The use of acidiphilic, chemolithotrophic iron- and sulfur-oxidizing microbes in processes to recover metals from certain types of copper, uranium, and gold-bearing minerals or mineral concentrates is now well established. During these processes insoluble metal sulfides are oxidized to soluble metal sulfates. Mineral decomposition is believed to be mostly due to chemical attack by ferric iron, with the main role of the microorganisms being to reoxidize the resultant ferrous iron back to ferric iron. Currently operating industrial biomining processes have used bacteria that grow optimally from ambient to 50 degrees C, but thermophilic microbes have been isolated that have the potential to enable mineral biooxidation to be carried out at temperatures of 80 degrees C or higher. The development of higher-temperature processes will extend the variety of minerals that can be commercially processed.

Biomining, the use of micro-organisms to recover precious and base metals from mineral ores and concentrates, has developed into a successful and expanding area of biotechnology. While careful considerations are made in the design and engineering of biomining operations, microbiological aspects have been subjected to far less scrutiny and control. Biomining processes employ microbial consortia that are dominated by acidophilic, autotrophic iron-or sulfur-oxidizing prokaryotes. Mineral biooxidation takes place in highly aerated, continuous-flow, stirred-tank reactors or in irrigated dump or heap reactors, both of which provide an open, non-sterile environment. Continuous-flow, stirred tanks are characterized by homogeneous and constant growth conditions where the selection is for rapid growth, and consequently tank consortia tend to be dominated by two or three species of micro-organisms. In contrast, heap reactors provide highly heterogeneous growth environments that change with the age of the heap, and these tend to be colonized by a much greater variety of micro-organisms. Heap micro-organisms grow as biofilms that are not subject to washout and the major challenge is to provide sufficient biodiversity for optimum performance throughout the life of a heap. This review discusses theoretical and pragmatic aspects of assembling microbial consortia to process different mineral ores and concentrates, and the challenges for using constructed consortia in non-sterile industrial-scale operations.

Microbiology (1 999), 145, 5-1 3 Printed in Great Britain REVIEW ARTICLEReasons why ' Leptospirillum '-like species rather than Thiobacillus ferrooxidans are the dominant iron-oxidizing bacteria in many commercial processes for the biooxidation of pyrite and related ores

Iron-oxidizing bacteria belonging to the genus Leptospirillum are of great importance in continuous-flow commercial biooxidation reactors, used for extracting metals from minerals, that operate at 40°C or less. They also form part of the microbial community responsible for the generation of acid mine drainage. More than 16 isolates of leptospirilla were included in this study, and they were clearly divisible into two major groups. Group I leptospirilla had G؉C moles percent ratios within the range 49 to 52% and had three copies of rrn genes, and based on 16S rRNA sequence data, these isolates clustered together with the Leptospirillum ferrooxidans type strain (DSM2705 or L15). Group II leptospirilla had G؉C moles percent ratios of 55 to 58% and had two copies of rrn genes, and based on 16S rRNA sequence data, they form a separate cluster. Genome DNA-DNA hybridization experiments indicated that three similarity subgroups were present among the leptospirilla tested, with two DNA-DNA hybridization similarity subgroups found within group I. The two groups could also be distinguished based on the sizes of their 16S-23S rRNA gene spacer regions. We propose that the group II leptospirilla should be recognized as a separate species with the name Leptospirillum ferriphilum sp. nov. Members of the two species can be rapidly distinguished from each other by amplification of their 16S rRNA genes and by carrying out restriction enzyme digests of the products. Several, but not all, isolates of the group II leptospirilla, but none from group I (L. ferrooxidans), were capable of growth at 45°C. All the leptospirilla isolated from commercial biooxidation tanks in South Africa were from group II.

Acidithiobacillus ferrooxidans has an arsenic resistance operon that is controlled by an As(III)-responsive transcriptional repressor, AfArsR, a member of the ArsR/SmtB family of metalloregulators. AfArsR lacks the As(III) binding site of the ArsRs from plasmid R773 and Escherichia coli, which have a Cys 32 -Val-Cys 34 -Asp-Leu-Cys 37 sequence in the DNA binding site. In contrast, it has three cysteine residues, Cys 95 , Cys 96 , and Cys 102 , that are not present in the R773 and E. coli ArsRs. The results of direct As(III) binding measurements and x-ray absorption spectroscopy show that these three cysteine residues form a 3-coordinate As(III) binding site. DNA binding studies indicate that binding of As(III) to these cysteine residues produces derepression. Homology modeling indicates that As(III) binding sites in AfArsR are located at the ends of antiparallel C-terminal helices in each monomer that form a dimerization domain. These results suggest that the As(III)-S 3 binding sites in AfArsR and R773 ArsR arose independently at spatially distinct locations in their three-dimensional structures.The metalloid arsenic is a ubiquitous environmental toxin that is ranked first on Superfund List of Hazardous Substances (www.atsdr.cdc.gov/cercla/05list.html). Nearly every organism has genes for arsenic detoxification (1). In bacteria, arsenic resistance is most frequently conferred by chromosomal or plasmid-encoded ars operons that are controlled by members of the ArsR/SmtB family of transcriptional repressors (2, 3). The well characterized plasmid R773 and Escherichia coli chromosomal ArsRs are homodimers that have a Cys 32 -Val-Cys 34 -Asp-Leu-Cys 37 sequence at the start of the helix-loop-helix DNA binding site (4) (Fig. 1). The three sulfur thiolates of the cysteine residues form an S 3 binding site for As(III), and binding of metalloid is presumed to cause a conformational change in the repressor leading to dissociation from the DNA and hence derepression (5). This site is congruent with the 4-coordinate (S 4 ) Cd(II) binding site of the CadC repressor (6), and we have termed these Type 1 metal binding site. CadC also has a C-terminal non-regulatory Zn(II) binding site that is formed by Asp 101 and His 103 from one monomer and His 114 Ј and Glu 117 Ј from the other monomer at the dimerization domain. This site corresponds to the Zn(II) regulatory site of SmtB, and we have termed these Type 2 metal binding sites. The Type 1 and Type 2 sites have also been called ␣3N and ␣5 sites (7), but these designations are imprecise because they were based on an incomplete structure of SmtB.Recently Butcher et al. (8) identified an ars operon in Acidithiobacillus ferrooxidans in which arsRC genes are transcribed divergently from arsBH genes. They termed the ArsR homologue "atypical" because it does not contain the CXC(X) 2 C motif in the DNA binding site (9) (Fig. 1). Deletion of the last 19 amino acid residues had no effect on arsenic regulation, but deletion of an additional 28 residues resulted in loss of regulation from ...

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Plasmids belonging to Escherichia coli incompatibility group Q are relatively small (approximately 5 to 15 kb) and able to replicate in a remarkably broad range of bacterial hosts. These include gram-positive bacteria such as Brevibacterium and Mycobacterium and gram-negative bacteria such as Agrobacterium, Desulfovibrio, and cyanobacteria. These plasmids are mobilized by several self-transmissible plasmids into an even more diverse range of organisms including yeasts, plants, and animal cells. IncQ plasmids are thus highly promiscuous. Recently, several IncQ-like plasmids have been isolated from bacteria found in environments as diverse as piggery manure and highly acidic commercial mineral biooxidation plants. These IncQ-like plasmids belong to different incompatibility groups but have similar broad-host-range replicons and mobilization properties to the IncQ plasmids. This review covers the ecology, classification, and evolution of IncQ and IncQ-like plasmids

The chromosomal arsenic resistance genes of the acidophilic, chemolithoautotrophic, biomining bacterium Thiobacillus ferrooxidans were cloned and sequenced. Homologues of four arsenic resistance genes, arsB, arsC, arsH, and a putative arsR gene, were identified. The T. ferrooxidans arsB (arsenite export) and arsC (arsenate reductase) gene products were functional when they were cloned in an Escherichia coli ars deletion mutant and conferred increased resistance to arsenite, arsenate, and antimony. Therefore, despite the fact that the ars genes originated from an obligately acidophilic bacterium, they were functional in E. coli. Although T. ferrooxidans is gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functional trxA (thioredoxin) gene was required for ArsC-mediated arsenate resistance in E. coli; this finding confirmed the gram-positive ArsC-like status of this resistance and indicated that the division of ArsC molecules based on Gram staining results is artificial. Although arsH was expressed in an E. coli-derived in vitro transcription-translation system, ArsH was not required for and did not enhance arsenic resistance in E. coli. The T. ferrooxidans ars genes were arranged in an unusual manner, and the putative arsR and arsC genes and the arsBH genes were translated in opposite directions. This divergent orientation was conserved in the four T. ferrooxidans strains investigated.Thiobacillus ferrooxidans is an acidophilic (optimum pH, 1.8 to 2.5), obligately chemolithotrophic bacterium that obtains its energy through oxidation of ferrous iron to ferric iron or oxidation of reduced inorganic sulfur compounds to sulfuric acid. It is a member of a consortium of bacteria (which includes Thiobacillus caldus and Leptospirillum ferrooxidans) that is used in commercial biooxidation processes to recover gold from arsenopyrite ores (22). Although recent analysis of microbial populations in continuous-flow biooxidation tanks has revealed that T. ferrooxidans may not be as dominant as was once thought, this organism is nevertheless usually present in such tanks (21). Total arsenic levels greater than 13 g liter Ϫ1 may be present in arsenopyrite biooxidation tanks, and therefore the microorganisms present must have a mechanism of resistance to arsenic (8).Plasmid-associated arsenic efflux resistance mechanisms have been known for many years and have been extensively reviewed (5,23,(30)(31)(32)35). Although the number of components of these systems varies, in the case of Escherichia coli plasmids R773 and R46, as well as Acidiphilium multivorum plasmid pKW301 (34), as many as five genes (arsRDABC) are present. In the case of R773, the genes are transcribed in a single operon. The arsR and arsD genes encode repressors that control the basal and upper levels of ars operon expression, while the arsABC genes encode the structural components of the arsenic resistance mechanism. ArsA is an ATPase which forms a complex with ArsB, the transmembrane arsenite efflux p...