Construction of a shuttle vector based on the small cryptic plasmid pJY33 from Weissella cibaria 33

• Published in: Plasmid Vol. 79; pp. 30 - 36
• Main Authors: Park, Ji Yeong, Jeong, Seon-Ju, Sa, Hyun Deok, Lee, Jae Yong, Liu, Xiaoming, Cho, Min Jeong, Lee, Kang Wook, Kim, Jeong Hwan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2015
• Subjects: Base Composition; DNA Replication; DNA, Bacterial - genetics; Erythromycin - pharmacology; Escherichia coli - genetics; Genetic Vectors; Lactobacillus brevis; Lactobacillus plantarum; Lactobacillus plantarum - genetics; Lactococcus lactis; Lactococcus lactis - genetics; Leuconostoc - genetics; Leuconostoc citreum; Leuconostoc mesenteroides; Open Reading Frames; pJY33E; Plasmids - genetics; Rolling circle replication; Sequence Analysis, DNA; Shuttle vector; Weissella; Weissella - genetics; Weissella cibaria; Weissella confusa; Weissella cibaria; Shuttle vector; pJY33E; Rolling circle replication
• ISSN: 0147-619X; 1095-9890
• DOI: 10.1016/j.plasmid.2015.03.008

•A cryptic plasmid, pJY33 (2365 bp), was isolated from Weissella cibaria 33.•pJY33 was used for the construction of a LAB-Escherichia coli shuttle vector, pJY33E.•pJY33E replicates in Lactococcus, Lactobacillus, Leuconostoc, and Weissella.•pJY33 replicates via RCR and aga gene was expressed by using pJY33E. A cryptic plasmid, pJY33, from Weissella cibaria 33 was characterized. pJY33 was 2365 bp in size with a GC content of 41.27% and contained two putative open reading frames (ORFs). orf1 encoded a putative hypothetical protein of 134 amino acids. orf2 was 849 bp in size, and its putative translation product exhibited 87% identity with a replication initiation factor from a plasmid from W. cibaria KLC140. A Weissella-Escherichia coli shuttle vector, pJY33E (6.5 kb, Emr), was constructed by ligation of pJY33 with pBluescript II SK(−) and an erythromycin resistance gene (Emr). pJY33E replicated in Lactococcus lactis, Leuconostoc citreum, Lactobacillus brevis, Lactobacillus plantarum, and Weissella confusa. A single-stranded DNA intermediate was detected from Lb. brevis 2.14 harbouring pJY33E, providing evidence for rolling-circle replication of pJY33. Most Lb. brevis 2.14 cells (85.9%) retained pJY33E after one week of daily culturing in MRS broth without Em. An aga gene encoding α-galactosidase (α-Gal) from Leuconostoc mesenteroides was successfully expressed in Lb. brevis 2.14 using pJY33E, and the highest level of α-Gal activity (36.13 U/mg protein) was observed when cells were grown on melibiose.