title: [Development of a genetic modification system for caerulomycin producer Actinoalloteichus sp. WH1-2216-6].
abstract: OBJECTIVE
  In order to enable the caerulomyicn biosynthetic study by in vivo gene disruptions, it is crucial to develop a genetic modification system for the producer Actinoalloteichus sp. WH1-2216-6.
  
  
  METHODS
  The spore germination timing and the concentration of MgSO4 in the medium were investigated for the optimal conjugal transfer of exotic pSET152 DNA into Actinoalloteichus sp. WH1-2216-6. Using the PCR-targeting system, we disrupted a putative caerulomycin 2,3-dihydroxybenzoate-AMP ligase gene by "in-frame deletion" in E. coli, to afford the cosmid pCSG2104, which was then transferred into Actinoalloteichus sp. WH1-2216-6 by conjugation under optimized conditions.
  
  
  RESULTS
  The putative caerulomycin 2,3-dihydroxybenzoate-AMP ligase in Actinoalloteichus sp. WH1-2216-6 was successfully disrupted by in-frame replacement with the aac3IV gene cassette. The resulting mutant strain was unable to produce caerulomycins.
  
  
  CONCLUSIONS
  The presence of high concentration of MgSO4 in the medium can promote the conjugation efficiency between E. coli and Actinoalloteichus sp. WH1-2216-6 and lead to the successful development of a genetic modification system for Actinoalloteichus sp. WH1-2216-6, enabling the functional characterization of caerulomycin biosynthetic genes in vivo. A positive example was provided for other Actinobacteria recalcitrant to genetic modification.
authors.affiliation: Key Laboratory of Marine Bio-resources Sustainable Utilization, RNAM Center for Marine Microbiology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China. linqinheng@163.com
authors.forename: Qinheng
authors.initials: Q
authors.lastname: Lin
authors.forename: Guangtao
authors.initials: G
authors.lastname: Zhang
authors.forename: Sumei
authors.initials: S
authors.lastname: Li
authors.forename: Haibo
authors.initials: H
authors.lastname: Zhang
authors.forename: Jianhua
authors.initials: J
authors.lastname: Ju
authors.forename: Weiming
authors.initials: W
authors.lastname: Zhu
authors.forename: Changsheng
authors.initials: C
authors.lastname: Zhang
mesh_terms: D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000378:metabolism; D000192:Actinomycetales / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D014871:Water Microbiology
chemical_list: D000900:Anti-Bacterial Agents; C043637:cerulomycin; D015082:2,2'-Dipyridyl