High frequency transformation of the Amphotericin-producing bacterium Streptomyces nodosus

Nikodinovic, Jasmina; Barrow, Kevin D.; Chuck, Jo-Anne

doi:10.1016/s0167-7012(03)00160-x

Cited by 18 publications

(17 citation statements)

References 22 publications

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“…When the number of the donor cells was up to 10 9 , the conjugation efficiency reached the highest efficiency of (2.3 ± 0.24) × 10 −3 , which is 34 times greater than that obtained with 10 7 donor cells. In contrast with the results of Streptomyces coelicolor [21] and Streptomyces nodosus [25], where the donor-to-recipient ratio did not affect the conjugal efficiency, our results indicated that the maximum efficiency was received with a certain donor-to-recipient ratio of 100:1. Similar results had been observed in Kitasatospora setae [32], Streptomyces lincolnensis [24], and Streptomyces diastatochromogenes 1628 [31], Streptomyces natalensis [33] in which a certain ratio of donor to recipient cell number was required to achieve highest frequency.…”

Section: Resultscontrasting

confidence: 99%

“…To select the appropriate medium for intergeneric conjugation, five agar media (MS, ISP4, 2CMY, YMS and TSA) containing 10 mmol/L MgCl 2 were assessed as conjugative medium. Among them, MS, ISP4 and 2CMY has been used as conjugal medium for several Streptomyces [1,21,25,26,27,28]; TSA is usually applied in the growth of Streptomyces species [1,22]; and YMS is the suitable medium for the growth and spore formation of S. noursei xinao-4. S. noursei xinao-4 grow and sporulate fast within 7 days on YMS agar at 28 °C.…”

Section: Resultsmentioning

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%

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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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Section: Resultscontrasting

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…Recent progress in genetic manipulation of polyene-producing streptomycetes indicates that a wider range of analogues can be produced by engineered biosynthesis. The biosynthetic gene clusters have been sequenced for nystatin, pimaricin, amphotericin, candicidin/FR008 and rimocidin, and methods have been developed for manipulation of the producing organisms (Aparicio et al, 2003;Chen et al, 2003;Seco et al, 2004;Nikodinovic et al, 2003). These advances have led to the construction of the first genetically modified strains that produce novel polyene analogues (Aparicio et al, 2003;Brautaset et al, 2002;Byrne et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques

Carmody

Byrne

Murphy

et al. 2004

Gene

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Amphotericin B is a medically important antifungal antibiotic that is produced by Streptomyces nodosus. Genetic manipulation of this organism has led to production of the first amphotericin analogues by engineered biosynthesis. Here, these studies were extended by sequencing the chromosomal regions flanking the amphotericin polyketide synthase genes, and by refining the phage KC515 transduction method for disruption and replacement of S. nodosus genes. A hybrid vector was constructed from KC515 DNA and the Escherichia coli plasmid pACYC177. This vector replicated as a plasmid in E. coli and the purified DNA yielded phage plaques on Streptomyces lividans after polyethylene glycol (PEG)-mediated transfection of protoplasts. The left flank of the amphotericin gene cluster was found to include amphRI, RII, RIII and RIV genes that are similar to regulatory genes in other polyene biosynthetic gene clusters. One of these regulatory genes, amphRI, was found to have a homologue, amphRVI, located in the right flank at a distance of 127 kbp along the chromosome. However, disruption of amphRVI using the hybrid vector had no effect on the yield of amphotericin obtained from cultures grown on production medium. The hybrid vector was also used for precise deletion of the DNA coding for two modules of the AmphC polyketide synthase protein. Analysis by UV spectrophotometry revealed that the deletion mutant produced a novel pentaene, with reduced antifungal activity but apparently greater water-solubility than amphotericin B. This shows the potential for use of the new vector in engineering of this and other biosynthetic pathways in Streptomyces.

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“…ciency [17], exconjugant frequency [18], transconjugation frequency [19], or transfer frequency [20]. We have used the efficiency of conjugation here to mean the number of transconjugants per donor cell.…”

Section: Figmentioning

confidence: 99%

Conjugation in Escherichia coli

Phornphisutthimas¹,

Thamchaipenet²,

Panijpan³

2007

Biochem Molecular Bio Educ

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Bacterial conjugation is a genetic transfer that involves cell-to-cell between donor and recipient cells. With the current method used to teach students in genetic courses at the undergraduate level, the transconjugants are identified using bacterial physiology and/or antibiotic resistance. Using physiology, however, is difficult for both first-year undergraduates and special science students at the high school levels, who do not have the basic knowledge. We have developed a laboratory exercise that comprises a simple and rapid technique for transferring bacterial DNA by conjugation and examining the transconjuants using only antibiotic resistance on agar. The identity of the right transconjuants is confirmed by agarose gel electrophoresis. This exercise is designed to help students understand how horizontal gene transfer occurs in bacteria by conjugation using Escherichia coli as a hands-on learning model. Students should be able to draw concept maps of three DNA transfer methods on their own after carrying out the experiment and getting some additional information.

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High frequency transformation of the Amphotericin-producing bacterium Streptomyces nodosus

Cited by 18 publications

References 22 publications

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

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

Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques

Conjugation in Escherichia coli

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