Strain polymorphism of the plasmid profiles in Acidithiobacillus ferrooxidans

Plasmid profiles were studied in 27 Acidithiobacillus ferrooxidans strains isolated from different geographic zones and substrates differing in the composition of the main sulfide minerals, and also in experimentally obtained strains with acquired enhanced resistance to the ions of heavy metals (Fe, Ni, Cu, Zn, As). In 16 out of 20 strains isolated from different substrates, one to four 2- to 20-kb and larger plasmids were revealed. Plasmids were found in all five strains isolated from gold-containing pyrite-arsenopyrite ores and concentrates, in nine of 11 strains isolated from the ores and concentrates containing nonferrous metals, and in two of four strains isolated from the oxidation substrates of simple composition (mine waters, pyritized coals, active sludge). Changes in the plasmid profiles in some A. ferrooxidans strains (TFZ, TFI-Fe, TFV-1-Cu) with experimentally enhanced resistance to Zn2+, Fe3+, and Cu2+, respectively, were noted as compared with the initial strains. After 30 passages on S0-containing medium, strain TFBk showed changes in the copy number of plasmids. The role of plasmids in the processes of oxidation of energy substrates and in the acquired enhanced resistance to the heavy metal ions is discussed.     

Phenotypic Characteristics of Thiobacillus ferrooxidansStrains

Phenotypicpolymorphism of Thiobacillus ferrooxidansstrains isolated from various ecological niches was studied. The strains differed both in rates of growth and oxidation of Fe²⁺, S⁰, FeS₂, and sulfide minerals contained in concentrate. Each strain, irrespective of its original environment, required a period of adaptation to a new substrate. Strains TFN-d, TFBk, TFO, and TFL-2, isolated from ores and concentrates rich in oxidizable substrates, showed an equal adaptation rate (five culture transfers) but differed in their adaptation efficiency. Strain TFV-1, isolated from low-grade ore and showing the lowest rates of growth and oxidation of all the substrates, required five culture transfers to adapt to S⁰and FeS₂and seven culture transfers to adapt to the concentrate. It is concluded that the phenotypic properties of the strains correlate with their genotypic polymorphism and the environmental conditions under which their microevolution took place.     

Plasmid Profiles of Acidithiobacillus ferrooxidans Strains Adapted to Different Oxidation Substrates

Plasmid profiles were studied in five Acidithiobacillus ferrooxidans strains of various origin cultivated on a medium with Fe²⁺, as well as adapted to such oxidation substrates as S⁰, FeS₂, and sulfide concentrate. The method used revealed plasmids in all A. ferrooxidans strains grown on a medium with Fe²⁺. One plasmid was found in strain TFL-2; two plasmids, in strains TFO, TFBk, and TFV-1; and three plasmids were detected in strain TFN-d. The adaptation of strain TFN-d to sulfide concentrate and the adaptation of strain TFV-1 to S⁰, FeS₂, or sulfide concentrate resulted in a change in the number of plasmids occurring in cells. In cells of strain TFN-d adapted to sulfide concentrate, the number of plasmids decreased from three to two. The number of plasmids in cells of strain TFV-1 adapted to different substrates varied from three to six depending on the energy source present in the medium: three plasmids were found after growth on FeS₂, four after growth on S⁰, and six after growth on sulfide concentrate. The possible role of plasmids in the adaptation of A. ferrooxidans to new energy substrates and in the regulation of the intensity of their oxidation is discussed.     

Interaction of Chromosomal and Plasmid DNA in Acidithiobacillus ferrooxidans Strains Adapted to Different Oxidation Substrates

Restriction analysis of plasmids pTFK1 and pTFK2 of theAcidithiobacillus ferrooxidans strain TFBk was carried out, and the sizes of these plasmids were determined (13.5 and 30 kb, respectively). A macrorestriction map was built for plasmid pTFK1. DNA–DNA hybridization revealed that the plasmids contained homologous nucleotide sequences. Plasmid pTFK2 labeled with ³²P was used as a probe for Southern hybridization with blots of XbaI-generated fragments of the chromosomal DNA of A. ferrooxidans strains grown on a medium containing Fe²⁺ or adapted to different oxidation substrates. Low-intensity hybridization signals were observed for many fragments of the chromosomal DNA of the strains studied. In the process of adaptation to new oxidation substrates, the localization of bands producing the low-intensity hybridization signals changed in a number of cases. Certain fragments of the chromosomal DNA of the strains adapted to different oxidation substrates produced strong hybridization signals with pTFK2. The data obtained are discussed in terms of the possible role of IST elements and plasmids in the adaptation of A. ferrooxidans to new energy substrates, microevolution, and strain polymorphism.     

Theczc operon ofAlcaligenes eutrophus CH34: from resistance mechanism to the removal of heavy metals

Summary The plasmid-borneczc operon ensures for resistance to Cd²⁺, Zn²⁺ and Co²⁺ ions through a tricomponent export pathway and is associated to various conjugative plasmids ofA. eutrophus strains isolated from metal-contaminated industrial areas. Theczc region of pMOL30 was reassessed especially for the segments located upstream and downstream the structural genesczc CBA. In cultures grown with high concentrations of heavy metals,czc-mediated efflux of cations is followed by a process of metal bioprecipitation. These observations led to the development of bioreactors designed for the removal of heavy metals from polluted effluents.     

Plasmids in the bacterial assemblage of a dystrophic Lake: Evidence for plasmid-encoded nickel resistance

Sixty-two aerobic bacterial strains isolated from the unproductive dystrophic Lake Skärshultsjön (South Sweden) were screened for plasmids. The lake is considered to be an extreme environment because of its high concentration of persistent but nontoxic humic compounds. One-third of the isolates harbored multiple plasmids usually of similar high molecular weights (>25 Mdal). The plasmid-bearing strains were members of the common aquatic taxaPseudomonas spp.,Acinetobacter sp.,Alcaligenes sp.,Aeromonas/Vibrio group, andEnterobacteriaceae (taxonomy is tentative). The majority of isolates displayed multiple resistance to antibiotics and heavy metals. Some of them were capable of degrading aromatic compounds. Three isolates were chosen for curing experiments. Only strain S-68, anAlcaligenes sp., could be cured of one of its two plasmids. It harbored the two cryptic plasmids pQQ32 and pQQ70 of 32 and ca. 70 Mdal, and the latter was segregated during ethidium bromide treatment. Parental strain S-68 was capable of degrading some of nonchlorinated phenolic compounds and displayed resistance to a broad spectrum of antibiotics and the heavy metals Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, and Hg²⁺. Derivative strain S-68-41 lost its resistance to nickel, suggesting segregated plasmid PQQ70 coded for nickel resistance. Transformation experiments to restore nickel resistance in the cured derivative strain were not successful.     

Effect of heavy metals on the glutathione status in different ectomycorrhizal <Emphasis Type="Italic">Paxillus involutus</Emphasis> strains

The glutathione (GSH) status and heavy metal tolerance were investigated in four Paxillus involutus strains isolated from different heavy-metal-polluted and non-polluted regions of Europe. The heavy metal burden in the habitats did not affect significantly either the heavy metal (Cr₂O₇²⁻, Cd²⁺, Hg²⁺, Pb²⁺, Zn²⁺, Cu²⁺) tolerance and accumulation or the GSH production of the strains tested. Exposures to heavy metals increased the intracellular GSH concentrations in 12 from 24 experimental arrangements (four strains exposed to six heavy metals) independently of the habitats of the strains. The importance of GSH in heavy metal tolerance (high MIC values, ability to accumulate heavy metals and to grow in the presence of heavy metals) was thus demonstrated in this ectomycorrhizal fungus.     

Restriction Profiles of the Chromosomal DNA from Acidithiobacillus ferrooxidans Strains Adapted to Different Oxidation Substrates

Restriction profiles of chromosomal DNA were studied in different Acidithiobacillus ferrooxidans strains grown on medium with Fe²⁺ and further adapted to another oxidation substrate (S⁰, FeS₂, or sulfide ore concentrates). The restriction endonuclease XbaI digested the chromosomal DNA from different strains into different numbers of fragments of various sizes. Adaptation of two strains (TFBk and TFN-d) to new oxidation substrates resulted in structural changes in XbaI-restriction patterns of their chromosomal DNA. Such changes in the DNA restriction patterns occurred in strain TFBk after the adaptation to precyanidated gravitational pyrite-arsenopyrite concentrate (no. 1) from the Nezhdaninskoe deposit or to copper-containing ore from the Udokanskoe deposit and also in strain TFN-d adapted to untreated pyrite-arsenopyrite concentrate (no. 2) from the Nezhdaninskoe deposit. No changes in the number or size of the XbaI-restriction patterns of chromosomal DNA were revealed in either strain TFBk cultivated on media with pyrite from the Angren and Tulun deposits or in strains TFN-d and TFO grown on media with S⁰ and pyrite. Neither were changes observed in the XbaI-restriction patterns of the DNA from strain TFV-1, isolated from the copper ore of the Volkovskoe deposit, when Fe²⁺ was substituted with alternative substrates—S⁰, pyrite or concentrate no. 2 from the ore of the Nezhdaninskoe deposit. In strain TFO, no differences in the XbaI-restriction patterns of the chromosomal DNA were revealed between the culture grown on medium containing concentrate no. 2 or the concentrate of surface-lying ore from the Olimpiadinskoe deposit and the culture grown on medium with Fe²⁺. When strain TFO was cultivated on the ore concentrate from deeper horizons of the Olimpiadinskoe deposit, which are characterized by lower oxidation degrees and high antimony content, mutant TFO-2 differing from the parent strain in the chromosomal DNA structure was isolated. The correlation between the lability of the chromosomal DNA structure in A. ferrooxidans strains and the physical and chemical peculiarities of the isolation substrate and habitat is discussed.     

Differential Influence of Ions on the Copy Number of Plasmids in Thiobacillus ferrooxidans

Thiobacillus ferrooxidans MAL4-1, an isolate from Malanjkhand copper mines, India, was adapted to grow in the presence of high concentration (30 gL⁻¹) of Cu²⁺, resulting in a 15-fold increase in its tolerance to Cu²⁺. While wild-type T. ferrooxidans MAL4-1 contained multiple plasmids, cultures adapted to Cu²⁺ concentrations of 20 gL⁻¹ or more showed a drastic reduction in the copy number of the plasmids. The reduction for three of the plasmids was estimated to be over 50-fold. Examination of the plasmid profiles of the strains adapted to high concentration of SO₄ ²⁻ anion (as Na₂SO₄ or ZnSO₄) indicated that the reduction in plasmid copy number is not owing to SO₄ ²⁻ anion, but is specific for Cu²⁺. The effect of mercury on the plasmids was similar to that of copper. Deadaptation of the Cu²⁺- or Hg²⁺-adapted T. ferrooxidans resulted in restoration of the plasmids to the original level within the first passage. The fact that the plasmid copy number, in general, is drastically reduced in Cu²⁺-adapted T. ferrooxidans suggests that resistance to copper is chromosome mediated. This is the first report of a selective negative influence of copper ions on the copy number of plasmids in T. ferrooxidans.     

Acidithiobacillus ferrooxidans and its potential application

The widely distributed Acidithiobacillus ferrooxidans (A. ferrooxidans) lives in extremely acidic conditions by fixing CO₂ and nitrogen, and by obtaining energy from Fe²⁺ oxidation with either downhill or uphill electron transfer pathway and from reduced sulfur oxidation. A. ferrooxidans exists as different genomovars and its genome size is 2.89–4.18 Mb. The chemotactic movement of A. ferrooxidans is regulated by quorum sensing. A. ferrooxidans shows weak magnetotaxis due to formation of 15–70 nm magnetite magnetosomes with surface functional groups. The room- and low-temperature magnetic features of A. ferrooxidans are different from other magnetotactic bacteria. A. ferrooxidans has potential for removing sulfur from solids and gases, metals recycling from metal-bearing ores, electric wastes and sludge, biochemical production synthesizing, and metal workpiece machining.

     

Physiological aspects of hydrocarbon emulsification, metal resistance and DNA profile of biodegrading bacteria isolated from oil polluted sites

The production of biosurfactants was evaluated for seven bacterial strains isolated from different oil contaminated sites by the Emulsification Index using diesel oil as the hydrocarbon source. Minimum Inhibitory Concentrations of Mg²⁺, Cr³⁺ and Cu²⁺ were determined to identify the less sensitive bacteria in order to select the best strains for bioremediation. Plasmid extraction was also performed in order to search for gene sequences involved with biosurfactant synthesis. All strains were able to emulsify diesel oil. Rhodococcus ruber AC239 presented the best index (58%), followed by other Rhodococcus strains. Pseudomonas aeruginosa, R. ruber AC239, AC87 and R. erytropolis AC272 presented smallest sensitivities to heavy metals used, being suitable for use in sites contaminated with high concentrations of them. No plasmid DNA was detected showing that biosurfactant coding genes should be in the chromosomal DNA.     

Molecular characterization of Acidithiobacillus ferrooxidans and A. thiooxidans strains isolated from mine wastes in Brazil

Nineteen strains of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, including 12 strains isolated from coal, copper, gold and uranium mines in Brazil, strains isolated from similar sources in other countries and the type strains of the two species were characterized together with the type strain of A. caldus by using a combination of molecular systematic methods, namely ribotyping, BOX- and ERIC-PCR and DNA-DNA hybridization assays. Data derived from the molecular fingerprinting analyses showed that the tested strains encompassed a high degree of genetic variability. Two of the Brazilian A. ferrooxidans organisms (strains SSP and PCE) isolated from acid coal mine waste and uranium mine effluent, respectively, and A. thiooxidans strain DAMS, isolated from uranium mine effluent, were the most genetically divergent organisms. The DNA-DNA hybridization data did not support the allocation of Acidithiobacillus strain SSP to the A. ferrooxidans genomic species, as it shared only just over 40% DNA relatedness with the type strain of the species. Acidithiobacillus strain SSP was not clearly related to A. ferrooxidans in the 16S rDNA tree.     

Occurrence and diversity of plasmids in populations of streptomycetes in soil

Studies were made of naturally occurring plasmids hosted in Streptomyces strains isolated from two different terrestrial ecosystems: an agricultural field and a protected forest area. Six out of the 147 screened isolates contained plasmids. The strains containing these plasmids were all isolated from the agricultural soil. Plasmids were not found among the strains isolated from the forest area. Cross hybridization of the six newly isolated plasmids revealed very high similarities between four of them. However, no similarities were found between the six newly isolated plasmids and well studied streptomycete plasmids such as pIJ101 and SCP2^*. The host strains of the four similar plasmids belonged to three different species S. anulatus, S. rochei and S. diastaticus. This implies a possible conjugative transfer of these plasmids within the streptomycete population in the agricultural area. The reason for the absence of streptomycete plasmids from the populations derived from the forest area is discussed.     

Metals tolerance in moderately thermophilic isolates from a spent copper sulfide heap, closely related to Acidithiobacillus caldus, Acidimicrobium ferrooxidans and Sulfobacillus thermosulfidooxidans

Selective enrichments enabled the recovery of moderately thermophilic isolates with copper bioleaching ability from a spent copper sulfide heap. Phylogenetic and physiological characterization revealed that the isolates were closely related to Sulfobacillus thermosulfidooxidans, Acidithiobacillus caldus and Acidimicrobium ferrooxidans. While isolates exhibited similar physiological characteristics to their corresponding type strains, in general they displayed similar or greater tolerance of high copper, zinc, nickel and cobalt concentrations. Considerable variation was found between species and between several strains related to S. thermosulfidooxidans. It is concluded that adaptation to metals present in the bioleaching heap from which they were isolated contributed to but did not entirely explain high metals tolerances. Higher metals tolerance did not confer stronger bioleaching performance, suggesting that a physical, mineralogical or chemical process is rate limiting for a specific ore or concentrate.     

Metal-Resistant Heterotrophic Bacteria in Coastal Waters of Primorye

Colony-forming heterotrophic bacteria were isolated from several locations in coastal waters of Primorye that differ in the extent and character of heavy metal pollution. Statistically significant differences in the minimum inhibitory concentrations of some metals were found for bacteria isolated from the environments with different levels of pollution. Bacterial strains that were highly tolerant of several heavy metals also showed resistance to a wide spectrum of antibiotics, and some strains bore plasmids of varying numbers and sizes. Bacteria of the family Enterobacteriaceae and the genus Pseudomonas were the dominant metal-resistant bacterial forms in the polluted coastal wasters of Primorye. No strict correlation was found between metal resistance and taxonomic identity of bacteria.Original Russian Text Copyright © 2005 by Biologiya Morya, Bezverbnaya, Buzoleva, Khristoforova.     

Isolation and Molecular Characterization of Heavy Metal-Resistant Azotobacter chroococcum from Agricultural Soil and Their Potential Application in Bioremediation

Pollution of soil with heavy metals, herbicides, antibiotics and other chemicals is known to have a negative effect on microbial activities. Therefore, the aim of this study was to isolate cultures of Azotobacter sp. from polluted and unpolluted soils and to study the effect of these pollutants on their growth. A total of 120 Azotobacter sp. were isolated from soils irrigated with wastewater (contaminated soils) and groundwater (uncontaminated soils). These isolates were screened for resistance to heavy metals, herbicide and antibiotics. Also, the soils from which the cultures were isolated were analyzed for the concentrations of Zn²⁺, Cd²⁺, Cu²⁺, Pb²⁺ and Mn²⁺ they contained. Contaminated soil showed high levels of heavy metals as compared to uncontaminated soil. The size of the Azotobacter population in contaminated soil was lower than that in uncontaminated soil. Of the Azotobacter isolates, 64 that were recovered from contaminated soil exhibited high resistance to heavy metals (Hg²⁺, Cd²⁺, Cu²⁺, Cr³⁺, Co²⁺, Ni²⁺, Zn²⁺ and Pb²⁺) and herbicide 2,4-D compared to 56 isolates from uncontaminated soil. Also, isolates from contaminated soil showed high resistance to chloramphenicol, nitrofurantoin and co-trimoxazole compared to those isolated from uncontaminated soil. The majority of Azotobacter isolates from contaminated soil showed multiple-resistance to different metal ions and antibiotics. All isolates failed to grow at pH less than 6. Salt concentration (5%) was found to be inhibitory to all isolates. The most potent isolates from contaminated soil that showed multiresistance to all substances tested were identified on the basis of morphological and biochemical characteristics, and 16S rRNA as A. chroococcum. These resistant isolates could be employed in contaminated soils and/or bioremediation.     

溶磷性大豆根瘤内生菌的筛选、抗性及系统发育和促生

对采自河南省不同地区的大豆根瘤进行内生菌分离纯化、溶磷性筛选试验。根据能否产生溶磷圈及溶磷圈直径(D)、菌落直径(d)和D/d值大小确定菌株溶磷能力,采用钼锑抗比色法测定培养液中有效磷含量;平板筛选法对筛选菌株进行耐盐性、耐酸碱、重金属等抗性测定,并对筛选菌株进行理化特性、16S r DNA、rec A序列和系统发育分析。结果表明,从分离纯化的324株内生菌中筛选出36株具有溶磷特性,其中20株有较强溶磷性。菌株DD291发酵液中可溶性磷含量最高(452 mg/L),发酵液p H与对照相比均有不同程度下降,最大降幅达2.92。大部分溶磷性内生菌具有较强耐盐碱性,对Pb2+、Cr6+和Cu2+有较高耐受性,对Ni2+和Hg2+抗性较弱。结合细胞形态、生理生化、16S r DNA、rec A序列和系统发育分析结果,菌株确定为Bacillus cereus,Enterobacter cancerogenus,E.cloacae和Pseudomonas putida。部分溶磷菌株对大豆的生长有促进作用,显示出潜在的应用前景。     

Chromosome-encoded inducible copper resistance inPseudomonas strains

NinePseudomonas strains were selected by their high copper tolerance from a population of bacteria isolated from heavy-metal polluted zones. Copper resistance (Cu ^r ) was inducible by previous exposure of cultures to subinhibitory amounts of copper sulfate. All nine strains possessed large plasmids, but transformation and curing results suggest that Cu ^r is conferred by chromosomal genes. Plasmid-lessPseudomonas aeruginosa PAO-derived strains showed the same level of Cu ^r as environmental isolates and their resistance to copper was also inducible. Total DNA from the environmentalPseudomonas, as well as fromP. aeruginosa PAO strains, showed homology to a Cu ^r P. syringae cop probe at low-stringency conditions but failed to hybridize at high-stringency conditions.     

Thiobacillus ferrooxidans response to copper and other heavy metals: growth, protein synthesis and protein phosphorylation

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.     

Quorum-sensing system in Acidithiobacillus ferrooxidans involved in its resistance to Cu²⁺

Aims: The purpose of this study was to search for the relationship between quorum sensing (QS) and Cu²⁺ resistance in Acidithiobacillus ferrooxidans. Methods and Results: Resistance to Cu²⁺ of A. ferrooxidans significantly decreased with the treatment dose of a synthetic QS blocker (5Z)‐4‐bromo‐5‐(bromomethylene)‐2(5H)‐furanone (FUR). Relative differences in expression of the QS genes afeI, afeR and Cu²⁺ resistance‐associated genes afe0329, afe0454 were examined in the presence of Cu²⁺ and/or FUR compound. The expression of QS genes afeI and afeR increased significantly with 50 mmol l^?1 Cu²⁺ in the culture, while for samples treated with both 50 mmol l^?1 Cu²⁺ and 0·01 μg ml^?1 FUR compound, they showed little changes compared with control, and the expression of afe0329 and afe0454 genes increased slightly either. These results showed that QS system was positively related to the mechanism of Cu²⁺ resistance. Conclusions: QS system in A. ferrooxidans involved in its resistance to Cu^2+. Significance and Impact of the Study: The mechanisms of Cu²⁺ resistance in A. ferrooxidans could be revealed on a population level rather than on a single‐cell level. Our work also provides useful data for further selection of A. ferrooxidans strains with suitable Cu²⁺ resistance that could probably increase the bioleaching efficiency.