Development of an intergeneric conjugal transfer system for rimocidin-producing<i>Streptomyces rimosus</i>

Phornphisutthimas, Somkiat; Sudtachat, N.; Bunyoo, Chakrit; Chotewutmontri, P.; Panijpan, Bhinyo; Thamchaipenet, Arinthip

doi:10.1111/j.1472-765x.2010.02835.x

Cited by 19 publications

(16 citation statements)

References 31 publications

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“…3 and Table S3 in the supplemental material); this prevents cluster-calling errors, which are likely when most reads begin with an identical sequence derived from within the transposon phyla, including the Firmicutes (26) and Actinobacteria (27,28). In addition to the introduction of the genes for the transposon delivery, however, consideration must be given to the ability of the strain to express the transposase and the selectable marker.…”

Section: Resultsmentioning

confidence: 99%

Rapid Transposon Liquid Enrichment Sequencing (TnLE-seq) for Gene Fitness Evaluation in Underdeveloped Bacterial Systems

Fels

Zane

Blake

et al. 2013

Appl Environ Microbiol

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cWhole-genome fitness analysis in microbes that uses saturating transposon mutagenesis combined with massively parallel sequencing (Tn-seq) is providing a measure of the contribution of each gene to a given growth condition. With this technique, gene fitness profiles and essential genes are discovered by simultaneous analyses of whether the absence of each gene product alters the growth kinetics of the bacterium. Here we modify the standard Tn-seq procedure to simplify and shorten the process by including delivery of the transposon through conjugation and liquid culture enrichment of the mutant pool, creating transposon liquid enrichment sequencing (TnLE-seq). To illustrate the success of these modifications and the robustness of the procedure, analyses of gene fitness of two cultures of the strictly anaerobic bacterium Desulfovibrio vulgaris Hildenborough were performed, with growth on lactate as the electron donor and sulfate as the electron acceptor. These data demonstrate reproducibility and provide a base condition for analysis of fitness changes in deletion mutants and in various growth conditions. The procedural modifications will facilitate the application of this powerful genetic analysis to microbes lacking a facile genetic system. Pilot studies produced 2.5 ؋ 10 5 and 3.4 ؋ 10 5 unique insertion mutants in the anaerobe Desulfovibrio vulgaris Hildenborough grown under typical laboratory conditions in rich medium. These analyses provided two similar high-resolution maps of gene fitness across the genome, and the method was also applied to growth in minimal medium. These results were also compared to the coverage obtained with a ca. 13,000-member cataloged transposon library constructed by sequencing transposon insertion sites in individual mutants.

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Section: Resultsmentioning

confidence: 99%

Rapid Transposon Liquid Enrichment Sequencing (TnLE-seq) for Gene Fitness Evaluation in Underdeveloped Bacterial Systems

Fels

Zane

Blake

et al. 2013

Appl Environ Microbiol

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“…Theoretically, the described procedure provides a promising approach for the ultimate site-specific homologous recombination between this cassette and the bacterial chromosome to generate targeted gene knockouts. However, in practice, it does not work properly for every Streptomyces species, and further optimization is often required to successfully conjugate E. coli with refractory species (62)(63)(64)(65). Various species-dependent parameters which strongly affected the transformation efficiency were identified.…”

Section: Discussionmentioning

confidence: 99%

“…strain C5 conjugation efficiencies of 1.5 ϫ 10 Ϫ4 per recipient cell or 1.1 ϫ 10 Ϫ5 exconjugants per recipient spore, respectively, were calculated (67). Phornphisutthimas et al (62) obtained high efficiencies of conjugation (10 Ϫ2 to 10 Ϫ3 ) for S. rimosus with a procedure based on heat treatment of the spores at 40°C for 10 min prior to their being mixed with E. coli ET12567(pUZ8002/pIJ8600). In that study, tryptic soy agar medium containing 10 mmol/liter MgCl 2 was the preferred medium for conjugation.…”

Section: Discussionmentioning

confidence: 99%

An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

Netzker

Schroeckh

Gregory

et al. 2016

Appl Environ Microbiol

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Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces rapamycinicus. Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca 2؉ ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate. IMPORTANCE The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species. Since the discovery of streptomycin in 1943 (1), streptomycetes have been shown to produce thousands of compounds with possibly beneficial features, e.g., antibiotics, immunosuppressants, or anticancer drugs. Actinomycete-derived metabolites comprise over two-thirds of all known antibiotic compounds (2), and recent genome sequencing programs revealed that their biosynthesis potential has been underestimated. In their 8-to 12-Mb genomes, approximately 20 to 30 gene clusters encode the biosynthesis of secondary metabolites (3-5). One of the most important Streptomyces-derived compounds is the mechanistic target of rapamycin (mTOR) inhibitor rapamycin (sirolimus), a "billion dollar molecule" and, after cyclosporine, the most widely used immunosuppressant of microbial origin (6). Rapamycin was previously reported as an antifungal antibiotic produced by Streptomyces hygroscopicus ATCC 29253 (7), later renamed Streptomyces rapamycinicus (8). The rapamycin gene cluster in this strain has been sequenced (9), the biosynthetic pathway has been extensively characterized (10-14), and engineering of the cluster has yielded an impressive range of bioactive modified rapamycins (rapalogs) (15, 16), some in multigram amounts. So far, two other rapamycin-producing species are known: the taxonomically closely related Streptomyces iranensis HM 35 (5, 17) and Actinoplanes sp. strain N902-109 (18). To elucidate the molecular biology of rapamycin formation in these strains and to further exploit the physiological and pharmacological capability of rapamycin derivatives, genetic manipulation of these altern...

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“…Streptomyces are soil-dwelling Gram-positive bacteria, and produce various bioactive molecules including half of microbial origin antibiotics employed in the field of medicine and agriculture [1,2]. Peptidyl nucleoside antibiotics are a group of secondary metabolites with similar chemical structures, which possess impressive antitumoral, antiviral, antibacterial, and antifungal activities [3,4].…”

Section: Introductionmentioning

confidence: 99%

“…Intergeneric conjugation between Escherichia coli and Streptomyces which was firstly reported in 1989 [19], was proved to be a reliable method. Especially the employment of methylation-deficient E. coli as donor and the construction of various E. coli shuttle vectors [20] improved this method, so that it was used widely in the Streptomyces [1,21,22,23,24,25,26]. Although transformation systems for several Streptomyces strains have been developed since 1989 [1,21,22,23,24,25,26], there is still no universal protocol applicable to all Streptomyces strains.…”

Section: Introductionmentioning

confidence: 99%

Development of an Intergeneric Conjugal Transfer System for Xinaomycins-Producing Streptomyces noursei Xinao-4

Sun

Luo

Shu

et al. 2014

IJMS

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To introduce DNA into Streptomyces noursei xinao-4, which produces xinaomycins, we explored an intergeneric conjugal transfer system. High efficiency of conjugation (8 × 10−3 exconjugants per recipient) was obtained when spores of S. noursei xinao-4 were heat-shocked at 50 °C for 10 min, mixed with Escherichia coli ET12567 (pUZ8002/pSET152) in the ratio of 1:100, plated on 2CMY medium containing 40 mmol/L MgCl2, and incubated at 30 °C for 22 h. With this protocol, the plasmids pKC1139 and pSET152 were successfully transferred from E. coli ET12567 (pUZ8002) with different frequencies. Among all parameters, the ratio of donor to recipient cell number had the strongest effect on the transformation efficiency. In order to validate the above intergeneric conjugal transfer system, a glycosyltransferase gene was cloned and efficiently knocked out in S. noursei xinao-4 using pSG5-based plasmid pKC1139.

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Development of an intergeneric conjugal transfer system for rimocidin-producingStreptomyces rimosus

Cited by 19 publications

References 31 publications

Rapid Transposon Liquid Enrichment Sequencing (TnLE-seq) for Gene Fitness Evaluation in Underdeveloped Bacterial Systems

Rapid Transposon Liquid Enrichment Sequencing (TnLE-seq) for Gene Fitness Evaluation in Underdeveloped Bacterial Systems

An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

Development of an Intergeneric Conjugal Transfer System for Xinaomycins-Producing Streptomyces noursei Xinao-4

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