A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica
JournalArticle (Originalarbeit in einer wissenschaftlichen Zeitschrift)
 
ID 156258
Author(s) Kaniga, K; Delor, I; Cornelis, G R
Author(s) at UniBasel Cornelis, Guy R.
Year 1991
Title A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica
Journal Gene
Volume 109
Number 1
Pages / Article-Number 137-41
Keywords RECOMBINANT DNA, LUXAB, DOMINANT STREPTOMYCIN-RESISTANCE, AMPICILLIN-RESISTANCE, NONPOLAR MUTATIONS, SACB
Abstract A new suicide vector (pKNG101) that facilitates the positive selection of double recombination events in Gram-bacteria has been developed. It contains a conditional origin of replication (oriR6K), the strAB genes encoding the streptomycin phosphotransferase (SmR), an origin of transfer (mobRK2), the sacB gene mediating sucrose sensitivity, and multiple cloning sites. It was used to mutate the blaA gene of Yersinia enterocolitica, by marker-exchange mutagenesis. To do this, we have first cloned into the suicide vector pKNG101, a 2.5-kb fragment of Y. enterocolitica chromosomal DNA encoding the 20-kDa beta-lactamase A. Gene blaA was then mutated in vitro by insertion of luxAB, which resulted in pKNG105. The disrupted blaA gene was then reintroduced into Y. enterocolitica chromosome by homologous recombinations in two steps. First, E. coli SM10 lambda pir (pKNG105) was mated with strains of Y. enterocolitica. This led to the integration of pKNG105 into the chromosome, by a single homologous recombination event. The transconjugants, selected for SmR, were sensitive to sucrose due to the synthesis of levans (toxic compounds), catalysed by levansucrase, the product of sacB. For the second step, a single colony from the first step was grown in rich medium deprived of antibiotic, allowing the occurrence of a second crossing-over that replaced the wild-type allele blaA with the mutant one, and then excised the plasmid-borne sacB from the chromosome. Such blaA mutants were selected on their ability to grow on TSA medium containing 5% sucrose.(ABSTRACT TRUNCATED AT 250 WORDS)
Publisher Elsevier/North-Holland
ISSN/ISBN 0378-1119
edoc-URL http://edoc.unibas.ch/dok/A5259243
Full Text on edoc No
Digital Object Identifier DOI 10.1016/0378-1119(91)90599-7
PubMed ID http://www.ncbi.nlm.nih.gov/pubmed/1756974
ISI-Number WOS:A1991GX09900018
Document type (ISI) Journal Article
 
   

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