The two isoforms of copper metallothionein (CuMT) gene of a copper resistant ciliate, Tetrahymena tropicalis lahorensis (Ttl), have been isolated and characterized. The molecular cloning and nucleotide sequencing of cDNAs coding for the two CuMT isoforms revealed that TtlCuMT1 gene has 300, while TtlCuMT2 has 327 nucleotides, both with ATG as the initiation codon and TGA as the translational termination codon. TAG codes for glutamine in TtlCuMT2 gene which is peculiar to Tetrahymena. The deduced or translated TtlCuMT1 and TtlCuMT2 peptide sequences contain 100 and 108 amino acid residues including 28 and 32 cysteine residues, respectively. The amino acid sequences of TtlCuMT1 and TtlCuMT2 have special features of two and three CXCXXCXCXXCXC intragenic tandem repeats with a conserved structural pattern of cysteine, respectively. The predicted tertiary structures of these two isoforms indicate two domains. Domain I and the initial part of domain II showed >98% homology with other Tetrahymena CuMT. On the basis of the differences in the domain II, the metallothionein subfamily 7b can be divided into two groups, one (TtlCuMT1) comprising >100 amino acids and the other (TtlCuMT2) comprising <100 amino acids. This is a novel finding of the present study as no such report on this type of classification exists at the moment. TtlCuMT1 has 95%, while TtlCuMT2 has 97% resemblance with the previously reported CuMT genes of Tetrahymena spp. SDS-PAGE analysis using fluorescent probe as well as coomassie brilliant blue staining also confirmed the presence of metallothionein.

In this study, the survival of genetically engineered microorganisms (GEMs) and their interactions with the environmental microbiota of a tropical river was investigated. Diffusion chambers were used for the in situ survival experiments with a nonplasmid containing Escherichia coli DH7 strain and two model GEMs, E. coli JM103 containing a 2.6 kilobase plasmid (pUC9) and E. coli DH1 with a 4.8 kb plasmid (pWTAla5'). Pure culture survival studies indicated that after a week in the environment a 1.0 log,, decrease in bacterial numbers occurred for both E. coli DH1, while a 3.0 log,, reduction was observed for E. coli JM103. However, a reduction of 4.0 log,, was observed for the E. coli DH1 (pWTAla5') when placed in a chamber conjointly with the resident microbiota. The data suggest that the presence of a plasmid makes no difference on the survival time of GEMs, whereas the presence of competing bacteria is ultimately what limits the survival time of GEMs in the environment. 0 7996 by John Wiley & Sons, Inc.

The most prevalent airborne human pathogenic fungus Aspergillus fumigatus is ubiquitous in soil and like other ascomycotous fungi it can utilize a variety of nitrogen sources. Nitrogen metabolite repression (NMR) may induce virulence in A. fumigatus as in pathogenic fungi but the mechanism of regulation and its key components are not identified. The present study focuses on the molecular modeling and in silico characterization of the main player of this regulation, the NmrA of A. fumigatus. Physiochemical and structural characterization using various sequence and structure based predictors and quality assessment of the proposed two- and three-dimensional models were carried out. The characteristic motifs such as glycine-rich NAD(P)-binding motif (GxxGxxG) and altered active site motif (HxxxK) were located in NmrA along with DNA-binding residues (T11, R39, D40 and A45). The results obtained using bioinformatics tools indicated that the protein was hydrophilic in nature, stable in vitro and had very low disorder probability. Based on the quality score, the proposed secondary and tertiary structures were correct and extremely good to represent NmrA of A. fumigatus. Phylogenetic analysis signified its close relation with NMR regulatory protein of opportunistic human pathogens A. lentulus and A. novofumigatus.

Metals are environmental pollutants of major concern due to their ecological, sanitary and even economic consequences. Both prokaryotes and eukaryotes inhabiting such environments carry cellular systems that maintain the metal homeostasis. The ciliate protists tolerate elevated concentrations of metals, which are accumulated, bound to metallothioneins (MTs) peculiar to these organisms. Copper is one of such contaminant found in the wastewater of local industries. The concentrations of copper which caused 50% reduction (LC 50 ) in the cell population of Tetrahymena sp RT1, and two Euplotes spp. RE-1 and RE-2, isolated from the industrial waste, were found to be 60, 48 and 49 ppm, respectively, compared to those of the cultures without copper in the media. RT-1 showed significantly high tolerance to copper ions and could uptake 52.66% of the copper ions from the medium. The axenic culture of RT-1 could uptake 61.2% of copper from the medium compared to 68.41 and 59.16% by the ATCC culture of Tetrahymena thermophila and T. pyriformis, respectively. RT-1 tolerated about 500 µM copper in the medium without affecting its movement. This ciliate showed promise as a member of the consortium used for bioremediation of copper contaminated wastewater.

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