1887

Abstract

Given the lack of functional transposons for use in spp., an alternative method of insertional mutagenesis using natural transformation was developed. High efficiencies of transformation were only obtained with species-specific DNA. This feature was a key element in the construction of mutant libraries of this bacterium. A chromosomal library of 81116 DNA was made in shuttle vector pUOA18. Next, a kanamycin-resistance (Km) cassette was ligated into the inserts of the plasmids. 81116 was then transformed with the resulting products to allow homologous recombination between genomic fragments present in the shuttle vector and the chromosome. Transformants were pooled and chromosomal DNA from these transformants was used to retransform 81116. This resulted in transformants containing the Km cassette in the chromosome but lacking the vector. In order to evaluate this approach for the construction of a mutant bank, the Km insertional mutants were screened for loss of motility. Partial characterization of 11 non-motile mutants indicated that the inserted genes are involved in motility. Four mutants had the Km cassette inserted in genes involved in flagella biosynthesis, namely and , and produced incomplete or no flagella. Four mutants had the Km cassette inserted in genes possibly involved in flagella motor function: and downstream of the gene. Three mutants had the Km cassette inserted in genes that are homologous to genes encoding hypothetical proteins of

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/content/journal/micro/10.1099/13500872-145-8-2145
1999-08-01
2019-11-15
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http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-145-8-2145
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