Development of a Markerless Genetic Exchange System in Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency Page: 2 of 36
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1 ABSTRACT:
2 In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio
3 vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current
4 marker exchange deletion method does not allow for easy selection of multiple sequential gene
5 deletions in a single strain because of the limited number of selectable markers available in D.
6 vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless
7 deletion system has been developed. The counterselectable marker that makes this deletion
8 system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by
9 upp. In wild-type D. vulgaris, growth was shown to be inhibited by the toxic pyrimidine analog
10 5-fluorouracil (5-FU); whereas, a mutant bearing a deletion of the upp gene was resistant to 5-
11 FU. When a plasmid containing the wild-type upp gene expressed constitutively from the
12 aph(3')-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the
13 upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a
14 two-step integration and excision strategy for the deletion of genes of interest. Since this in-
15 frame deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated
16 in a single strain without the accumulation of genes conferring antibiotic resistances. We used
17 this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I
18 restriction-modification system, that we designated JW7035. The transformation efficiency of
19 the JW7035 strain was found to be 100 to 1000 times greater than that of the wild-type strain
20 when stable plasmids were introduced via electroporation.2
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Keller, Kimberly L.; Bender, Kelly S. & Wall, Judy D. Development of a Markerless Genetic Exchange System in Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency, article, July 21, 2009; Berkeley, California. (https://digital.library.unt.edu/ark:/67531/metadc831543/m1/2/: accessed March 28, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.