Developmental genetic studies on Thiobacillus ferrooxidans

Thiobacillus ferrooxidans is an industrially important bacterium which is used in bioleaching operations. The work reported in this investigation extends current knowledge of the genetics of this organism. Conjugation was attempted as a means for plasmid DNA transfer to T. ferrooxidans. Recombinant T. ferrooxidans plasmids, pDER401 and pDER405, were shown to code for mobilization and replication functions in Escherichia coli and Thiobacillus novellus strains. The plasmids were mobilizable at high frequency by the IncP plasmid, R68.45. Attempts to transfer the T. ferrooxidans recombinant plasmids directly from E. coli to T. ferrooxidans were unsuccessful. In multistage mating experiments, plasmid DNA was transferred from E. coli to T. novellus, and from T. novellus to Thiobacillus intermedius. However, in subsequent matings, plasmid transfer from these thiobacilli to T. ferrooxidans could not be shown. A genomic library of T. ferrooxidans ATCC 33020 was constructed in the plasmid vector, pEcoR251, for the purpose of cloning a recA-like gene from this organism. The library consisted of approximately 1,78 X 10⁴ clones carrying chromosomal DNA fragments of about 3-12 kilobases (kb). The library was successfully screened for functional complementation of E. coli auxotrophic mutants. Clones that conferred resistance to methyl methane sulfonate (MMS), a DNA-damaging agent, were isolated in an E. coli recA⁻ mutant. In an attempt to clone a homologous marker, T. ferrooxidans ATCC 33020 was mutated to rifampicin resistance (Rifʳ) and DNA from the mutant strain was used in the construction of plasmid- and cosmid-based libraries. The plasmid library contained approximately 1,35 X 10⁴ clones with inserts of about 1-13 kb. The cosmid library consisted of approximately 8.2 X 10³ colonies, 4.0 X 10⁴ in vitro packaged cosmids, and an amplified in vivo-packaged cosmid lysate containing approximately 1.82 X 10¹¹ infectious particles, carrying inserts of about 35-55 kb. Complementation of E. coli auxotrophic mutants was observed with the plasmid and cosmid library of the T. ferrooxidans Rifʳ strain. Screening both libraries for a Rifʳ marker was unsuccessful. Three recombinant plasmids, pRSR100, pRSR101, and pRSR102, each containing the functional analogue of the E. coli recA gene, were isolated from the plasmid-based genomic library of T. ferrooxidans ATCC 33020. The plasmid, pRSR100, was used for further characterization of the cloned recA-like gene. pRSR100 complemented defects in DNA repair and homologous recombination in an E. coli recA- strain. Antiserum raised against E. coli RecA⁻ protein reacted with two protein bands with an apparent Mᵣ of approximately 40 000 and 38 000 in extracts of the recA deletion mutant, E. coli JK696, containing pRSR100. A single band with an apparent Mᵣ of approximately 40 000 was detected in T. ferrooxidans cell extracts with the E. coli RecA antiserum. The nucleotide sequence of the T. ferrooxidans recA gene has been determined. No SOS box characteristic of LexA- regulated promoters could be identified in the 196-bp region upstream of the coding region. 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 P. 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 ATPase and constitutive protease activity have been substituted, the cloned protein has retained these activities. The cloned recA gene was expressed in E. coli from both the λ Pᵣ and lac promoters. However, no expression from the 2.2 kb T. ferrooxidans DNA preceding the gene was evident

The pio Operon Is Essential for Phototrophic Fe(II) Oxidation in Rhodopseudomonas palustris TIE-1

Phototrophic Fe(II)-oxidizing bacteria couple the oxidation of ferrous iron [Fe(II)] to reductive CO2 fixation by using light energy, but until recently, little has been understood about the molecular basis for this process. Here we report the discovery, with Rhodopseudomonas palustris TIE-1 as a model organism, of a three-gene operon, designated the pio operon (for phototrophic iron oxidation), that is necessary for phototrophic Fe(II) oxidation. The first gene in the operon, pioA, encodes a c-type cytochrome that is upregulated under Fe(II)-grown conditions. PioA contains a signal sequence and shares homology with MtrA, a decaheme c-type cytochrome from Shewanella oneidensis MR-1. The second gene, pioB, encodes a putative outer membrane beta-barrel protein. PioB is a homologue of MtrB from S. oneidensis MR-1. The third gene, pioC, encodes a putative high potential iron sulfur protein (HiPIP) with a twin-arginine translocation (Tat) signal sequence and is similar to the putative Fe(II) oxidoreductase (Iro) from Acidithiobacillus ferrooxidans. Like PioA, PioB and PioC appear to be secreted proteins. Deletion of the pio operon results in loss of Fe(II) oxidation activity and growth on Fe(II). Complementation studies confirm that the phenotype of this mutant is due to loss of the pio genes. Deletion of pioA alone results in loss of almost all Fe(II) oxidation activity; however, deletion of either pioB or pioC alone results in only partial loss of Fe(II) oxidation activity. Together, these results suggest that proteins encoded by the pio operon are essential and specific for phototrophic Fe(II) oxidation in R. palustris TIE-1

Análisis genómico comparativo de dos nuevos plásmidos de la cepa PQ33 de Acidithiobacillus ferrivorans

Acidithiobacillus ferrivorans is a psychrotolerant acidophile capable of growing and oxidizing ferrous and sulphide substrates at low temperatures. To date, six genomes of this organism have been characterized; however, evidence of a plasmid in this species has been reported only once, whereby there is no conclusive role of the plasmids in the species. Herein, two novel plasmids of A. ferrivorans PQ33 were molecularly characterized and compared at a genomic scale. The genomes of two plasmids (12 kbp and 10 kbp) from A. ferrivorans PQ33 (NZ_LVZL01000000) were sequenced and annotated. The plasmids, named pAfPQ33-1 (NZ_CP021414.1) and pAfPQ33-2 (NZ_CP021415.1), presented 9 CDS and 13 CDS, respectively. In silico analysis showed proteins involved in conjugation (TraD, MobA, Eep and XerD), toxin-antitoxin systems (HicA and HicB), replication (RepA and DNA binding protein), transcription regulation (CopG), chaperone DnaJ, and a virulence gene (vapD). Furthermore, the plasmids contain sequences similar to phosphate-selective porins O and P and a diguanylate cyclase-phosphodiesterase protein. The presence of these genes suggests the possibility of horizontal transfer, a regulatory system of plasmid maintenance, and adhesion to substrates for A. ferrivorans species and PQ33. This is the first report of plasmids in this strain.Acidithiobacillus ferrivorans es un acidófilo psicrotolerante capaz de hacer crecer y oxidar sustratos ferrosos y sulfurosos a bajas temperaturas. Hasta la fecha se han caracterizado seis genomas de este organismo; sin embargo, la evidencia de un plásmido en esta especie ha sido informado solo una vez, por lo que no hay un rol concluyente de los plásmidos en la especie. Aquí, dos plásmidos novedosos de A. ferrivorans PQ33 se caracterizaron molecularmente y se compararon a escala genómica. Se secuenciaron y anotaron los genomas de dos plásmidos (12 kpb y 10 kpb) de A. ferrivorans PQ33 (NZ_LVZL01000000). Los plásmidos, denominados pAfPQ33-1 (NZ_CP021414.1) y pAfPQ33-2 (NZ_CP021415.1), presentaron 9 CDS y 13 CDS, respectivamente. El análisis in silico mostró proteínas involucradas en la conjugación (TraD, MobA, Eep y XerD), sistemas de toxina-antitoxina (HicA y HicB), replicación (RepA y proteína de unión al ADN), regulación de la transcripción (CopG), chaperona DnaJ y un gen de virulencia (vapD). Además, los plásmidos contienen secuencias similares a las porinas selectivas de fosfato O y P y una proteína diguanilato ciclasa-fosfodiesterasa. La presencia de estos genes sugiere la posibilidad de transferencia horizontal, un sistema regulador de mantenimiento de plásmidos y adhesión a sustratos para especies de A. ferrivorans y PQ33. Este es el primer informe de plásmidos en esta cepa

The study of two genes involved in the maintenance of intracellular redox potentials in Thiobacillus ferrooxidans

Bibliography: p. 128-155.The thioredoxin and ϒ-glutamylcysteine synthetase genes of Thiobacillus ferrooxidans were isolated by complementation of an Escherichia coli trxA gshA double mutant for growth on minimal medium lacking glutathione. Transduction of a T. ferrooxidans genome cos mid library into the E. coli mutant resulted in the identification of two groups of complementing cosmids. One of these groups was able to complement the TrxA⁺ requirement of an E. coli trxA met mutant for conversion of methionine sulfoxide to methionine. The nucleotide sequence of a 1.1 kbp Hindiii-Psti T.ferrooxidans chromosomal DNA fragment containing the cloned trxA gene, was determined. An open reading frame (ORF) corresponding to the thioredoxin gene and part of an ORF corresponding to the N-terminal region of the rho gene were identified. A 14 kDa protein corresponding to the T. ferrooxidan thioredoxin was synthesised in an E. coli-derived in vitro transcription/translation system. The predicted amino acid sequence of the T.ferrooxidans trxA gene showed 70.6% identity to that of E. coli. The cloned T. ferrooxidans trxA gene was able to support T7 phage replication in an E. coli trxA mutant to almost the same level as a TrxA⁺ wild type strain. However, it was not able to satisfy the thioredoxin requirement of M13 phage. Crude extracts of the E. coli trxA gshA mutant containing the cloned T. ferrooxidans trxA gene were able to reduce insulin far more rapidly those that did not contain the cloned gene, indicating that the T.ferrooxidans thioredoxin has disulfide reductase activity. DNA:RNA hybridisation analysis was carried out on transcripts prepared from the E. coli trxA gshA double mutant, the mutant containing the cloned trxA gene and T. ferrooxidans cells. A single transcript of about 0.5 kbp was obtained for RNA from T. ferrooxidans cells. Several transcripts were produced from the cloned T. ferrooxidans trxA gene in E. coli. One of these transcripts corresponded in size to the 0.5 kbp transcript produced by T. ferrooxidans cells, whereas the other transcripts were larger (1.35, 1.40, 1.60 and 1.80 kbp) and presumably represented transcription products originating from the lac promoter of the vector. Primer extension analysis of the thioredoxin gene with RNA prepared from T. ferrooxidans gave three transcription start sites. RNA derived from the cloned trxA gene in E. coli only gave two transcription start sites, which were identical to two of the three found with T. ferrooxidans RNA. The second group of cosmids which was able to complement the gshA trxA double mutant for growth on minimal media but which did not allow growth of the E. coli trxA met mutant on minimal media plus methionine sulfoxide was also examined. The nucleotide sequence of a 2.3 kbp Clai-BamHI T. ferrooxidans chromosomal DNA fragment containing the complementing DNA, was determined. Two ORFs, separated by 9 bp, corresponding to a citrate synthase gene and a ϒ-glutamylcysteine synthetase gene were identified. The predicted amino acid sequence of the T. ferrooxidans gltA gene showed 37% identity to that of E. coli, whereas the predicted amino acid sequence of the T. ferrooxidans gshA gene only showed 18% identity to that of E. coli. The low homology of the gshA gene products is not abnormal, as the ϒ-glutamylcysteine synthetases studied to date differ widely in amino acid sequence and structure. Partial sequencing of the cloned T. ferrooxidans chromosomal DNA upstream of the gltA gene indicated regions of homology to the genes of the pyruvate dehydrogenase complex. Synthesis of proteins corresponding to the T. ferrooxidans citrate synthase (43 kDa) and ϒ-glutamylcysteine synthetase (49 kDa) enzymes were confirmed using an E. coli-derived in vitro transcription/translation system. The identity of the proposed citrate synthase gene was confirmed by complementation of an E. coli gltA mutant. Similarly, the DNA upstream of the gltA gene, was found to complement an E. coli mutant for the pyruvate dehydrogenase complex, confirming that this region contained the T. ferrooxidans pyruvate dehydrogenase complex, as predicted from the partial sequence analysis. The identity of the T. ferrooxidans ϒ-glutamylcysteine synthetase gene was confirmed by determining the levels of glutathione in crude extracts of the E. coli gshA trxA double mutant, the mutant containing the cloned T. ferrooxidans gshA gene, and an E. coli parental strain. Crude extracts prepared from E. coli double mutant contained much lower levels of glutathione than crude extracts prepared from the E.coli mutant containing the cloned gshA gene, indicating that the gene from T. ferrooxidans encoded a protein with ϒ-glutamylcysteine synthetase activity. RNA:DNA hybridisation analysis was carried out using probes specific to the gshA gene, the gltA gene and the aceF gene (pyruvate dehydrogenase transacetylase gene). All three probes gave transcripts of 9 kbp and a number of smaller transcripts with RNA derived from T. ferrooxidans cells. The 9 kbp transcripts indicated transcriptional linkage of the y- glutamylcysteine synthetase gene, the citrate synthase gene and the genes of the pyruvate dehydrogenase complex in T.ferrooxidans

An investigation of the replication of the broad-host-range plasmid pTF-FC2

Plasmid pTF-FC2, a 12.4 kilobase pairs (kb) cryptic plasmid, was originally isolated from the acidophilic chemoautotroph Thiobacillus ferrooxidans and subsequently cloned into the pMB1-based vector, pBR325. Deletion of the pBR325 origin of replication revealed that pTF-FC2 was able to replicate autonomously in a number of Gram-negative bacteria besides T. ferrooxidans. Constructs carrying the pTF-FC2 origin were able to replicate independently of DNA polymerase I (Pol I) in Escherichia coli and this feature was used to distinguish between replication from the T. ferrooxidans origin and the pMB1-derived origins of the vectors. A 3.2 kb Sau3A partial fragment was obtained which had retained the ability to replicate in a E. coli polA- mutant and also in Pseudomonas aeruginosa. A series of deletions of this fragment was used to identify the minimal replicon, the vegetative origin of replication (orzV) and the areas determining plasmid incompatibility. The copy number of pTF-FC2 in E. coli was estimated at 12 - 15 copies per chromosome and a deletion plasmid was identi.fied which replicated at a reduced copy number. An area which affected the ability of the replicon to replicate in P. aeruginosa, was identified. The nucleotide sequence of the 3.2 kb minimal replicon of pTF-FC2 was determined from overlapping DNA sequence obtained from a series of sequential deletions from each end of the fragment. Analysis of the orzV sequence revealed three, tandemly repeated 22 base pairs (bp) DNA sequences and two sets of complementary inverted repeats. The 22 bp direct repeats appeared to be essential for replication and also for incompatibility. The role of one of the two sets of complementary inverted repeats was unclear. The deletion plasmid previously shown to replicate at reduced copy number was found to have half of the second set of repeats deleted

Studies on Thiobacillus ferrooxidans ATCC 33020 ATP genes and gene products, using Escherichia coli

An atp gene cluster from the extreme acidophile Thiobacillus ferrooxidans ATCC 33020 was cloned by complementation of Escherichia coli unc mutants. Eight different E. coli unc mutants were screened with T. ferrooxidans ATCC 33020 pEcoR251 plasmid and pHC79 cosmid gene banks. The ability of the transformants/transductants to grow on succinate as the sole non-fermentable carbon source was used to select mutants with a functional F₁F₀ ATPsynthase. Many F₁ -complementing plasmids and cosmids were isolated from the four E. coli F₁ unc point mutants screened. No plasmids or cosmids which complemented an E. coli fΔunc strain or any of the three E. coli F₀ mutants screened, were isolated

Studies of the cloning and expression of Thiobacillus Ferrooxidans Plasmid and Nitrogenase genes

Bibliography : pages 145-156.This dissertation forms part of a fundamental investigation into the molecular biology of the industrially important bacterium, Thiobacillus ferrooxidans. The expression of T. ferrooxidans plasmid encoded functions, as well as the identification, cloning, sequencing and expression in a variety of heterotrophic bacteria and in vitro systems of the T. ferrooxidans nitrogenase structural genes were studied

The development of genetic systems for iron-oxidizing, acidophilic moderate thermophiles

Acidophilic, moderately thermophilic, Gram positive bacteria which are able to oxidize iron and solubilize sulphide ores are likely to be of industrial importance for the leaching of metals from mineral ores. Genetic manipulation of these bacteria might produce improved strains with regard to desirable leaching characteristics for industry. This work describes initial attempts to develop a host:vector system for the moderate thermophiles. The bacteria were found to be microaerophilic and a pour plate technique and filter disc assay were used to assess the comparative sensitivities of strains ALV, BC1 and TH3 to antibiotics and metals. Chloramphenicol and kanamycin were further investigated as potential selection agents for transformation. The latter was found to be unstable at pH 1.7 and 45°C in a ferrous iron medium. A large plasmid which migrated more slowly than chromosomal DNA during agarose gel electrophoresis was identified in strain 1X2, along with comparatively small plasmids in strains 1X2., IXL, TH1 and BC1. The latter plasmid (pBCl) was cloned into E. coli vectors pACYC177, pBR325 and pMTL20C. These recombinant vectors were used to investigate the host range of pBCl and in vitro expression from the pBCl DNA in an E. coli system. Recombinant vectors containing pBCl did not transform B. subtilis 168 but expressed a polypeptide with apparent M_ of 42,000 as determined by SDS-PAGE following in vitro transcription and translation in an E. coli system. The complete nucleotide sequence of pBCl (2,617 bp) was obtained and encoded four putative open reading frames (ORFs A, B, C and Z) which corresponded to polypeptides of Mp 41,112, 14,227, 8,228, and 6,538 respectively. Analysis of the nucleotide and predicted ORF amino acid sequences Indicated that pBCl belonged to the pC194/pUB110 family of interrelated plasmids from Gram positive bacteria and replicated by a rolling-circle mechanism via a ssDNA intermediate. Evidence for this was the similarity between ORF A of pBCl and other plasmid replication proteins and the identification of a conserved region within the ORF A product (Rep) containing a tyrosine residue which has been shown elsewhere to bind to DNA during replication. Furthermore, a second possible DNA-binding domain was identified within Rep and single- stranded DNA was isolated from strain BC1. A region of the pBCl sequence upstream of Rep was similar to the nick-site within the origin of plus strand replication of pC194 and pUBHO and homology with the minus origins (MO) of these plasmids suggested the presence of an MO in pBCl. A large putative secondary structure of about 100 bases was predicted from the pBCl DNA sequence and was positioned about 250 bases upstream of ORF A and within ORF C. Methodology was developed for the electroporation of strains ALV and BC1 but no electro transformants were isolated. However, a novel method was developed which indicated that plasmid DNA was transferred into the bacteria during electroporation