Intergeneric conjugation between Escherichia coli and Streptomyces species

Mazodier, Philippe; Petter, R; Thompson, Charles J.

doi:10.1128/jb.171.6.3583-3585.1989

Cited by 287 publications

(180 citation statements)

References 14 publications

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“…Escherichia coli strains used in plasmid transformations or for phage propagation were grown as described by Sambrook et al (1989). The DNA-methylation-deficient E. coli strain ET12567(pUZ8002) used in conjugal transfer of plasmids from E. coli to S. venezuelae was grown on LB agar supplemented as required with ampicillin (Ap; 50 mg ml 21 ), Cm (25 mg ml 21 ), Am (50 mg ml 21 ), Ts (25 mg ml 21 ) and kanamycin (Km, 50 mg ml 21 ) for specific selections (Mazodier et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

2004

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Five ORFs were detected in a fragment from the Streptomyces venezuelae ISP5230 genomic DNA library by hybridization with a PCR product amplified from primers representing a consensus of known halogenase sequences. Sequencing and functional analyses demonstrated that ORFs 11 and 12 (but not ORFs 13-15) extended the partially characterized gene cluster for chloramphenicol (Cm) biosynthesis in the chromosome. Disruption of ORF11 (cmlK) or ORF12 (cmlS) and conjugal transfer of the insertionally inactivated genes to S. venezuelae gave mutant strains VS1111 and VS1112, each producing a similar series of Cm analogues in which unhalogenated acyl groups replaced the dichloroacetyl substituent of Cm. 1 H-NMR established that the principal metabolite in the disrupted strains was the a-N-propionyl analogue. The sequence of CmlK implicated the protein in adenylation, and involvement in halogenation was inferred from biosynthesis of analogues by the cmlK-disrupted mutant. A role in generating the dichloroacetyl substituent was supported by partial restoration of Cm biosynthesis when a cloned copy of cmlK was introduced in trans into VS1111. Complementation of the mutant also indicated that inactivation of cmlK rather than a polar effect of the disruption on cmlS expression had interfered with dichloroacetyl biosynthesis. The deduced CmlS sequence resembled sequences of FADH 2 -dependent halogenases. Conjugal transfer of cmlK or cmlS into S. venezuelae cml-2, a chlorination-deficient strain with a mutation mapped genetically to the Cm biosynthesis gene cluster, did not complement the cml-2 lesion, suggesting that one or more genes in addition to cmlK and cmlS is needed to assemble the dichloroacetyl substituent. Insertional inactivation of ORF13 did not affect Cm production, and the products of ORF14 and ORF15 matched Streptomyces coelicolor A3(2) proteins lacking plausible functions in Cm biosynthesis. Thus cmlS appears to mark the downstream end of the gene cluster.

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Section: Methodsmentioning

confidence: 99%

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

2004

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“…The resultant plasmid (pLST9734) was initially propagated in E. coli NM522, before being introduced into E. coli S17-1, the donor strain used for conjugal transfer to Streptomyces spp. (modified from Bierman et al, 1992;Mazodier et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

Stimulation of polyketide metabolism in Streptomyces fradiae by tylosin and its glycosylated precursors

Fish¹,

Cundliffe

1997

Microbiology

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Three glycosyltransferases are involved in tylosin biosynthesis in Streptomyces fradiae. The first sugar to be added to the polyketide aglycone (tylactone) is mycaminose and the gene encoding mycaminosyltransferase is oH2* (tyhW2). However, targeted disruption of od2* did not lead to the accumulation of tylactone under conditions that normally favour tylosin production; instead, the synthesis of tylactone was virtually abolished. This may, in part, have resulted from a polar effect on the expression of genes downstream of oH2*, particularly od4* (ccr) which encodes crotonyl-CoA reductase, an enzyme that supplies 4-carbon extender units for polyketide metabolism. However, that cannot be the entire explanation, since tylosin production was restored a t about 10% of the wild-type level when oH2* was re-introduced into the disrupted strain. When glycosylated precursors of tylosin were fed to the disrupted strain, they were converted to tylosin, confirming that two of the three glycosyltransferase activities associated with tylosin biosynthesis were still intact. Interestingly, however, tylactone also accumulated under such conditions and, to a much lesser extent, when tylosin was added to similar fermentations. It is concluded that glycosylated macrolides exert a pronounced positive effect on polyketide metabolism in S. fradiae. ;ity OfKeywords : tylosin production, glycosyltransferase, polyketide metabolism, Streptomyces fradiae INTRODUCTIONThe macrolide antibiotic, tylosin (Fig. l ) , is produced by Streptomyces fradiae via a combination of polyketide and 6-deoxyhexose metabolism. Glycosylation of tylactone, the cyclized polyketide product, always begins with the addition of mycaminose followed, in a preferred but not obligatory order, by deoxyallose and then mycarose to generate demethyl-macrocin. Stepwise bis 0-methylation then converts the deoxyallose moiety to mycinose as macrocin is produced and converted to tylosin (Baltz et al., 1983). Characterization of the tylosin biosynthetic pathway (Fig. 2) was facilitated by the availability of non-producing mutants of S. fradiae, generated using NTG (Baltz & Seno, 1981). Collectively, these exhibited nine distinct phenotypes in cross-feeding Abbreviations: OMT, 0-mycaminosyl-tylonolide; DMT, demycinosyltyl osi n. experiments and allowed 13 genetic loci (tylA-M) to be mapped (Fig. 3) following complementation studies using cloned fragments of tyl DNA (Beckmann et al.,

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“…The recombinant DNA may be introduced into K. setae by an E. coli/ Streptomyces conjugation using an IncP-plasmid (RK2) origin transfer. 34,35 However, we found that the transformation of K. setae KM-6054 was improved by electroporation (approximately 10-to 100-fold higher than the conjugation with E. coli and IncP-plasmid). K. setae generated submerged spores in the liquid culture and DNA was introduced into the submerged spores by electroporation.…”

Section: Characterization Of Bafilomycin Biosynthesismentioning

confidence: 83%

Characterization of bafilomycin biosynthesis in Kitasatospora setae KM-6054 and comparative analysis of gene clusters in Actinomycetales microorganisms

et al. 2017

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(setamycin) produced by Kitasatospora setae KM-6054 belong to the plecomacrolide family, which exhibit antibacterial, antifungal, antineoplastic and immunosuppressive activities. An analysis of gene clusters from K. setae KM-6054 governing the biosynthesis of bafilomycins revealed that it contains five large open reading frames (ORFs) encoding the multifunctional polypeptides of bafilomycin polyketide synthases (PKSs). These clustered PKS genes, which are responsible for bafilomycin biosynthesis, together encode 11 homologous sets of enzyme activities, each catalyzing a specific round of polyketide chain elongation. The region contains an additional 13 ORFs spanning a distance of 73 287 bp, some of which encode polypeptides governing other key steps in bafilomycin biosynthesis. Five ORFs, BfmB, BfmC, BfmD, BfmE and BfmF, were involved in the formation of methoxymalonyl-acyl carrier protein (ACP). Two possible regulatory genes, bfmR and bfmH, were found downstream of the above genes. A gene-knockout analysis revealed that BfmR was only a transcriptional regulator for the transcription of bafilomycin biosynthetic genes. Two genes, bfmI and bfmJ, were found downstream of bfmH. An analysis of these gene-disruption mutants in addition to an enzymatic analysis of BfmI and BfmJ revealed that BfmJ activated fumarate and BfmI functioned as a catalyst to form a fumaryl ester at the C21 hydroxyl residue of bafilomycin A 1 . A comparative analysis of bafilomycin gene clusters in K. setae KM-6054, Streptomyces lohii JCM 14114 and Streptomyces griseus DSM 2608 revealed that each ORF of both gene clusters in two Streptomyces strains were quite similar to each other. However, each ORF of gene cluster in K. setae KM-6054 was of lower similarity to that of corresponding ORF in the two Streptomyces species.

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Intergeneric conjugation between Escherichia coli and Streptomyces species

Cited by 287 publications

References 14 publications

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

Stimulation of polyketide metabolism in Streptomyces fradiae by tylosin and its glycosylated precursors

Characterization of bafilomycin biosynthesis in Kitasatospora setae KM-6054 and comparative analysis of gene clusters in Actinomycetales microorganisms

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