Electrotransformation of Thiobacillus ferrooxidans with plasmids containing a mer determinant

Abstract

The mer operon from a strain of ThiobaciUus ferrooxidans (C. Inoue, K. Sugawara, and T. Kusano, Mol. Microbiol. 5:2707-2718 consists of the regulatory gene merR and an operator-promoter region followed by merC and merA structural genes and differs from other known gram-negative mer operons. We have constructed four potential shuttle plasmids composed of a T. ferrooxidans-borne cryptic plasmid, a pUC18 plasmid, and the above-mentioned mer determinant as a selectable marker. Mercury ion-sensitive T. ferro. oxidans strains were electroporated with constructed plasmids, and one strain, Y4-3 (of 30 independent strains tested), was found to have a transformation efficiency of 120 to 200 mercury-resistant colonies per ,ug of plasmid DNA. This recipient strain was confirmed to be T. ferrooxidans by physiological, morphological, and chemotaxonomical data. The transformants carried a plasmid with no physical rearrangements through 25 passages under no selective pressure. Cell extracts showed mercury ion-dependent NADPH oxidation activity.

IST1 insertional inactivation of the resB gene: implications for phenotypic switching in Thiobacillus ferrooxidans

“…Unfortunately, the genetic manipulation of T. ferrooxidans is still at a rudimentary stage of development. Although techniques for introducing DNA into T. ferrooxidans have been described [19,20] they have not found widespread application. In the absence of such techniques it becomes di¤cult to generate genetic support for models of FeII oxidation and the use of native insertion sequences such as IST1 o¡ers a promising alternative route for the elucidation of not only the FeII oxidizing pathway of T. ferrooxidans but also of other biochemical functions.…”

Development of a Markerless Gene Replacement System for Acidithiobacillus ferrooxidans and Construction of a pfkB Mutant

“…Besides the allelic exchange, other strategies to generate marker-free mutations in A. ferrooxidans had also been considered, such as bacteriophage -Red and Cre-loxP recombination. Due to the low efficiency and poor reproducibility of plasmid transfer by electrotransformation (22) and the obligate chemolithoautotrophic characteristics of A. ferrooxidans, we eventually focused on the I-SceI-based homologous recombination system. Transfers of both the suicide plasmid and the helper plasmid containing the I-SceI gene from E. coli to A. ferrooxidans ATCC 23270 were performed using the wellestablished method of conjugation (28).…”

Construction of arsB and tetH Mutants of the Sulfur-Oxidizing Bacterium Acidithiobacillus caldus by Marker Exchange

“…Nevertheless, plasmid transfer and the expression of heterologous genes have been reported for three species of acidithiobacilli, Acidithiobacillus ferrooxidans (16,19,22,23), Acidithiobacillus thiooxidans (13,30), and A. caldus (15,18,32). In spite of this, there has been only one report of the construction of a null mutant among these bacteria, a recA mutant of A. ferrooxidans (19).…”