STUDIES ON COPPER BINDING PROTEINS IN MICROORGANISMS

The use of microorganisms to treat aqueous streams for the removal, concentration and recovery of toxic and valuable heavy metals, recently received increased attention, specifically for microbial populations that are able to grow in the presence of, and at the same time accumulate, heavy metals.\ud \ud Until now, Saccharomyces cerevisiae is the only model organism used for searching the metal binding protein and copper metallothionein (MT) genes were only reported in the bakers yeast. But surprisingly same type of MT-like proteins have been found in Rhodotorula pilimanae which confers a maximum resistance upto 2o mM CuSO4 for the organism, while Cu-MT only confers a tolerance upto 2 mM for metal tolerant S. cerevisiae yeast strains.\ud \ud The extent of genetic diversity in R. pilimanae with respect to metal resistance was assessed by using metal tolerant s. cerevisiae F762 strain as a control. Minimal metal ion concentrations inhibitory to the growth of the strains were first determined. The maximum tolerance level of R. pilimanae were found to be 2o, 55, 15, 25 and 300 mM against Cu, Cd, Co, V and Mn respectively, which were significantly higher than the tolerance of F762. While the resistance against Ag, Pb and Hg were approximately equal for both strains. But R. pilimanae was less resistant to Fe and Zn comparing to F762, and both the strains were resistant to all concentrations of LiClz. To study the mechanism leading to development of resistance to copper as well as some other metal ions in R. pilimanae, protein analysis has been done, by using s. cerevisiae, YEP and F762 yeast strains which contain Cu-MT gene as a control. Comparison of coomassie stained protein bands on SDS-polyacrylamide gel electrophoresis (PAGE) between the strains, to find out the similarity or dissimilarity, indicated that more proteins are synthesized in S. cerevisiae strains. Silver stained SDS-PAGE also showed differences of some proteins in the experimental and control strains, and the result clearly indicated the presence of new protein bands after exposing R. pilimanae cells to Cu, Ag and Fe. To check either that new bands are cysteine-rich protein like MT or not, See-cysteie labeled protein experiment was performed and the autoradiogram revealed the presence of cysteine-rich protein in the case of R. pilimanae after treating with copper and that protein is higher in molecular weight than Cu-MT of the control. To find out the tendency of that Cu-MT like protein of pigmented yeast strain to bind some of the other metals, See-cysteine labeled protein was examined after treating R. pilimanae cells with different metals. The results indicated the presence of two types of expression in R. pilimanae, constitutive and inducible, the constitutive expression showed more intensity in the case of some metals than others. Inducible MT-like protein was observed only in the case of copper and that inducible protein is higher in molecular weight than the constitutive protein. Some other inducible protein was observed in less quantity at highest level in the case of Fe.\ud \ud Because the half life of MT is a function of metal content i.e. can detoxify elements with more half-life, therefore, the stability of Cu-MT like protein of pink yeast strain was investigated by See-cysteine labeling experiment and the autoradiogram clearly showed the stability of that protein during whole time of the investigation (6 hours).\ud \ud An interesting property of metallothioneins is their induction by the same heavy metal ions to which they bind (Kagi and Nordberg 1979). Thus it was of interest to firstly determine whether the higher molecular weight, cysteine rich proteins of R. pilimanae are related to metallothionein family or not, by studying the homology between mRNA of that strain, after exposing to different metal ions, and normal Cu-MT probe containing sequences in the presence of F762 as a control. Secondly to study the mRNA from R. pilimanae is inducible to nay extent by Li, Zn, Co, Fe, Cu, Cd and Ag. The autoradiogram of the northern blot revealed that mRNA of R. pilimanae have the same sequences of normal Cu-MT probe and consequently that mRNA has translated to the higher molecular weight cysteine-rich protein from the same metallothionein family. Also the aultoradiogram indicated that mRNA of R. pilimanae can induced by Cu, Cd and Ag but not by the other metals. It is possible that, metallothionein-like protein responsible for high metal tolerance in R. pilimanae will exhibit greater homology to mammalian metallothionein.\ud \ud Increased resistance presumably would depend upon the metal inducing MT gene for producing MT protein. To determine whether Cu-MT is conserved DNA segment, the homology and structure of this region was examined from R. pilimanae and F762 by Southern blot hybridization analysis of restriction endonuclease digest of genomic DNA. The hybridization probe used in this study was MT plasmid with a unique EcoRI fragment which contains the entire coding region for Cu-MT gene. The autoradiogram represented that F762 and R. pilimanae yeasts had reproducible yield of EcoRI pieces which showed significant differences in size, R pilimanae has longer piece with more Cu-MT coding sequences than F672.\ud \ud Thus our studies confirm earlier observations that R. pilimanae can tolerate high metal concentrations and suggest that inducible, with long half-life, and constitutive MT-like proteins in addition to high copy number of Cu-MT like DNA sequences inside the cells, at least in part, is a requirement for cellular resistance to the cytotoxic effects of copper and some other metals.\ud \ud MNNG treatment successfully induced mutations in R. pilimanae resulting in loss of survival against lower doses of Cu, Cd and few other metals. Loss of pink pigment was also achieved in one of the mutant strain. For looking the protein bands which mediates metal resistance to the wild type and is absent from the mutant cells of R. pilimanae, total proteins were isolated from normal and mutant cells before and after treating with copper or cadmium ions and SDS-PAGE of silver staining showed differences in some proteins of normal and mutant strains