λ Red-Mediated Genetic Manipulation of Antibiotic-Producing Streptomyces

Gust, Bertolt; Chandra, Govind; Jakimowicz, Dagmara; Tian, Yun; Bruton, Celia J.; Chater, Keith F.

doi:10.1016/s0065-2164(04)54004-2

Cited by 244 publications

(251 citation statements)

References 59 publications

Supporting

Mentioning

249

Contrasting

Order By: Relevance

“…E. coli strains were grown and manipulated by standard methods 40, 41, 42. For generating spore stocks, S. venezuelae and S. coelicolor were grown on MYM‐TAP agar43 and mannitol soya flour (SFM) agar,44 respectively.…”

Section: Methodsmentioning

confidence: 99%

Discovery of Unusual Biaryl Polyketides by Activation of a SilentStreptomyces venezuelaeBiosynthetic Gene Cluster

et al. 2016

Self Cite

View full text Add to dashboard Cite

Comparative transcriptional profiling of a ΔbldM mutant of Streptomyces venezuelae with its unmodified progenitor revealed that the expression of a cryptic biosynthetic gene cluster containing both type I and type III polyketide synthase genes is activated in the mutant. The 29.5 kb gene cluster, which was predicted to encode an unusual biaryl metabolite, which we named venemycin, and potentially halogenated derivatives, contains 16 genes including one—vemR—that encodes a transcriptional activator of the large ATP‐binding LuxR‐like (LAL) family. Constitutive expression of vemR in the ΔbldM mutant led to the production of sufficient venemycin for structural characterisation, confirming its unusual biaryl structure. Co‐expression of the venemycin biosynthetic gene cluster and vemR in the heterologous host Streptomyces coelicolor also resulted in venemycin production. Although the gene cluster encodes two halogenases and a flavin reductase, constitutive expression of all three genes led to the accumulation only of a monohalogenated venemycin derivative, both in the native producer and the heterologous host. A competition experiment in which equimolar quantities of sodium chloride and sodium bromide were fed to the venemycin‐producing strains resulted in the preferential incorporation of bromine, thus suggesting that bromide is the preferred substrate for one or both halogenases.

show abstract

Section: Methodsmentioning

confidence: 99%

Discovery of Unusual Biaryl Polyketides by Activation of a SilentStreptomyces venezuelaeBiosynthetic Gene Cluster

et al. 2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…Fusion genes expressing SsgB-eGFP or SsgB-mCherry were introduced into the genome by gene replacement, so as to replace the wild-type ssgB. For this, we cloned egfp directly behind the coding region of ssgB in the vector pIJ786, and gene replacement was carried out using the Redirect method developed by Gust et al (2004) (for details, see Gust et al 2004 and references therein). An mCherry variant of pIJ786 was constructed by replacing the gene for eGFP with that for mCherry; for this, we amplified the gene for mCherry with oligonucleotides pIJ786-mCherry-Fw and pIJ786-mCherry-Rev and inserted the fragment into pIJ786 as an NdeI-HindIII fragment so as to replace egfp, thus generating pGWS510.…”

Section: Bacterial Strains and Constructsmentioning

confidence: 99%

“…Subsequently, SsgB-786Fw and SsgB-786Rev were used to amplify the entire insert of either pIJ786 or pGWS510, generating DNA fragments encompassing a cassette of either egfp or mcherry followed by the apramycin resistance cassette aacC4, which is inserted between the last seven codons of ssgB and the first 20 nucleotides (nt) of the ssgB downstream region. Using highefficiency recombination achieved in a l red background (Gust et al 2004 and references therein) derivatives of cosmid L2, which contains an ;40-kb section of the S. coelicolor chromosome including ssgB, were obtained with either egfp or mcherry fused immediately behind ssgB. Using apramycin resistance for selection of the desired recombinants, the wild-type ssgB gene was replaced with the respective fusion genes according to routine methods.…”

Section: Bacterial Strains and Constructsmentioning

confidence: 99%

Positive control of cell division: FtsZ is recruited by SsgB during sporulation ofStreptomyces

Willemse¹,

Borst²,

Waal³

et al. 2011

Genes Dev.

171

201

View full text Add to dashboard Cite

In bacteria that divide by binary fission, cell division starts with the polymerization of the tubulin homolog FtsZ at mid-cell to form a cell division scaffold (the Z ring), followed by recruitment of the other divisome components. The current view of bacterial cell division control starts from the principle of negative checkpoints that prevent incorrect Z-ring positioning. Here we provide evidence of positive control of cell division during sporulation of Streptomyces, via the direct recruitment of FtsZ by the membrane-associated divisome component SsgB. In vitro studies demonstrated that SsgB promotes the polymerization of FtsZ. The interactions are shown in vivo by time-lapse imaging and Fö rster resonance energy transfer and fluorescence lifetime imaging microscopy (FRET-FLIM), and are corroborated independently via two-hybrid studies. As determined by fluorescence recovery after photobleaching (FRAP), the turnover of FtsZ protofilaments increased strongly at the time of Z-ring formation. The surprising positive control of Z-ring formation by SsgB implies the evolution of an entirely new way of Z-ring control, which may be explained by the absence of a mid-cell reference point in the long multinucleoid hyphae. In turn, the localization of SsgB is mediated through the orthologous SsgA, and premature expression of the latter is sufficient to directly activate multiple Z-ring formation and hyperdivision at early stages of the Streptomyces cell cycle.

show abstract

“…For this purpose, the betalactamase (bla) gene on the backbone of 31C2 was replaced with an integration cassette of pIJ787 (23,44) containing the attP attachment site and the integrase gene (int) of phage ⌽C31, a tetracycline resistance gene (tet) and an origin of transfer (oriT) using -Red recombination. The generated cosmid cpzLK09 was introduced into S. coelicolor M512 by polyethylene glycolmediated protoplast transformation (20) and three kanamycin ϩ for CPZ A and B at Rt ϭ 24.37 min).…”

Section: Identification and Cloning Of The Caprazamycin Gene Cluster-mentioning

confidence: 99%

Identification and Manipulation of the Caprazamycin Gene Cluster Lead to New Simplified Liponucleoside Antibiotics and Give Insights into the Biosynthetic Pathway

Kaysser

Lutsch

Siebenberg

et al. 2009

Journal of Biological Chemistry

Self Cite

127

View full text Add to dashboard Cite

λ Red-Mediated Genetic Manipulation of Antibiotic-Producing Streptomyces

Cited by 244 publications

References 59 publications

Discovery of Unusual Biaryl Polyketides by Activation of a SilentStreptomyces venezuelaeBiosynthetic Gene Cluster

Discovery of Unusual Biaryl Polyketides by Activation of a SilentStreptomyces venezuelaeBiosynthetic Gene Cluster

Positive control of cell division: FtsZ is recruited by SsgB during sporulation ofStreptomyces

Identification and Manipulation of the Caprazamycin Gene Cluster Lead to New Simplified Liponucleoside Antibiotics and Give Insights into the Biosynthetic Pathway

Contact Info

Product

Resources

About