Douglas E. Rawlings

Mining with Microbes

Microbes are playing increasingly important roles in commercial mining operations, where they are being used in the “bioleaching” of copper, uranium, and gold ores. Direct leaching is when microbial metabolism changes the redox state of the metal being harvested, rendering it more soluble. Indirect leaching includes redox chemistry of other metal cations that are then coupled in chemical oxidation or reduction of the harvested metal ion and microbial attack upon and solubilization of the mineral matrix in which the metal is physically embedded. In addition, bacterial cells are used to detoxify the waste cyanide solution from gold-mining operations and as “absorbants” of the mineral cations. Bacterial cells may replace activated carbon or alternative biomass. With an increasing understanding of microbial physiology, biochemistry and molecular genetics, rational approaches to improving these microbial activities become possible

The poison-antidote stability system of the broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2.

In plasmid pTF‐FC2, three small open reading frames (ORFs) are situated between the repB (primase) gene and the repA (helicase) gene of its IncQ‐type replicon. Disruption of each of the three ORFs followed by tests for plasmid stability and host cell growth indicated that the ORFs encoded a poison–antidote plasmid stability system. The three genes were named pasA, pasB and pasC (plasmid addiction system), in which PasA is the antidote, PasB the toxin and PasC a protein that appears to enhance the ability of the antidote to neutralize the toxin. Disruption of the pasA gene resulted in two different spontaneous deletions, which inactivated the stability system but did not alter the host range or plasmid copy number. This indicated that the three small ORFs were not involved in plasmid replication. When placed behind a tac promoter, induction of pasB was found to be highly lethal to host cells, which suggests that the Pas system acts by killing plasmid‐free host cells rather than by retarding the growth of plasmid‐free segregants, as occurs in the ParD system of R1. In spite of this, the presence of the Pas poison–antidote system resulted in a relatively modest threefold stabilization of the pTF‐FC2 host replicon and a similar increase in the stabilization of an unstable heterologous R1 plasmid replicon. The Pas system is a poison–antidote plasmid stability module, which appears to have become integrated within the pTF‐FC2 replicon module.

Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia coli

The nucleotide sequence of the recA gene of Thiobacillus ferrooxidans has been determined. No SOS box characteristic of LexA-regulated promoters could be identified in the 196-bp region upstream from the coding region. The cloned T. ferrooxidans recA gene was expressed in Escherichia coli from both the lambda pR and lac promoters. It was not expressed from the 2.2-kb of T. ferrooxidans DNA preceding the gene. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and Pseudomonas aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with proteolytic activity have been substituted, the cloned protein has retained protease activity towards the lambda and E. coli LexA repressors.

Nucleotide sequence of the glutamine synthetase gene and its controlling region from the acidophilic autotroph Thiobacillus ferrooxidans

A 2089-bp chromosomal DNA segment containing the Thiobacillus ferrooxidans glnA gene has been sequenced. Putative glnAp1-type promoter sequences, a consensus ntrC-gene-product-binding site and a catabolite-activating protein consensus recognition sequence were detected upstream of the structural gene. The glnA gene was followed by a sequence resembling a Rho-independent termination sequence. The complete amino acid sequence (468 residues) of the glutamine synthetase (GS) has been deduced, and comparisons are made with reported amino acid sequences of GS from other organisms.

Construction of arsenic-resistant Thiobacillus ferrooxidans recombinant plasmids and the expression of autotrophic plasmid genes in a heterotrophic cell-free system

Recombinant plasmids between Thiobacillus ferrooxidans and Escherichia coli which contain arsenic and antibiotic resistance genes and unique restriction sites have been constructed. The expression of autotrophic T. ferrooxidans genes in a heterotrophic E. coli cell-free system was demonstrated.

Identification and cloning of Thiobacillus ferrooxidans structural nif genes in Escherichia coli

The presence of nucleotide sequences which are homologous to the nifHDK genes of Klebsiella pneumoniae was demonstrated in total DNA preparations from five different iron-oxidizing Thiobacillus ferrooxidans strains. A non-iron-oxidizing Thiobacillus novellus strain and a heterotrophic Acidiphilium strain, which occurs in close association with T. ferrooxidans, did not contain DNA homologous to the nifHDK genes. The T. ferrooxidans ATCC33020 nifHDK genes were cloned and their arrangement was characterized on a 6.7-kb insert in pIMP16.

Cloning and sequencing of the gene for the Thiobacillus ferrooxidans ATCC33020 glutamate synthase (GOGAT) small subunit and complementation of an Escherichia coli gltD mutant

A recombinant plasmid which contains the gltD gene coding for the glutamate synthase (GOGAT) small subunit was isolated from a Thiobacillus ferrooxidans ATCC33020 gene bank by complementation of an Escherichia coli gltD mutant. The sequence of gltD was determined. The deduced amino acid sequence shows strong similarity to the two other prokaryote gltD sequences available, namely those of E. coli and A. brasilense (53% and 45% identity, respectively). A cosmid containing the gltBD region was isolated from a T. ferrooxidans cosmid gene bank, but was unable to complement an E. coli gltB mutant.

Sequence and structural analysis of the α- and β-dinitrogenase subunits of Thiobacillus ferrooxidans

Abstract The structural genes ( nifD and nifK ) for the α andβ subunits of the molybdenum-iron (MoFe) protein of the Thiobacillus ferrooxidans dinitrogenase have been sequenced. The M r values deduced from the nucleotide sequences are 54919 and 57901 for the α andβ subunits, respectively. The amino acid sequences of both subunits were quantitatively compared with the equivalent subunits from other bacteria. Distinct areas of amino acid homology were found between the α andβ subunits of T. ferrooxidans .

Regulation of mobilization of the broad‐host‐range plasmid pTF‐FC2

The mobilization region of the broad‐host‐range plasmid pTF‐FC2 consists of an oriT and five genes arranged in two operons that are divergently transcribed from the oriT. The transcriptional starts of both operons were identified and the quantity of transcript from the mobC‐mobE promoter (PI) was at least 10‐fold greater than that from the mobA‐mobB promoter (P2). A translational fusion between the first protein of each operon and a lacZ reporter gene was constructed and used to demonstrate that mob gene expression was autoregulated. Analysis of the oriT resulted in the detection of a putative integration host factor (IHF)‐binding site and an intrinsically bent region. In the absence of IHF, the mobilization frequency and expression from P1 were reduced. The presence of ssi sites on both strands within the oriT region was demonstrated by using an M13 phage mutant, defective in its mechanism for priming DNA replication. Initiation of DNA synthesis at the oriT did not require a plasmid‐encoded primase.