Affiliation:
1. University of Missouri, Columbia, Missouri
2. Virtual Institute of Microbial Stress and Survival, Berkeley, California
Abstract
ABSTRACT
In recent years, the genetic manipulation of the sulfate-reducing bacterium
Desulfovibrio vulgaris
Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in
D. vulgaris
. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by
upp
. In wild-type
D. vulgaris
, growth was shown to be inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU), whereas a mutant bearing a deletion of the
upp
gene was resistant to 5-FU. When a plasmid containing the wild-type
upp
gene expressed constitutively from the
aph
(3′)-II promoter (promoter for the kanamycin resistance gene in Tn
5
) was introduced into the
upp
deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this in-frame deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (
hsdR
, DVU1703) of a type I restriction-modification system that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1,000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
76 articles.
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