Engineered nickel bioaccumulation in Escherichia coli by NikABCDE transporter and metallothionein overexpression

Mine wastewater often contains dissolved metals at concentrations too low to be economically extracted by existing technologies, yet too high for environmental discharge. The most common treatment is chemical precipitation of the dissolved metals using limestone and subsequent disposal of the sludge in tailing impoundments. While it is a cost-effective solution to meet regulatory standards, it represents a lost opportunity. In this study, we engineered Escherichia coli to overexpress its native NikABCDE transporter and a heterologous metallothionein to capture nickel at concentrations in local effluent streams. We found the engineered strain had a 7-fold improvement in the bioaccumulation performance for nickel compared to controls, but also observed a drastic decrease in cell viability due to metabolic burden or inducer (IPTG) toxicity. Growth kinetic analysis revealed the IPTG concentrations used based on past studies lead to growth inhibition, thus delineating future avenues for optimization of the engineered strain and its growth conditions to perform in more complex environments.     

Characterization of translation products of the polyadenylated RNA of free and membrane-bound polyribosomes of rat forebrain.

Poly(A)+ RNA (polyadenylated RNA) isolated from membrane-bound and free polyribosomes was translated in reticulocyte lysates, and the products were analysed by two-dimensional gel electrophoresis. Several translation products were specific to membrane-bound polyribosomal mRNA, including polypeptides of 47kDa, 35kDa and 21 kDa, whereas others (e.g. of 37 kDa, 17 kDa and 14 kDa) were specific to free polyribosomal mRNA. Although many products were common to both mRNA species, cross-contamination could be ruled out on the basis of the presence of these and other specific products. The common products included a 68 kDa microtubule-associated protein, tubulin, actin, the brain form of creatine kinase, neuron-specific enolase and protein 14-3-3 and calmodulin, all of which were identified on the basis of two-dimensional gel and peptide analyses. The 35 kDa protein product of membrane-specific mRNA was co-translationally processed in vitro by microsomal membranes, resulting in its cleavage to 33 kDa (and partial glycosylation). The 33 kDa processed protein (but not the 35 kDa precursor) was integrated into both dog pancreas and rat brain microsomal membranes. The occurrence of the enzymes and calmodulin as products of membrane-bound polyribosomal mRNA is discussed in the light of their presence on rat brain synaptic plasma membranes [Lim, Hall, Leung, Mahadevan & Whatley (1983) J. Neurochem. 41, 1177-1182] and their existence in a specific component of axonal flow. It is suggested that some of these translation products of the rough endoplasmic reticulum may represent proteins destined for the plasma membrane. However, the identity and location of the 35 kDa membrane-specific product (or its processed form) still remain unestablished.     

Negative correlation between protozoal and bacterial levels in rumen samples and its relation to the determination of dietary effects on the rumen microbial population.

The bacterial protein content and protozoal protein content of unfractionated samples from the liquid-small particle phase of the rumen were determined on the basis of direct microscopic measurement of bacteria numbers and protozoa numbers and cell volumes. Standard values of 8.7 X 10(-11) mg of protein per bacterial cell and 5.9 X 10(-11) mg/micron 3 of protozoa cell volume, obtained from analysis of isolated cells, were used to convert the microscopic measurements to an estimate of the protein content of the rumen sample. When the correlation between bacterial and protozoal protein levels was examined within groups of animals, a highly significant negative correlation between these two parameters was found (P less than 0.001). The variation among animals for total (bacterial plus protozoal) microbial protein was smaller than the variation among animals for bacterial or protozoal protein alone. There was also a highly significant positive correlation (P less than 0.001) between protozoal protein level and total microbial protein level. The variation found among animals in total microbial protein level could be reduced by using a regression equation determined for bacterial versus protozoal protein to correct for the different population dynamics of the two groups.     

Methane synthesis by membrane vesicles and a cytoplasmic cofactor isolated from Methanobacterium thermoautotrophicum.

Methanobacterium thermoautotrophicum when grown on ordinary culture medium has a tough cell wall which is lysozyme-resistant and difficult to disrupt by physical means. The cell wall, however, can be weakened by the addition of D-sorbitol to the growth medium and the organisms form protoplasts after lysozyme addition. This technique allowed the isolation of two types of intracellular small vesicles: (a) isolated by disruption of the total cell population (lysozyme-sensitive and lysozyme-resistant cells) by ultrafrequency sound and (b) isolated by osmotic lysis of protoplasts. For the first time, a small vesicle fraction isolated as in (a) was capable of synthesizing methane from CO2 and H2 without cytoplasm. There was, however, an absolute requirement for a small, heat-stable, oxygen-sensitive cofactor which was isolated from the cytoplasm. Methane synthesis with this vesicle fraction was inhibited by the detergent deoxycholate, and by the protonophores 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Mg2+-ATPase appeared to be located on the outer or cytoplasmic surface of the small vesicle fraction isolated as in (b). The results were consistent with a previously made suggestion [Sauer, Erfle & Mahadevan (1981) J. Biol. Chem. 256, 9843-9848] that the interior of the small intracellular vesicles becomes acid during methane synthesis.     

Trehalose Toxicity in Cuscuta reflexa: Cell Wall Synthesis Is Inhibited upon Trehalose Feeding

α,α-Trehalose induced a rapid blackening of the terminal 2.5-centimeter region of excised Cuscuta reflexa Roxb. vine. The incorporation of radioactivity from [¹⁴C]glucose into alkali-insoluble fraction of shoot tip was markedly inhibited by 12 hours of trehalose feeding to an excised vine. This inhibition was confined to the apical segment of the vine in which cell elongation occurred. The rate of blackening of shoot tip explants was hastened by the addition of gibberellic acid A₃, which promoted elongation growth of isolated Cuscuta shoot tips. The symptom of trehalose toxicity was duplicated by 2-deoxyglucose, which has been shown to be a potent inhibitor of cell wall synthesis in yeast. The observations suggest that trehalose interferes with the synthesis of cell wall polysaccharides, the chief component of which was presumed to be cellulose.     

Influence of a carbamate pesticide on growth,respiration (14C)-carbon metabolism and symbiosis of aRhizobium sp.

Summary Addition of aldicarb (2 methyl-2(methyl thio) propionaldehyde-0-methyl carbamoyl oxime) in the growth medium enhanced the growth ofRhizobium sp. (cowpea group) at 2 ppm level while an inhibition was observed at the normal (5 ppm) and higher (10 ppm) concentrations. Respiration of the cells was also inhibited by 5 and 10 ppm levels of the chemical eventhough a stimulation was observed at 2 ppm (lower) concentration. The insecticide, when incorporated at 5 and 10 ppm levels in the medium increased the¹⁴C-glucose incorporation and considerably altered the assimilation of the radioactive carbon in different fractions of rhizobium cells. Soil application of this insecticide (Temik 10 G) reduced the number of nodules formed and the total nitrogen content in cowpea plants inoculated with theRhizobium sp. but enhanced the dry matter production of cowpea plants.Based on the M. Sc. thesis submitted by the first author to the Tamil Nadu Agricultural University, Coimbatore-3.     

Purification and properties of urease from bovine rumen.

Urease (urea amidohydrolase, EC 3.5.1.5) was extracted from the mixed rumen bacterial fraction of bovine rumen contents and purified 60-fold by (NH4)2SO4 precipitation, calcium phosphate-gel adsorption and chromatography on hydroxyapatite. The purified enzyme had maximum activity at pH 8.0. The molecular weight was estimated to be 120000-130000. The Km for urea was 8.3 X 10(-4) M+/-1.7 X 10(-4) M. The maximum velocity was 3.2+/-0.25 mmol of urea hydrolysed/h per mg of protein. The enzyme was stabilized by 50 mM-dithiothreitol. The enzyme was not inhibited by high concentrations of EDTA or phosphate but was inhibited by Mn2+, Mg2+, Ba2+, Hg2+, Cu2+, Zn2+, Cd2+, Ni2+ and Co2+. p-Chloromercuribenzenesulfphonate and N-ethylmaleimide inhibited the enzyme almost completely at 0.1 mM. Hydroxyurea and acetohydroxamate reversibly inhibited the enzyme. Polyacrylamide-gel electrophoresis showed that the mixed rumen bacteria produce ureases which have identical molecular weights and electrophoretic mobility. No multiple forms of urease were detected.