Open AccessMolecular cloning and restriction endonuclease mapping of the glutamate synthase gene from salt‐tolerant Bradyrhizobium spp. strain WR1001
Author(s) -
Hua SuiSheng T,
Lawrence David A
Publication year - 1999
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1999.tb13456.x
Subject(s) - cosmid , ecori , biology , restriction enzyme , microbiology and biotechnology , genomic library , southern blot , plasmid , hindiii , restriction map , molecular cloning , restriction fragment , restriction site , genomic dna , gene , pbr322 , psti , genetics , gene expression , peptide sequence
In order to clone the glutamate synthase gene ( glt ) from salt‐tolerant Bradyrhizobium spp. strain WR1001, a genomic cosmid library was constructed using the vector pHC79. The library was mass‐conjugated into Escherichia coli ET1194 ( glt − ) in a triparental mating, using pRK2013 as a mobilizing plasmid. Transconjugants of ET1194 which grew on minimal medium and had Glt + phenotype were selected for the isolation of recombinant cosmids. Plasmid pHL27, which contained a 40‐kb insert of WR1001 DNA and complemented ET1194 to Glt + phenotype, was chosen to delimit the glutamate synthase gene. The region that encoded glt in cosmid pHL27 was determined by Southern blot analysis using the cloned glt gene of E. coli DH1 as a probe. An 11‐kb Hin dIII DNA fragment from pHL27 contained the fully functional glt gene. Partial Pst I and Eco RI restriction enzyme digests of cosmid pHL27 were subcloned into the vectors pBS + and pUC8 and transformed into ET1194 to test for Glt + phenotype. The glt coding region was defined by aligning the physical maps of the subclones