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

Abstract

Acidithiobacillus caldus is a moderately thermophilic, acidophilic bacterium that has been reported to be the dominant sulfur oxidizer in stirred-tank processes used to treat gold-bearing arsenopyrite ores. It is also widely distributed in heap reactors used for the extraction of metals from ores. Not only are these bacteria commercially important, they have an interesting physiology, the study of which has been restricted by the nonavailability of defined mutants. A recently reported conjugation system based on the broad-host-range IncW plasmids pSa and R388 was used to transfer mobilizable narrow-host-range suicide plasmid vectors containing inactivated and partially deleted chromosomal genes from Escherichia coli to A. caldus. Through the dual use of a selectable kanamycin resistance gene and a hybridization probe made from a deleted portion of the target chromosomal gene, single-and double-recombinant mutants of A. caldus were isolated. The functionality of the gene inactivation system was shown by the construction of A. caldus arsB and tetH mutants, and the effects of these mutations on cell growth in the presence of arsenic and by means of tetrathionate oxidation were demonstrated.

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

“…The apparent recombination frequency of the mobilizable suicide plasmid pKOT was approximately 10 Ϫ7 ; that is, single-recombination events occurred at a rate of 10 Ϫ7 single recombinants per recipient or 10 Ϫ3 per transconjugant. This is consistent with the results in E. coli that homologous recombination occurs at a frequency of about 10 Ϫ3 to 10 Ϫ4 less than the plasmid transfer frequency (13,41). Although the initial crossover event occurred at a relatively low efficiency, potential single recombinants were generated and screened with little effort because of the availability of the kanamycin-selectable marker that was integrated into the chromosome of the recipient.…”

The Two-Component System RsrS-RsrR Regulates the Tetrathionate Intermediate Pathway for Thiosulfate Oxidation in Acidithiobacillus caldus

“…Gene transfer methods, conjugation, and electroporation, have been developed for A. caldus , and mutants were constructed by a marker replacement knockout method (Liu et al, 2007; Zyl et al, 2008; Chen et al, 2010, 2012). However, previously reported gene knockout methods are extremely difficult and not reproducible.…”

The Use of Extremophilic Microorganisms in the Industrial Recovery of Metals

“…Effective molecular tools for the study of biomining microorganism genetics and physiology are still scarce (Liu et al, 2000(Liu et al, , 2007van Zyl et al, 2008). To achieve efficiency and reproducibility of these procedures is still a rather difficult task.…”