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BackgroundAcidithiobacillus caldus is a sulfur oxidizing extreme acidophile and the only known mesothermophile within the Acidithiobacillales. As such, it is one of the preferred microbes for mineral bioprocessing at moderately high temperatures. In this study, we explore the genomic diversity of A. caldus strains using a combination of bioinformatic and experimental techniques, thus contributing first insights into the elucidation of the species pangenome.Principal findingsComparative sequence analysis of A. caldus ATCC 51756 and SM-1 indicate that, despite sharing a conserved and highly syntenic genomic core, both strains have unique gene complements encompassing nearly 20% of their respective genomes. The differential gene complement of each strain is distributed between the chromosomal compartment, one megaplasmid and a variable number of smaller plasmids, and is directly associated to a diverse pool of mobile genetic elements (MGE). These include integrative conjugative and mobilizable elements, genomic islands and insertion sequences. Some of the accessory functions associated to these MGEs have been linked previously to the flexible gene pool in microorganisms inhabiting completely different econiches. Yet, others had not been unambiguously mapped to the flexible gene pool prior to this report and clearly reflect strain-specific adaption to local environmental conditions.SignificanceFor many years, and because of DNA instability at low pH and recurrent failure to genetically transform acidophilic bacteria, gene transfer in acidic environments was considered negligible. Findings presented herein imply that a more or less conserved pool of actively excising MGEs occurs in the A. caldus population and point to a greater frequency of gene exchange in this econiche than previously recognized. Also, the data suggest that these elements endow the species with capacities to withstand the diverse abiotic and biotic stresses of natural environments, in particular those associated with its extreme econiche.

Plasmids pRAS3.1 and pRAS3.2 are natural variants of the IncQ-2 plasmid family, that except for two differences, have identical plasmid backbones. Plasmid pRAS3.1 has four 22-bp iterons in its oriV region, while pRAS3.2 has only three 6-bp repeats and pRAS3.1 has five 6-bp repeats in the promoter region of the mobB-mobA/repB genes and pRAS3.2 has only four. In previous work, we showed that the overall effect of these differences was that when the plasmid was in an Escherichia coli host, the copy numbers of pRAS3.1 and pRAS3.2 were approximately 41 and 30, respectively. As pRAS3.1 and pRAS3.2 are likely to have arisen from the same ancestor, we addressed the question of whether one of the variants had an evolutionary advantage over the other. By constructing a set of identical plasmids with the number of 22-bp iterons varying from three to seven, it was found that plasmids with four or five iterons displaced plasmids with three iterons even though they had lower copy numbers. Furthermore, the metabolic load that the plasmids placed on E. coli host cells compared with plasmid-free cells increased with copy number from 10.9% at a copy number of 59 to 2.6% at a copy number of 15. Plasmid pRAS3.1 with four 22-bp iterons was able to displace pRAS3.2 with three iterons when both were coresident in the same host. However, the lower-copy-number pRAS3.2 placed 2.8% less of a metabolic burden on an E. coli host population, and therefore, pRAS3.2 has a competitive advantage over pRAS3.1 at the population level, as pRAS3.2-containing cells would be expected to outgrow pRAS3.1-containing cells. doi: 10.1128/JB.00176-10

Plasmids pRAS3.1 and pRAS3.2 are two closely related, natural variants of the IncQ-2 plasmid family that have identical plasmid backbones except for two differences. Plasmid pRAS3.1 has five 6-bp repeat sequences in the promoter region of the mobB gene and four 22-bp iterons in its oriV region, whereas pRAS3.2 has only four 6-bp repeats and three 22-bp iterons. Plasmid pRAS3.1 was found to have a higher copy number than pRAS3.2, and we show that the extra 6-bp repeat results in an increase in mobB and downstream mobA/repB expression. Placement of repB (primase) behind an arabinose-inducible promoter in trans resulted in an increase in repB expression and an approximately twofold increase in the copy number of plasmids with identical numbers of 22-bp iterons. The pRAS3 plasmids were shown to have a previously unrecognized toxin-antitoxin plasmid stability module within their replicons. The ability of the pRAS3 plasmids to mobilize the oriT regions of two other plasmids of the IncQ-2 family, pTF-FC2 and pTC-F14, suggested that the mobilization proteins pRAS3 are relaxed and can mobilize oriT regions with substantially different sequences. Plasmids pRAS3.1 and pRAS3.2 were highly incompatible with plasmids pTF-FC2 and pTC-F14, and this incompatibility was removed on inactivation of an open reading frame situated downstream of the mobCDE mobilization genes rather than being due to the 22-bp oriV-associated iterons. We propose that the pRAS3 plasmids represent a third, [gamma] incompatibility group within the IncQ-2 family plasmids. doi: 10.1128/JB.00864-09

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.

All strains of the moderately thermophilic, acidophilic, sulphur-oxidizing bacterium Acidithiobacillus caldus that have been tested contain a set of chromosomal arsenic resistance genes. Highly arsenic-resistant strains isolated from commercial arsenopyrite bio-oxidation tanks contain additional transposon-located (TnAtcArs) arsenic resistance genes. The chromosomal At. caldus ars genes were cloned and found to consist of arsR and arsC genes transcribed in one direction, and arsB in the opposite direction. The arsRC genes were co-transcribed with ORF1, and arsB with ORF5 in both At. caldus and Escherichia coil, although deletion of ORFs 1 and 5 did not appear to affect resistance to arsenate or arsenite in E. coli. ORFs 1 and 5 have not previously been reported as part of the ars operons, and had high amino acid identity to hypothetical proteins from Polaromonas naphthalenivorus (76 %) and Legionella pneumophila (60 %), respectively. Reporter-gene studies showed that the arsenic operon of transposon origin (TnAtcArs) was expressed at a higher level, and was less tightly regulated in E. coli than were the At. caldus ars genes of chromosomal origin. Plasmid pSa-mediated conjugal transfer of TnAtcArs from E. coli to At. caldus strains lacking the transposon was successful, and resulted in greatly increased levels of resistance to arsenite. DOI: 10.1099/mic.0.29247-0

Studies were carried out to investigate genetic changes that had taken place when two sets of arsenic resistance genes were isolated from highly arsenic-resistant Leptospirillum ferriphilum Fairview strain. TnLfArs and TnAtcArs were sufficiently different for them not being transferred from one type of bacterium to the other in the biooxidation tank.

A transposon, TnAtcArs, that carries a set of arsenic-resistance genes was isolated from a strain of the moderately thermophilic, sulfur-oxidizing, biomining bacterium Acidithiobacillus caldus. This strain originated from a commercial plant used for the bio-oxidation of gold-bearing arsenopyrite concentrates. Continuous selection for arsenic resistance over many years had made the bacterium resistant to high concentrations of arsenic. Sequence analysis indicated that TnAtcArs is 12 444 bp in length and has 40 bp terminal inverted repeat sequences and divergently transcribed resolvase and transposase genes that are related to the Tn21-transposon subfamily. A series of genes consisting of arsR, two tandem copies of arsA and arsD, two ORFs (7 and 8) and arsB is situated between the resolvase and transposase genes. Although some commercial strains of At. caldus contained the arsDA duplication, when transformed into Escherichia coli, the arsDA duplication was unstable and was frequently lost during cultivation or if a plasmid containing TnAtcArs was conjugated into a recipient strain. TnAtcArs conferred resistance to arsenite and arsenate upon E. coil cells. Deletion of one copy of arsDA had no noticeable effect on resistance to arsenite or arsenate in E. coil. ORFs 7 and 8 had clear sequence similarity to an NADH oxidase and a CBS-domain-containing protein, respectively, but their deletion did not affect resistance to arsenite or arsenate in E. coli. TnAtcArs was actively transposed in E. coli, but no increase in transposition frequency in the presence of arsenic was detected. Northern hybridization and reporter gene studies indicated that although ArsR regulated the 10 kb operon containing the arsenic-resistance genes in response to arsenic, ArsR had no effect on the regulation of genes associated with transposition activity.

Two closely related but compatible plasmids of the IncQ-2[alpha] and IncQ-2[beta] groups pTF-FC2 and pTF-F14, were discovered in two acidiphilic chemolithotrophic bacteria. Cross-complementation and cross-regulation experiments by the replication proteins were carried out to discover what changes were necessary when the plasmids evolved to produce two incompatibility groups. The requirement of a pTC-F14 oriV for a RepC DNA-binding protein was plasmid specific, whereas the requirement for the RepA helicase and RepB primase was less specific and could be complemented by the IncQ-2[alpha] plasmid pTC-FC2, and the IncQ-1 [beta] plasmid plE1108. None of the IncQ-1[alpha] plasmid replication proteins could complement the pTC-F14 oriV, and pTC-F14 and RSF1010 were incompatible. This incompatibility was associated with the RepC replication protein and was not due to iteron incompatibility. Replication of pTC-F14 took place from a 5.7 kb transcript that originated upstream of the mobB gene located within the region required for mobilization. A pTC-F14 mobB-lacZ fusion was regulated by the pTC-F14 repB gene product and was plasmid specific, as it was not regulated by the RepB proteins of pTF-FC2 or the IncQ-1[alpha] and IncQ-1[beta] plasmids. Plasmid pTC-F14 appears to have evolved independently functioning iterons and a plasmid-specific RepC-binding protein; it also has a major replication transcript that is independently regulated from that of pTF-FC2. However, the RepA and RepB proteins have the ability to function with either replicon.

Plasmid pTC-F14 contains a plasmid stability system called pas (plasmid addiction system), which consists of two proteins, a PasA antitoxin and a PasB toxin. This system is closely related to the pas of plasmid pTF-FC2 (81 and 72% amino acid identity for PasA and PasB, respectively) except that the pas of pTF-FC2 contains a third protein, PasC. As both pTC-F14 and pTF-FC2 are highly promiscuous broad-host-range plasmids isolated from bacteria that share a similar ecological niche, the plasmids are likely to encounter each other. We investigated the relative efficiencies of the two stability systems and whether they had evolved apart sufficiently for each pas to stabilize a plasmid in the presence of the other. The three-component pTF-FC2pas was more efficient at stabilization of a heterologous tester plasmid than the two component pas of pTC-F14 in Escherichia coli host cells ([+ or -] 92% and [+ or -] 60% after 100 generations, respectively). The PasA antidote of each pas was unable to neutralize the PasB toxin of the other plasmid. The pas proteins of each plasmid autoregulated their own expression as well as that of the pas of the other plasmid. The pas of pTF-FC2 was more effective at repressing the pas operon of pTC-F14 than the pas of pTC-F14 was able to repress itself or the pas of pTF-FC2. This increased efficiency was not due to the PasC of pTF-FC2. The effect of this stronger repression was that pTF-FC2 displaced pTC-F14 when the two plasmids were coresident in the same E. coli host cell. Plasmid curing resulted in the arrest of cell growth but did not cause cell death, and plasmid stability was not influenced by the E. coli mazEF genes.

Plasmid pTC-F14 is a 14.2-kb plasmid isolated from Acidithiobacius caldus that has a replicon that is closely related to the promiscuous, broad-host-range IncQ family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins that were related to those of the DNA processing (Dtr or Tral) region of IncP plasmids rather than to the three-Mob-protein system of the IncQ group 1 plasmids (e.g., plasmid RSF1010 or R1162). Plasmid pTC-F14 is the second example of an IncQ family plasmid that has five mob genes, the other being pTF-FC2. The minimal region that was essential for mobilization included the mobA, mobB, and mobC genes, as well as the oriT gene. The mobD and mobE genes were nonessential, but together, they enhanced the mobilization frequency by approximately 300-fold. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3,500-fold less efficient than the mobilization of pTF-FC2. When both plasmids were coresident in the same E. coli host, pTC-F14 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-F14 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. Mob protein interaction at the oriT regions was unidirectionally plasmid specific in that a plasmid with the oriT region of pTC-F14 could be mobilized by pTF-FC2 but not vice versa. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.

Research has been conducted on the iron-oxidizing bacteria which belong to Leptospirillum genus and form microbial community responsible for the acid mine drainage generation. The diversity of the Leptospirillum isolates from different geographical locations has been investigated via different molecular techniques and the results indicate that two distinct Leptospirillum species are presented among these isolates.

Abstract 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 [...]

Escherichia coli incompatibility group Q (IncQ) and IncQ-like plasmids occur in a variety of bacteria and environment. Results suggest that the backbones of IncQ plasmids consist of G+C content to an extent of 60%, while those of IncQ-like plasmid backbones exhibit a mobilization region and a plasmid replication maintenance region.

Research reveals that the sulfur-oxidizing, chemolithotrophic, obligately acidophilic, and thermophilic bacterium Acidithiobacillus caldus exhibit a 14-kilodalton plasmid replicon, pTC-F14, which is related to IncQ plasmid replicon of Escherichia coli. Sequence analysis of the pTC-F14 reveals five open reading frames with a replicon like organization similar to IncQ.

Chromosomal arsenic resistance genes of Thiobacillus ferrooxidans are discussed. They have an unusual divergent arsCRBH operon strucure and give increased antimony and arsenic resistance to Escherichia coli. These genes were cloned and sequenced.

The downstream portion of the Thiobacillus ferrooxidans was studied to determine whether gene order is conserved in the region. A Tn7-like transposon, Tn5468, was discovered in the glmUS region of the bacterium. Results of the Southern blot hybridization revealed that this transposon is closely related to the Tn7 transposition gene. The research also led to the discovery of Tn7-like terminal sequences and open reading frames associated with the Tn7 transposition genes tnsA, tnsB, tnsC and tnsD.

A research on the plasmids of bioleaching bacteria Thiobacillus ferrooxidans was conducted to identify the electron transport gene it possesses that stimulate metronidazole. DNA extraction and sequencing were the methodology used. Plasmids were segregated using metronidazole, wherein plasmid pTF5, a gene expressing to have redox-active proteins, was isolated from the process. Other findings indicated that pTF5 has a segment of DNA which is also existing on the chromosome. Furthermore, the study provided that pTF5 plasmids are geographically segregated and bear ferredoxin-, prismane-, and NADH- oxidoreductase-like proteins.

The Tn5467 transposon present in the Thiobacillus ferrooxidans pTF-FC2 plasmid has two 38-bp inverted repeat sequences and open reading frames similar to a glutaredoxin-like protein, a MeR regulatory protein, and a multidrug-resistant membrane transport-like protein. One invert sequence is identical to and the other has a single-base pair mismatch with those of the Tn21 transposase. The transposase and resolvase genes of Tn5467 are biologically inactive. The Tn5467 gene encoding the glutaredoxin-like protein expressed in Escherichia coli complements E. coli thioredoxin mutants.

The Thiobacillus ferrooxidans thioredoxin gene, trxA, was isolated by its ability to complement an Escherichia coli gshA trxA mutant which was otherwise unable to grow on minimal medium lacking glutathione. The T. ferrooxidans thioredoxin also enabled the in vivo reduction by E. coli of methionine sulfoxide to methionine, as well as the in vitro reduction of insulin. When present in E. coli, the T. ferrooxidans thioredoxin supported the replication of phage T7, but not the growth of phage M13. The T. ferrooxidans trxA gene was sequenced and the thioredoxin was found to be most like that of E. coli (71 % identity) and Chromatium vinosum (70% identity). As in the case of E. coli, the gene was located immediately upstream of the gene for the rho transcriptional terminator. DNA:RNA blot hybridization and primer-extension analysis of the trxA gene in T. ferrooxidans and the cloned gene in E. coli indicated that it was transcribed as an independent unit and that the major transcriptional start sites were the same in both organisms.

ntrBC genes are isolated from the Thiobacillus ferrooxidans gene library by activation of the expression of the T. ferrooxidans nifH-lacZ gene fusion in an agar plating technique. The presence of cloned T. ferrooxidans ntrBC genes is revealed by the generation of yellow colonies at the end of the agar plating process. The process also involves the sequencing of the 4.47 kb region from the T. ferrooxidans chromosome.

Molecular genetics research on Thiobacillus ferrooxidans has focused largely on the nutrition and energy production processes of the bacterium's chemoautolithotrophic behavior. This involves the creation of genetic systems required to effect physiological research and if necessary to genetically alter commercial strains. T ferroxidans, a rod-shaped, gram-negative bacterium possesses physiology appropriate for development in an inorganic mining environment.

A study was conducted to purify and determine a cyanide-degrading enzyme from Bacillus pumilus Cl. Three polypeptides of 45.6, 44.6 and 41.2 kilodaltons constituted this enzyme. A multimeric, rod-shaped protein of about 9 to 56 nanometers was seen through electron microscopy. Reversible increase of enzymatic functioning through azide was optimal at 4.5 micrometers azide and enhanced with time. The activity of cyanide dihydratase from B. pumilis is very specific, and its stability and the way in which H can be easily purified show that it can be used in the biological detoxification of cyanide in an industrial procedure.

The mobilization region of broad-host-range plasmid pTF-FC2, isolated from Thiobacillus ferrooxidans, was sequenced and characterized. A 3.7-kb region, within a 5.3-kb fragment of pTF-FC2, was required for mobilization in cis. This mobilization region contained five open reading frames (ORFs) and the origin of transfer (oriT). Three ORFs were essential for mobilization while two affected the mobilization frequency. The mobilization region of pTF-FC2 showed similarities with the Tra1 region of Inc plasmid RP4. The plasmid also contained a Tn21-related transposon and an ORF with homology to merR (mercury reductase) genes.

Abstract Microorganisms are used in large-scale heap or tank aeration processes for the commercial extraction of a variety of metals from their ores or concentrates. These include copper, cobalt, gold and, in the past, uranium. The metal solubilization processes are considered to be largely chemical with the microorganisms providing the chemicals and the space (exopolysaccharide layer) where the mineral dissolution reactions occur. Temperatures at which these processes are carried out can vary from ambient to 80°C and the types of organisms present depends to a large extent on the process temperature used. Irrespective of the operation temperature, biomining microbes have several characteristics in common. One shared characteristic is their ability to produce the ferric iron and sulfuric acid required to degrade the mineral and facilitate metal recovery. Other characteristics are their ability to grow autotrophically, their acid-tolerance and their inherent metal resistance or ability to acquire metal resistance. Although the microorganisms that drive the process have the above properties in common, biomining microbes usually occur in consortia in which cross-feeding may occur such that a combination of microbes including some with heterotrophic tendencies may contribute to the efficiency of the process. The remarkable adaptability of these organisms is assisted by several of the processes being continuous-flow systems that enable the continual selection of microorganisms that are more efficient at mineral degradation. Adaptability is also assisted by the processes being open and non-sterile thereby permitting new organisms to enter. This openness allows for the possibility of new genes that improve cell fitness to be selected from the horizontal gene pool. Characteristics that biomining microorganisms have in common and examples of their remarkable adaptability are described.

The inactivation of the chromosomally located arsenic resistance gene arsB in Acidithiobacillus caldus [KU.sup.T] is described by using marker exchange mutagenesis to serve as a model system for the knockout of other genes from the microorganism. The functionality of the gene inactivation system is shown by constructing A. caldus arsB and tetH mutants and the effects of these mutations on cell growth are shown in the presence of arsenic and tetrathionate oxidation.

An in vitro transposition system that delivers a kanamycin-selectable marker and an Escherichia coli plasmid origin of replication was used to identify three large cryptic plasmids from different isolates of Acidithiobacillus caldus. All three plasmids showed acquired additional genes with unknown function, that are present on a 26-kb common region.

Two plasmids of 28,878 bp and 28,012 bp are isolated from Leptospirillum ferrooxidans ATCC 49879. A total of 67 open reading frames (ORFs) are identified on both plasmids, of which 32 has predicted products with high homology to proteins of known function while 11 ORFs has predicted products with homology to previously identified proteins of unknown function.

Research has been conducted on chromosomal arsenic-resistane Acidithiobacillus ferrooxidans operon ars. Results demonstrate that A. ferrooxidans ArsR-like protein is a repressor of the microorganism's ars genes, and that gene expression induction can take place only with addition of arsetine or arsenate.