Isolation and characterization of a highly effective bacterium Bacillus cereus b-525k for hexavalent chromium detoxification.

The chromate resistant Gram-positive Bacillus cereus strain b-525k was isolated from tannery effluents, demonstrating optimal propagation at 37 °C and pH 8. The minimum inhibitory concentration (MIC) test showed that B. cereus b-525k can tolerate up to 32 mM Cr6+, and also exhibit the ability to resist other toxic metal ions including Pb2+ (23 mM), As3+ (21 mM), Zn2+ (17 mM), Cd2+ (5 mM), Cu2+ (2 mM), and Ni2+ (3 mM) with the resistance order as Cr 6+ > Pb2+ > As3+ >Zn2+ >Cd2+ >Ni2+ >Cu2+. B. cereus b-525k showed maximum biosorption efficiency (q) of 51 mM Cr6+/g after 6 days. Chromate stress elicited pronounced production of antioxidant enzymes such as catalase (CAT) 191%, glutathione transferase (GST) 192%, superoxide dismutase (SOD) 161%, peroxidase (POX) 199%, and ascorbate peroxidase (APOX) (154%). Within B. cereus b-525k, the influence of Cr6+ stress (2 mM) did stimulate rise in levels of GSH (907%) and non-protein thiols (541%) was measured as compared to the control (without any Cr6+ stress) which markedly nullifies Cr6+ generated oxidative stress. The pilot scale experiments utilizing original tannery effluent showed that B. cereus b-525k could remove 99% Cr6+ in 6 days, thus, it could be a potential candidate to reclaim the chromate contaminated sites.

Multiple upstream start codons (AUG) in 5' untranslated region enhance translation efficiency of cry2Ac11 without helper protein.

Cry2Ac11, a 65 kDa insecticidal protein produced by Bacillus thuringiensis, shows toxicity against dipteran and lepidopteran larvae. It is encoded by cry2Ac11 gene ( orf3), which is part of an operon comprising orf1, orf2, and orf3. Orf2, a helper protein, helps in proper folding and prevents aberrant aggregation of newly produced molecules. In this study, we have elucidated the effect of different mutations in translation initiation region (TIR), particularly the ribosomal binding site and the start codon (RBS-ATG) on cry2Ac11 gene expression without helper protein. All recombinant constructs were expressed in acrystalliferous B. thuringiensis subsp israelensis 4Q7 under the control of strong chimeric promoter cyt1AP/STAB. Of all the mutants, mut/RBS2, with two consecutive AUGs after the spacer region in TIR, exhibited 89- and 2246-fold higher transcript levels compared with 4Q7-operSalI/RBS ( cry2Ac11 operon) and 4Q7-w-RBS ( cry2Ac11 gene), respectively. The analysis of mut/RBS2 messenger RNA (mRNA) structure in the RBS-AUG region showed the presence of RBS in the single-stranded part of the moderately stable hairpin loop. The high expression efficiency of Cry2Ac11 mutant without helper protein is a cumulative and cooperative result of chimeric promoter cyt1AP/STAB-SD with the optimal context of RBS-AUG region provided by multiple AUGs and stabilizer sequence at 3' ends.

Cadmium resistance and uptake by bacterium, Salmonella enterica 43C, isolated from industrial effluent.

Cadmium resistant bacterium, isolated from industrial wastewater, was characterized as Salmonella enterica 43C on the basis of biochemical and 16S rRNA ribotyping. It is first ever reported S. enterica 43C bared extreme resistance against heavy metal consortia in order of Pb(2+)>Cd(2+)>As(3+)>Zn(2+)>Cr(6+)>Cu(2+)>Hg(2+). Cd(2+) stress altered growth pattern of the bacterium in time dependent manner. It could remove nearly 57 % Cd(2+) from the medium over a period of 8 days. Kinetic and thermodynamic studies based on various adsorption isotherm models (Langmuir and Freundlich) depicted the Cd(2+) biosorption as spontaneous, feasible and endothermic in nature. Interestingly, the bacterium followed pseudo first order kinetics, making it a good biosorbent for heavy metal ions. The S. enterica 43C Cd(2+) processivity was significantly influenced by temperature, pH, initial Cd(2+) concentration, biomass dosage and co-metal ions. FTIR analysis of the bacterium revealed the active participation of amide and carbonyl moieties in Cd(2+) adsorption confirmed by EDX analysis. Electron micrographs beckoned further surface adsorption and increased bacterial size due to intracellular Cd(2+) accumulation. An overwhelming increase in glutathione and other non-protein thiols levels played a significant role in thriving oxidative stress generated by metal cations. Presence of metallothionein clearly depicted the role of such proteins in bacterial metal resistance mechanism. The present study results clearly declare S. enterica 43C a suitable candidate for green chemistry to bioremediate environmental Cd(2+).

Molecular characterization, metal uptake and copper induced transcriptional activation of efflux determinants in copper resistant isolates of Klebsiella pneumoniae.

An efflux system, comprising cus determinants, plays an important role in pumping out this metal in gram negative bacteria exposed to high concentration of copper. Cus determinants comprise two operons, one regulatory (cusRS) and the other structural (cusCFBA). Although the efflux system has been described in quite a few members of Enterobacteriaceae, little is known about this system in Klebsiella spp. We are describing cus determinants in Klebsiella pneumoniae for the first time and also providing evidence for their metal-induced expression, both under aerobic and anaerobic conditions. Copper resistant K. pneumoniae, capable of copper uptake and later efflux of excessive copper, was isolated from industrial waste water. Expression of both cusRS and cusCFBA was quantified at transcriptional level through real time PCR. The results demonstrated that cus determinants were functional under both aerobic and anaerobic conditions. The mRNA level of both operons increased several fold in the presence of non-lethal as well as sub-lethal copper concentrations. The increase in cusCFBA transcripts was 74.8 fold 15 min after exposure to 3mM Cu(++) under aerobic conditions compared to the 16 fold increase in cusRS under the same conditions. Under anaerobic conditions the cusCFBA transcripts increased 32.65 fold and the cusRS five fold within 15 min after exposure to 3mM Cu(++). It is concluded that cus genetic determinants in K. pneumoniae comprise structural component (cusCFBA) and a regulatory component (cusRS), which show several fold expression under copper induction both under aerobic and anaerobic conditions. Under aerobic conditions, the structural genes express 4.7 fold more than the regulatory genes, whereas under anaerobic conditions, this expression is 6.5 fold. Finally, time course study revealed a novel pattern of immediate up-regulated expression followed by decreased and another increased in the transcript level of both operons of cus determinants in the presence of copper.

Functional analysis of cus operon promoter of Klebsiella pneumoniae using E. coli lacZ assay.

The transcriptional fusion of reporter lacZ gene with cusRS regulatory promoter of cus operon of Klebsiella pneumoniae enabled us to analyze the inductive effect of copper on promoter via lacZ assay. The stimulus response curve of promoter to a range of copper metal concentrations indicated a normal sigmoidal response profile with apparent Hill coefficient 1.0. There was a positive correlation of promoter response to the increasing concentration of copper in the medium. AC(50) value of copper was calculated to be 1mM, whereas the promoter response was exponential beyond 1mM and up to 2.5mM concentration. The promoter activity did not increase exponentially in copper concentration higher than 2.5mM. The promoter PcusRS requires two chromosomally encoded regulatory proteins, CusS and CusR, in trans for maximal in vitro activation. The PcusRS regulatory promoter sequence also contained regulatory -10 and -35 boxes along with CusR binding motif. The results supported the concept of cus operon regulation as an essential mechanism for maintaining the cellular homeostasis at very high (e.g. 3mM), and even toxic copper concentrations.