Pataki C. Banerjee

Extreme tolerance to cadmium and high resistance to copper, nickel and zinc in different Acidiphilium strains

An Acidiphilium cryptum strain and two Acidiphilium symbioticum strains exhibited extremely high tolerance to CdSO4, growing at a concentration of 700 mmol l‐1 or more in MGY medium (g l‐1 composition: KCl, 0.1; MgSO4.7H2O, 0.25; (NH4)2SO4, 2.0; K2HPO4, 0.25; glucose, 1.0; yeast extract, 0.1) of pH 3. Fairly high concentrations of ZnSO4 (200 > 125 mmol l‐1), NiSO4 (50 > 20 mmol l‐1) and CuSO4 (25 > 15 mmol l‐1) were also tolerated by these strains. Acidiphilium multivorum grew in the presence of NiSO4 (400 > 350 mmol l‐1), ZnSO4 (50 > 40 mmol l‐1), CdSO4 (40 > 20 mmol l‐1) and CuSO4 (20 > 10 mmol l‐1) while an Acidiphilium strain GS18h tolerated ZnSO4 (100 > 60 mmol l‐1), NiSO4 (50 > 30 mmol l‐1), CuSO4 (30 > 15 mmol l‐1) and CdSO4 (20 > 10 mmol l‐1) in the same medium. Acidiphilium angustum, on the other hand, was found to be metal sensitive—growth inhibition being observed at concentrations of 10 mmol l‐1 ZnSO4, 10 mmol l‐1 CuSO4, 20 mmol l‐1 NiSO4 and 0.2 mmol l‐1 CdSO4 in MGY medium. Acidiphilium organovorum also exhibited tolerance to these metal sulphates in this medium at the levels of 70 > 60 mmol l‐1 for Zn, 20 > 10 mmol l‐1 for Cd and Ni, and 10 > 5 mmol l‐1 for Cu. This strain, interestingly enough, showed a much higher level of tolerance in MAGY medium (g l‐1 composition: KCl, 0.15; MgSO4.7H2O, 3.36; K2HPO4, 0.15; (NH4)2SO4, 0.15; Al2(SO4)3.18H2O, 2.25; CaCl2.2H2O, 0.97; MnSO4.H2O, 0.12; glucose, 1.0; yeast extract, 0.1) of pH 3 than in MGY medium. Replacement of carbon source, however, did not affect the tolerance levels to any great extent.

Morphological changes in an acidophilic bacterium induced by heavy metals

The Acidiphilium strains inhabit acidic mine regions where they are subjected to occasional environmental stresses such as high and low temperatures, exposure to various heavy metals, etc. Change in morphology is one of the strategies that bacteria adopt to cope with environmental stresses; however, no study on this aspect has been reported in the case of Acidiphilium sp. This work is an attempt using the acidophilic heterotrophic bacterium Acidiphilium symbioticum H8. It was observed that the maximum alterations in size occurred when the bacterium was exposed to sub-inhibitory concentrations of Cu and Cd. Loosely packed coccobacillus-type normal cells formed characteristic chains of coccoidal lenticular shape with constrictions at the junctions between them in the presence of Cd; Cu induced transformation of cells to become round shaped; Ni caused the cells to aggregate, but Zn showed no effect. Respective metal depositions on the cell surface were confirmed by scanning electron microscopy equipped with energy dispersive X-ray analysis. Cell bound Ca2+ ions were replaced by these metal ions and measured by inductively coupled plasma mass spectrometry from the culture filtrate. Cell shape changed only after the addition of sub-inhibitory concentrations of the metals, but in growth inhibitory concentrations it was similar to the normal cells.

Resistance to cadmium and zinc in Acidiphilium symbioticum KM2 is plasmid mediated.

The acidophilic heterotroph, Acidiphilium symbioticum KM2, is highly resistant to several metals and harbors three plasmids of 3.8, 7.1, and 56 kb in size. The bacterium becomes extremely sensitive to metals when it is cured of its plasmids. A mini-plasmid library was constructed by ligating the plasmid DNA fragments generated by MboI partial digestion into the BamHI site of pBluescriptII KS+. The Lac−Ampr transformants of Escherichia coli DH5α, isolated after transformation with the library, were counter-selected on Cu2+, Cd2+, Ni2+, and Zn2+-containing plates. Only Cd2+- and Zn2+-resistant colonies were developed, and, after screening, four types of recombinant plasmids designated as pNM201 (7.2 kb), pNM206 (3.4 kb), pNM208 (4.5 kb), and pNM215 (4.9 kb) were obtained. The DNA insert in pNM206 hybridized strongly with the 3.8-kb plasmid and weakly with the 7.1-kb plasmid of Acidiphilium symbioticum KM2. The DNA insert in pNM215 hybridized only with the 7.1-kb plasmid. These results strongly suggested that resistance to cadmium and zinc in A. symbioticum KM2 is mediated by these plasmids. The smallest insert of 422 bp in pNM206 conferring metal resistance in E. coli has no sequence similarity with the reported metal-resistant genes. All the putative ORFs are significantly rich (up to 37%) in basic amino acids, mainly arginine.

Integration of Metal-Resistant Determinants from the Plasmid of an Acidocella Strain into the Chromosome of Escherichia coli DH5α*

Acidophilic bacteria of mine origin are ideal systems for studying microbial metal resistance because of their ability to grow in the presence of high concentrations of metal salts. We have previously shown that the metal-resistant transformants obtained after transformation of Escherichia coli DH5α with plasmid DNA preparation from Acidocella sp. strain GS19h did not contain any plasmid suggesting chromosomal integration of the plasmid(s) (Appl Environ Microbiol 1997; 63: 4523–4527). The present study provides evidence in support of this suggestion. The pulsed field gel electrophoresis (PFGE) pattern of genomic DNA of the plasmidless metal-resistant transformants differed markedly from that of the untransformed DH5α strain. Moreover, when the recombinant plasmids constructed by cloning plasmid DNA fragments of the Acidocella strain GS19h in the vector pBluescript II KS+ were used to transform E. coli DH5α strain, no plasmid DNA was detected in some of the zinc- and ampicillin-resistant (ZnrAmpr) clones. The PFGE pattern of genomic DNA of such a transformed clone also differed markedly from that of the parent strain, suggesting chromosomal integration of the recombinant plasmid(s) containing both ampicillin- and zinc-resistance determinants. This observation was further supported by hybridization of chromosomal DNA of the plasmidless ZnrAmprE. coli DH5α clone with the probes made from the plasmid DNA of strain GS19h and the vector DNA. Thus, this study corroborates our previous finding and documents the phenomenon of integration of metal-resistant determinants from the Acidocella GS19h plasmid(s) into the chromosome of E. coli DH5α.

Generation of Novel Plasmids in Escherichia coli S17-1(pSUP106)

When the highly metal-resistant acidophilic heterotrophic strain, Acidiphilium symbioticum KM2, was incubated with two Escherichia coli strains, viz. S17-1 (pSUP106) and K12, on a medium that supported growth of these two divergent species of different habitats, E. coli transconjugants were isolated that contained novel plasmids and were resistant to Zn2+ (48 mM), Cu2+ (12 mM), Ni2+ (12 mM), chloramphenicol (50 μg/ml), and tetracycline (25 μg/ml). The transconjugant plasmids did not hybridize with any of the A. symbioticum KM2 plasmids. After curing of the plasmids, the transconjugants became sensitive to 12 mM Zn2+, 12 mM Cu2+, and 12 mM Ni2+, but remained chloramphenicol and tetracycline resistant—the phenotypic markers that were originally present in pSUP106. That a part of pSUP106 was integrated into the chromosome of the transconjugants was evident from the hybridization of pSUP106 with chromosomal DNA of the cured derivatives of the transconjugants. Further, the transconjugant plasmids hybridized only with the chromosomal DNA of E. coli S17-1 and not with the chromosomal DNA of A. symbioticum KM2 or E. coli K12, suggesting their host chromosomal origin. Thus, the present study describes a unique event of genetic rearrangements in the E. coli strain S17-1 (pSUP106), resulting in the formation of novel plasmids conferring metal-resistance phenotypes in the cell.

Cloning of metal-resistance conferring genes from an Acidocella strain‡

Metal resistance is regarded as the most suitable phenotypic trait for selection of genetically engineered bioleaching bacteria. Since the expression of metal resistance conferring genes is very limited in heterologous systems, search for these genes from homologous and related biomining bacteria is envisaged to be rewarding for genetic manipulation of this group of microorganisms. Acidocella strain GS19h, a bacterium having high resistance to cadmium and zinc, was chosen for cloning of metal resistance genes from its plasmids. Purified plasmid preparation from this bacterium was partially digested with Sau 3AI, ligated at the Bam HI site of pBluescriptIIKS+/-, and transferred into E. coli DH5α strain by transformation. The E. coli derivatives containing cloned segments were tested for metal resistance and a few cadmium resistance colonies were found to contain a recombinant plasmid having a 0.8 kb insert DNA. This DNA fragment was sequenced and analyzed for common sequences in other sources from gene data banks. Although partial homologies with a part of various transposons, merR genes and others were detected, it appears that the cloned gene is a novel one with no apparent similarity with the existing cadmium, copper, nickel, or zinc resistance-conferring genes.