Biomedical subjects

Douglas E Rawlings

Publications and source records attributed to Douglas E Rawlings.

14 recordsLinked to original sources

PubMed2006

Cloning and characterization of the chromosomal arsenic resistance genes from Acidithiobacillus caldus and enhanced arsenic resistance on conjugal transfer of ars genes located on transposon TnAtcArs.

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 coli, 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.

Acidithiobacillus↗

PubMed2006

Resistance determinants of a highly arsenic-resistant strain of Leptospirillum ferriphilum isolated from a commercial biooxidation tank.

Two sets of arsenic resistance genes were isolated from the highly arsenic-resistant Leptospirillum ferriphilum Fairview strain. One set is located on a transposon, TnLfArs, and is related to the previously identified TnAtcArs from Acidithiobacillus caldus isolated from the same arsenopyrite biooxidation tank as L. ferriphilum. TnLfArs conferred resistance to arsenite and arsenate and was transpositionally active in Escherichia coli. TnLfArs and TnAtcArs were sufficiently different for them not to have been transferred from one type of bacterium to the other in the biooxidation tank. The second set of arsenic resistance genes conferred very low levels of resistance in E. coli and appeared to be poorly expressed in both L. ferriphilum and E. coli.

Arsenates↗

PubMed2005

Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates.

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 degrees 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.

Journal Article↗

PubMed2005

The evolution of pTF-FC2 and pTC-F14, two related plasmids of the IncQ-family.

Two plasmids, pTF-FC2 and pTC-F14, that belong to the IncQ-like plasmid family were isolated from two related bacteria, Acidithiobacillus ferrooxidans and Acidithiobacillus caldus, respectively. The backbone regions of the two plasmids share a sufficiently high amount of homology to indicate that they must have originated from the same ancestral plasmid. Although some of their replication proteins could complement each other, the plasmids have evolved sufficiently for their replicons to have become compatible. This compatibility has occurred by changes in the iteron sequence, RepC (iteron binding protein) specificity and the regulation properties of the RepB primase. Two of the five mobilization genes have remained highly conserved, whereas the other three genes appear to have evolved such that each plasmid is mobilized most efficiently by a different self-transmissible plasmid. Plasmids pTF-FC2 and pTC-F14 do not appear to compete at the level of mobilization. The antitoxins of the toxin-antitoxin (TA) plasmid stability systems were partly able to neutralize the toxins of the other plasmid and also to partly cross-regulate the TA systems of the other plasmid with the antitoxin of pTF-FC2 being the most effective cross-regulator. Other aspects of the evolution of the two plasmids are described and the danger of making the assumption that incompatibly of IncQ-like plasmids is a reflection of the degree of relatedness of two plasmids is discussed.

Acidithiobacillus↗

PubMed2005

An unusual Tn21-like transposon containing an ars operon is present in highly arsenic-resistant strains of the biomining bacterium Acidithiobacillus caldus.

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. coli cells. Deletion of one copy of arsDA had no noticeable effect on resistance to arsenite or arsenate in E. coli. 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.

Acidithiobacillus↗

PubMed2005

Isolation, sequence analysis, and comparison of two plasmids (28 and 29 kilobases) from the biomining bacterium Leptospirillum ferrooxidans ATCC 49879.

Two plasmids, of 28,878 bp and 28,012 bp, were isolated from Leptospirillum ferrooxidans ATCC 49879. Altogether, a total of 67 open reading frames (ORFs) were identified on both plasmids, of which 32 had predicted products with high homology to proteins of known function, while 11 ORFs had predicted products with homology to previously identified proteins of unknown function. Twenty-four ORFs had products with no homologues in the GenBank/NCBI database. An analysis of the ORFs and other features of the two plasmids, the first to be isolated from a bacterium of the genus Leptospirillum, is presented.

Bacteria↗

PubMed2004

Evolution of compatible replicons of the related IncQ-like plasmids, pTC-F14 and pTF-FC2.

Two closely related but compatible plasmids of the IncQ-2alpha and IncQ-2beta groups, pTF-FC2 and pTC-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-2alpha plasmid pTC-FC2, and the IncQ-1beta plasmid pIE1108. None of the IncQ-1alpha 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-1alpha and IncQ-1beta 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.

Bacterial Proteins↗

PubMed2004

Plasmid evolution and interaction between the plasmid addiction stability systems of two related broad-host-range IncQ-like plasmids.

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-FC2 pas was more efficient at stabilization of a heterologous tester plasmid than the two component pas of pTC-F14 in Escherichia coli host cells (+/- 92% and +/- 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.

Antidotes↗

PubMed2003

Biomineralization of metal-containing ores and concentrates.

Biomining is the use of microorganisms to extract metals from sulfide and/or iron-containing ores and mineral concentrates. The iron and sulfide is microbially oxidized to produce ferric iron and sulfuric acid, and these chemicals convert the insoluble sulfides of metals such as copper, nickel and zinc to soluble metal sulfates that can be readily recovered from solution. Although gold is inert to microbial action, microbes can be used to recover gold from certain types of minerals because as they oxidize the ore, they open its structure, thereby allowing gold-solubilizing chemicals such as cyanide to penetrate the mineral. Here, we review a strongly growing microbially-based metal extraction industry, which uses either rapid stirred-tank or slower irrigation technology to recover metals from an increasing range of minerals using a diversity of microbes that grow at a variety of temperatures.

Bacteria↗

PubMed2003

Analysis of the mobilization region of the broad-host-range IncQ-like plasmid pTC-F14 and its ability to interact with a related plasmid, pTF-FC2.

Plasmid pTC-F14 is a 14.2-kb plasmid isolated from Acidithiobacillus 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 Tra1) 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.

Bacterial Proteins↗

PubMed2002

Heavy metal mining using microbes.

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.

Bacteria↗

PubMed2002

The divergent chromosomal ars operon of Acidithiobacillus ferrooxidans is regulated by an atypical ArsR protein.

The chromosomal arsenic-resistance (ars) operon of Acidithiobacillus ferrooxidans is atypical in that it is divergent, with its arsCR and arsBH genes transcribed in opposite directions. Furthermore, the amino-acid sequence of the putative ArsR-like regulator of the ars operon is not conserved in regions that have been shown to be responsible for binding to arsenic. Instead, the ArsR-like protein of At. ferrooxidans is related to a group of unstudied ArsR-like proteins that have been found to be associated with chromosomal ars-like operons identified during genome-sequencing projects. Using arsB-lacZ, arsR-lacZ and arsC-lacZ fusions, it was shown that the ArsR-like protein of At. ferrooxidans is a repressor of the arsBH and arsRC genes of this organism, and that induction of gene expression took place when either AsIII (arsenite) or AsV (arsenate) were added. Deletion of 19 aa from the C terminus of the 118 aa ArsR protein did not affect the regulation of its activity, but deletion of an additional 28 aa inactivated ArsR. Northern-blot hybridization suggested that on induction of expression, the arsRC genes were transcribed in greater quantities than the arsBH genes, but that the level of induction was not affected by the form of arsenic added (AsIII or AsV).

Amino Acid Sequence↗

PubMed2002

Molecular relationship between two groups of the genus Leptospirillum and the finding that Leptospirillum ferriphilum sp. nov. dominates South African commercial biooxidation tanks that operate at 40 degrees C.

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 degrees 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 degrees C. All the leptospirilla isolated from commercial biooxidation tanks in South Africa were from group II.

Bioreactors↗

PubMed1997

A geographically widespread plasmid from Thiobacillus ferrooxidans has genes for ferredoxin-, FNR-, prismane- and NADH-oxidoreductase-like proteins which are also located on the chromosome.

During a search for genes encoding electron transport proteins from a Thiobacillus ferroxidans ATCC 33020 gene bank, a 19.8 kb plasmid, pTF5, which conferred increased sensitivity to the antimicrobial agent metronidazole upon an Escherichia coli mutant, was isolated and cloned in E. coli. The plasmid had an identical restriction enzyme map to a plasmid which has been found in T. ferrooxidans strains isolated from many different parts of the world. The plasmid was present at between two and four copies per genome and contained a region of approximately 5-6 kb which was also found on the chromosome. This region was sequenced and found to have four complete ORFs, which when translated had high percentage amino acid similarity to [3Fe-4S,4Fe-4S] ferredoxins, proteins of the FNR regulator family, prismane-like proteins and the NADH oxidoreductase subunit of a methane monooxygenase. In vitro protein analysis using an E. coli-derived transcription-translation system indicated that three of the four products (FdxA, PsmA and RedA) were expressed in the heterologous system. Ferredoxins, prismane-like proteins and NADH oxidoreductases are redox-active proteins and it is likely that the proteins on pTF5 represent an electron transport system of as yet unknown function. Surprisingly, although genes for redox-active proteins have been isolated from other bacteria by screening gene banks for increased sensitivity to metronidazole, the region of pTF5 containing the genes for these proteins was not responsible for the increase in metronidazole sensitivity conferred by the plasmid. The region of pTF5 which did confer increased metronidazole sensitivity to an E. coli metronidazole-resistant mutant was a 319 bp region of DNA close to the origin of plasmid replication. This region contained no ORFs and was identical to that previously reported for the replicon of a 9.8 kb T. ferrooxidans plasmid, pTF191.

Acidithiobacillus thiooxidans↗