Shelly M. Deane

The Chromosomal Arsenic Resistance Genes of Thiobacillus ferrooxidans Have an Unusual Arrangement and Confer Increased Arsenic and Antimony Resistance to Escherichia coli

ABSTRACT 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) andarsC (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. ferrooxidansis gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functionaltrxA (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 andarsC genes and the arsBH genes were translated in opposite directions. This divergent orientation was conserved in the four T. ferrooxidans strains investigated.

Resistance Determinants of a Highly Arsenic-Resistant Strain of Leptospirillum ferriphilum Isolated from a Commercial Biooxidation Tank

ABSTRACT 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.

Thiobacillus caldus and Leptospirillum ferrooxidans are widely distributed in continuous flow biooxidation tanks used to treat a variety of metal containing ores and concentrates

Using 16S rDNA-based techniques, several workers have reported that Leptospirillum ferrooxidans and Thiobacillus thiooxidans are the bacteria most commonly encountered in biooxidation processes for the treatment of copper by heap leaching as well as the treatment of zinc-lead or arsenopyrite concentrates by continuous-flow tank leaching. We have compared the 16S rRNA genes from four strains of Thiobacillus caldus and identified two restriction enzymes which permit the 16S rDNA from T. caldus to be distinguished from closely related strains of T. thiooxidans . DNA has been isolated directly from continuous-flow biooxidation tanks treating copper, nickel or arsenopyrite concentrates at 40–55 °.By examining the restriction enzyme patterns of the amplified 16S rDNA, we report that even at 40 °C it is the moderate thermophile T. caldus , rather than T. thiooxidans , which is the dominant sulphur-oxidising bacterium in these tanks. By examining the restriction enzyme patterns of the 16S rDNA from fifteen strains of Leptospirillum spp isolated from different parts of the world, we have confirmed that the leptospirilli can be divided into two major groups. Furthermore, we have found that the leptospirilli which dominate the iron-oxidising bacteria in the continuous-flow biooxidation tanks belong to the same sub-group as the L. ferrooxidans type strain (DSM2705).Restriction enzyme maps of the 16S rDNA which enable isolates of T. ferrooxidans, T. thiooxidans, T. caldus and the subgroups of the genus Leptospirillum to be readily distinguished from each other are presented.

Isolation of a New Broad-Host-Range IncQ-Like Plasmid, pTC-F14, from the Acidophilic Bacterium Acidithiobacillus caldus and Analysis of the Plasmid Replicon

A moderately thermophilic (45 to 50 degrees C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic Acidithiobacillus caldus strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the A. caldus cells revealed two plasmids of approximately 14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404, which suggested that pTC-F14 is a broad-host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids. Three of the open reading frames encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14 oriV region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 had a copy number of 12 to 16 copies per chromosome in both E. coli, and A. caldus. The rep gene products of pTC-F14 and pTF-FC2 were unable to functionally complement each others oriV regions, but replication occurred when the genes for each plasmids own RepA, RepB, and RepC proteins were provided in trans. Two smaller open reading frames were found between the repB and repA genes of pTC-F14, which encode proteins with high amino acid sequence identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF-FC2. This is the second time a plasmid stability system of this type has been found on an IncQ-like plasmid.

Production and Activation of an SDS-resistant Alkaline Serine Exoprotease of Vibrio alginolyticus

Vibrio alginolyticus produced an extracellular SDS-resistant protease (protease A) with an apparent M r of approximately 54000 when cultured in complex, proteinaceous media. Ca2+ was required for the activation and stability of this protease. Its activity was inhibited by EDTA and a serine protease inhibitor, but was not affected by an inhibitor of trypsin-like enzymes. Optimum protease activity occurred under alkaline conditions. Two SDS-resistant exoproteases, B and C, with apparent M r values of approximately 41000 and 37000 respectively, were also produced in complex proteinaceous media. Dialysis of cell-free supernatant samples, which contained predominantly protease A, against distilled water, resulted in increased B and C activity. Production of protease A, B and C activities was inhibited by o-phenanthroline, quinacrine and lack of aeration.

Proteomic Profiling of the Acid Stress Response in Lactobacillus plantarum 423

Acid tolerance is considered an important characteristic of probiotic bacteria. Lactobacillus plantarum 423 tolerates acidic pH and is the ideal candidate in which to study molecular mechanisms that acid-tolerant lactic acid bacteria employ to survive such conditions. In this study we recorded changes in the protein profile of L. plantarum 423 when exposed to pH 2.5 by using a gel-free nanoLC-MS/MS proteomics approach. In total, 97 proteins were detected as more abundant, and 12 proteins were detected solely when strain 423 was exposed to pH 2.5. General stress response proteins, the utilization of a variety of carbohydrate sources in a glucose rich environment, altered pyruvate metabolism, increased lysine biosynthesis, and a significant oxidative stress response was observed in acid-stressed cells. The accumulation of basic compounds also seemed to play an integral role in the response to acid stress. We observed a marked decrease in proteins involved in cell wall and phospholipid biosynthesis, transcription, translation, and cell division. The most abundant protein detected was an uncharacterized protein, JDM1_2142. Functional analysis revealed that this protein plays a role in survival during acid stress. Our results contribute to the growing body of knowledge on the molecular mechanisms employed by lactobacilli, in particular L. plantarum, to ensure survival in acidic conditions.

A transposon-located arsenic resistance mechanism from a strain of Acidithiobacillus caldus isolated from commercial, arsenopyrite biooxidation tanks

The moderately thermophilic, sulfur-oxidizing bacterium Acidithiobacillus caldus is an important role player in continuous-flow biooxidation processes associated with biomining. Biooxidation of arsenopyrite concentrates by a combination of Leptospirillum and At. caldus results in the release of soluble arsenic into the surrounding environment. Arsenate and arsenite are toxic to most organisms, yet At. caldus is able to thrive in this harsh environment. The purpose of this study was to identify and investigate arsenic resistance genes in At. caldus and compare its ars operon with those previously studied in other bacteria. A gene bank of a South African At. caldus strain #6 was constructed and transformed into an Escherichia coli arsenic-sensitive mutant strain to identify clones containing At. caldus arsenic resistance genes. A clone with an insert of 6.4 kb was selected and after two cycles of chromosome-walking a further 5.6 kb of DNA upstream of the original clone was isolated. This region was sequenced and found to have an unusual ars operon with a functional arsB gene, two arsD-like and two arsA-like genes. The arsenic resistance mechanism appears to be located on a Tn21-like transposon and includes transposase and resolvase-like genes with the entire assemblage being flanked by Tn21-like 38 bp inverted repeats (IR). Southern hybridization experiments using the arsB gene as a probe against total DNA from six different At. caldus strains indicated that the three South African strains which had been previously exposed to arsenic gave a positive hybridization signal, while three other At. caldus strains (KU, BC13 and C-SH12) did not. Southern hybridization of pulse field gels indicated that the arsB gene was chromosomally encoded. Genes encoding for NADH oxidase-like and IMP dehydrogenase-like enzymes were situated between the arsA and arsB genes. PCR and Southern hybridization experiments revealed that this unusual arrangement was not an artefact of cloning but that the 2.1 kb NADH oxidase-like and IMP dehydrogenase-like gene containing region was present between the arsA and arsB genes in all three South African strains. When transformed into the E. coli arsenic-sensitive mutant, the transposon-located system conferred increased resistance to arsenite, but not to arsenate. This indicated that the NADH oxidase-like gene did not function as an arsenate reductase in E. coli. It is possible that increased exposure to arsenic placed selection pressure on the three South African strains to acquire a Tn21-like transposon containing an ars operon to enhance their arsenic resistance.

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.

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.

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.