title: pheS*, an effective host-genotype-independent counter-selectable marker for marker-free chromosome deletion in Bacillus amyloliquefaciens.
abstract: Aside from applications in the production of commercial enzymes and metabolites, Bacillus amyloliquefaciens is also an important group of plant growth-promoting rhizobacteria that supports plant growth and suppresses phytopathogens. A host-genotype-independent counter-selectable marker would enable rapid genetic manipulation and metabolic engineering, accelerating the study of B. amyloliquefaciens and its development as both a microbial cell factory and plant growth-promoting rhizobacteria. Here, a host-genotype-independent counter-selectable marker pheS*was constructed through a point mutation of the gene pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase in Bacillus subtilis strain 168. In the presence of 5 mM p-chloro-phenylalanine, 100 % of B. amyloliquefaciens strain SQR9 cells carrying pheS*were killed, whereas the wild-type strain SQR9 showed resistance to p-chloro-phenylalanine. A simple pheS*and overlap-PCR-based strategy was developed to create the marker-free deletion of the amyE gene as well as a 37-kb bmy cluster in B. amyloliquefaciens SQR9. The effectiveness of pheS*as a counter-selectable marker in B. amyloliquefaciens was further confirmed through the deletion of amyE genes in strains B. amyloliquefaciens FZB42 and NJN-6. In addition, the potential use of pheS*in other Bacillus species was preliminarily assessed. The expression of PheS*in B. subtilis strain 168 and B. cereus strain ATCC 14579 caused pronounced sensitivity of both hosts to p-chloro-phenylalanine, indicating that pheS*could be used as a counter-selectable marker (CSM) in these strains.
authors.affiliation: Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural, Environment of Ministry of Agriculture, Nanjing Agricultural University, 6 Tongwei Road, Nanjing, Jiangsu, 210095, People's Republic of China.
authors.forename: Chaoyang
authors.initials: C
authors.lastname: Zhou
authors.affiliation: Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural, Environment of Ministry of Agriculture, Nanjing Agricultural University, 6 Tongwei Road, Nanjing, Jiangsu, 210095, People's Republic of China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Shi
authors.affiliation: Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural, Environment of Ministry of Agriculture, Nanjing Agricultural University, 6 Tongwei Road, Nanjing, Jiangsu, 210095, People's Republic of China.
authors.forename: Bin
authors.initials: B
authors.lastname: Ye
authors.affiliation: National Engineering Research Center for Organic-based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, 6 Tongwei Road, Nanjing, Jiangsu, 210095, People's Republic of China.
authors.forename: Haichao
authors.initials: H
authors.lastname: Feng
authors.affiliation: Jiangsu Key Laboratory for Chemistry of Low-Dimensional Materials, School of Chemistry and Chemical Engineering, Huaiyin Normal University, Huaian, 223300, People's Republic of China.
authors.forename: Ji
authors.initials: J
authors.lastname: Zhang
authors.affiliation: National Engineering Research Center for Organic-based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, 6 Tongwei Road, Nanjing, Jiangsu, 210095, People's Republic of China.
authors.forename: Ruifu
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural, Environment of Ministry of Agriculture, Nanjing Agricultural University, 6 Tongwei Road, Nanjing, Jiangsu, 210095, People's Republic of China. yanxin@njau.edu.cn.
authors.forename: Xin
authors.initials: X
authors.lastname: Yan
mesh_terms: D000900:Anti-Bacterial Agents / Q000633:toxicity; D000069977:Bacillus amyloliquefaciens / Q000235:genetics* / Q000502:physiology; D001412:Bacillus subtilis / Q000235:genetics / Q000502:physiology; D010134:Fenclonine / Q000633:toxicity; D055786:Gene Knockout Techniques / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D005838:Genotype; D050296:Microbial Viability / Q000187:drug effects; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D012641:Selection, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D050505:Mutant Proteins; D010652:Phenylalanine-tRNA Ligase; D010134:Fenclonine
keywords: B. amyloliquefaciens; Counter-selectable marker; Unmarked genetic manipulation; pheS *