title: A new recombineering system for Photorhabdus and Xenorhabdus.
abstract: Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed 'recombineering', in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5'-3' exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced recombineering efficiency. Results from bioinformatic analysis and recombineering assays suggest that Plu2934 may be functionally equivalent to Redγ, which inhibits the major endogenous E. coli nuclease, RecBCD. The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.
authors.affiliation: Hunan Provincial Key Laboratory for Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, College of Life Science, Hunan Normal University, 410081 Changsha, People's Republic of China Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China Department of Genomics, Dresden University of Technology, BioInnovations-Zentrum, Tatzberg 47-51, 01307 Dresden, Germany Helmholtz Institute for Pharmaceutical Research, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO Box 151150, 66041 Saarbrücken, Germany.
authors.forename: Jia
authors.initials: J
authors.lastname: Yin
authors.affiliation: Department of Protein Evolution, Max-Planck Institute for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Hunan Provincial Key Laboratory for Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, College of Life Science, Hunan Normal University, 410081 Changsha, People's Republic of China.
authors.forename: Liqiu
authors.initials: L
authors.lastname: Xia
authors.affiliation: Hunan Provincial Key Laboratory for Microbial Molecular Biology-State Key Laboratory Breeding Base of Microbial Molecular Biology, College of Life Science, Hunan Normal University, 410081 Changsha, People's Republic of China.
authors.forename: Xuezhi
authors.initials: X
authors.lastname: Ding
authors.affiliation: Helmholtz Institute for Pharmaceutical Research, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO Box 151150, 66041 Saarbrücken, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hoffmann
authors.affiliation: Helmholtz Institute for Pharmaceutical Research, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO Box 151150, 66041 Saarbrücken, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hoffmann
authors.affiliation: Helmholtz Institute for Pharmaceutical Research, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO Box 151150, 66041 Saarbrücken, Germany.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Bian
authors.affiliation: Helmholtz Institute for Pharmaceutical Research, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO Box 151150, 66041 Saarbrücken, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Müller
authors.affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China Department of Genomics, Dresden University of Technology, BioInnovations-Zentrum, Tatzberg 47-51, 01307 Dresden, Germany.
authors.forename: Jun
authors.initials: J
authors.lastname: Fu
authors.affiliation: Department of Genomics, Dresden University of Technology, BioInnovations-Zentrum, Tatzberg 47-51, 01307 Dresden, Germany.
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
authors.affiliation: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China zhangyouming@sdu.edu.cn.
authors.forename: Youming
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D043211:Exodeoxyribonuclease V / Q000037:antagonists & inhibitors; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D023281:Genomics; D008969:Molecular Sequence Data; D005810:Multigene Family; D009876:Operon; D020637:Photorhabdus / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D020593:Xenorhabdus / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D043211:Exodeoxyribonuclease V