

Streptomyces Hygroscopicus ◽

Biosynthetic Gene ◽

Thiopeptide Antibiotics ◽

Reading Frames ◽

Macrocyclic Structure

ABSTRACT Thiopeptide antibiotics are an important class of natural products resulting from posttranslational modifications of ribosomally synthesized peptides. Cyclothiazomycin is a typical thiopeptide antibiotic that has a unique bridged macrocyclic structure derived from an 18-amino-acid structural peptide. Here we reported cloning, sequencing, and heterologous expression of the cyclothiazomycin biosynthetic gene cluster from Streptomyces hygroscopicus 10-22. Remarkably, successful heterologous expression of a 22.7-kb gene cluster in Streptomyces lividans 1326 suggested that there is a minimum set of 15 open reading frames that includes all of the functional genes required for cyclothiazomycin production. Six genes of these genes, cltBCDEFG flanking the structural gene cltA, were predicted to encode the enzymes required for the main framework of cyclothiazomycin, and two enzymes encoded by a putative operon, cltMN, were hypothesized to participate in the tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure. This rigorous bioinformatics analysis based on heterologous expression of cyclothiazomycin resulted in an ideal biosynthetic model for us to understand the biosynthesis of thiopeptides.

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Gyrase Mutations Are Associated with Variable Levels of Fluoroquinolone Resistance in Mycobacterium tuberculosis

Journal of Clinical Microbiology ◽

10.1128/jcm.02775-15 ◽

2016 ◽

Vol 54 (3) ◽

pp. 727-733 ◽

Cited By ~ 41

Author(s):

Maha R. Farhat ◽

Karen R. Jacobson ◽

Molly F. Franke ◽

Devinder Kaur ◽

Alex Sloutsky ◽

...

Keyword(s):

Multidrug Resistant ◽

Open Reading Frames ◽

Fluoroquinolone Resistance ◽

Test Results ◽

Resistant Tuberculosis ◽

Multidrug Resistant Tuberculosis ◽

Disease Mutations ◽

The Individual ◽

Selection Of

Molecular diagnostics that rapidly and accurately predict resistance to fluoroquinolone drugs and especially later-generation agents promise to improve treatment outcomes for patients with multidrug-resistant tuberculosis and prevent the spread of disease. Mutations in thegyrgenes are known to confer most fluoroquinolone resistance, but knowledge about the effects ofgyrmutations on susceptibility to early- versus later-generation fluoroquinolones and about the role of mutation-mutation interactions is limited. Here, we sequenced the fullgyrAandgyrBopen reading frames in 240 multidrug-resistant and extensively drug-resistant tuberculosis strains and quantified their ofloxacin and moxifloxacin MIC by testing growth at six concentrations for each drug. We constructed a multivariate regression model to assess both the individual mutation effects and interactions on the drug MICs. We found thatgyrBmutations contribute to fluoroquinolone resistance both individually and through interactions withgyrAmutations. These effects were statistically significant. In these clinical isolates, severalgyrAandgyrBmutations conferred different levels of resistance to ofloxacin and moxifloxacin. Consideration ofgyrmutation combinations during the interpretation of molecular test results may improve the accuracy of predicting the fluoroquinolone resistance phenotype. Further, the differential effects ofgyrmutations on the activity of early- and later-generation fluoroquinolones requires further investigation and could inform the selection of a fluoroquinolone for treatment.

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Biosynthetic Gene Cluster for the Polyenoyltetramic Acid α-Lipomycin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00007-06 ◽

2006 ◽

Vol 50 (6) ◽

pp. 2113-2121 ◽

Cited By ~ 54

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.

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