Expression of metallothionein encoding gene bmtA in biofilm-forming marine bacterium Pseudomonas aeruginosa N6P6 and understanding its involvement in Pb(II) resistance and bioremediation.

The genetic basis and biochemical aspects of heavy metal endurance abilities have been precisely studied in planktonic bacteria; however, in nature, bacteria mostly grows as surface-attached communities called biofilms. A hallmark trait of biofilm is increased resistance to heavy metals compared with the resistance of planktonic bacteria. A proposed mechanism that contributes to this increased resistance is the enhanced expression of metal-resistant genes. bmtA gene coding for metallothionein protein is one such metal-resistant gene found in many bacterial spp. In the present study, lead (Pb) remediation potential of a biofilm-forming marine bacterium Pseudomonas aeruginosa N6P6 was explored. Biofilm-forming marine bacterium P. aeruginosa N6P6 possess bmtA gene and shows resistance towards many heavy metals, i.e., Pb, Cd, Hg, Cr, and Zn. The expression of metallothionein encoding gene bmtA is significantly high in 48-h-old biofilm culture (11. 4 fold) followed by 24-h-old biofilm culture of P. aeruginosa N6P6 (4.7 fold) (P < 0.05). However, in the case of planktonically grown culture of P. aeruginosa N6P6, the highest expression of bmtA gene was observed in 24-h-old culture. The expression of bmtA also increased significantly with increase in Pb concentration up to 800 ppm. CSLM analysis indicated significant reduction in the raw integrated density of biofilm-associated lipids and polysaccharides (PS) of P. aeruginosa N6P6 biofilm grown in Pb (sub-lethal concentration)-amended medium (P < 0.05), whereas no significant reduction was observed in the raw integrated density of EPS-associated protein. The role of bmtA gene as Pb(II)-resistant determinant was characterized by overexpressing the bmtA gene derived from P. aeruginosa N6P6 in Escherichia coli BL21(DE3). ESI-MS and SDS-PAGE analyses validated the presence of 11.5-kDa MT protein isolated from Pb(II)-induced recombinant E. coli BL21(DE3) harboring bmtA gene.

Metallothionein assisted periplasmic lead sequestration as lead sulfite by Providencia vermicola strain SJ2A.

Lead resistant Providencia vermicola strain SJ2A was isolated from the waste of a battery manufacturing industry which could tolerate upto 3.0mM lead nitrate in the minimal medium. Interestingly, this isolate showed presence of a plasmid borne metallothionein gene, bmtA that matched significantly (96%) with that of Pseudomonas aeruginosa. Scanning electron micrographs of bacterial cells exposed to lead revealed a unique alteration in the cell morphology from rods to long inter-connected filaments. On the other hand, electron dispersive X-ray spectroscopy (EDX) clearly indicated no significant lead adsorption therefore, we speculated intracellular sequestration in this bacterial strain. Transmission electron micrographs of the bacterial cells exposed to lead evidently demonstrated periplasmic sequestration of lead which was also supported by Fourier transformed infrared spectroscopic (FTIR) analysis. The bacterium internalised 155.12mg Pb2+/g biomass as determined by atomic absorption spectroscopy. Subsequently, the accumulated lead was identified as lead sulfite by X-ray diffraction studies. Therefore P. vermicola strain SJ2A has potential to bioremediate lead contaminated environmental sites.

Borreliacidal activity of Borrelia metal transporter A (BmtA) binding small molecules by manganese transport inhibition.

Borrelia burgdorferi, the causative agent of Lyme disease, utilizes manganese (Mn) for its various metabolic needs. We hypothesized that blocking Mn transporter could be a possible approach to inhibit metabolic activity of this pathogen and eliminate the infection. We used a combination of in silico protein structure prediction together with molecular docking to target the Borrelia metal transporter A (BmtA), a single known Mn transporter in Borrelia and screened libraries of FDA approved compounds that could potentially bind to the predicted BmtA structure with high affinity. Tricyclic antihistamines such as loratadine, desloratadine, and 3-hydroxydesloratadine as well as yohimbine and tadalafil demonstrated a tight binding to the in silico folded BmtA transporter. We, then, tested borreliacidal activity and dose response of the shortlisted compounds from this screen using a series of in vitro assays. Amongst the probed compounds, desloratadine exhibited potent borreliacidal activity in vitro at and above 78 µg/mL (250 µM). Borrelia treated with lethal doses of desloratadine exhibited a significant loss of intracellular Mn specifically and a severe structural damage to the bacterial cell wall. Our results support the possibility of developing a novel, targeted therapy to treat Lyme disease by targeting specific metabolic needs of Borrelia.

Mining genomes of marine cyanobacteria for elements of zinc homeostasis.

Zinc is a recognized essential element for the majority of organisms, and is indispensable for the correct function of hundreds of enzymes and thousands of regulatory proteins. In aquatic photoautotrophs including cyanobacteria, zinc is thought to be required for carbonic anhydrase and alkaline phosphatase, although there is evidence that at least some carbonic anhydrases can be cambialistic, i.e., are able to acquire in vivo and function with different metal cofactors such as Co(2+) and Cd(2+). Given the global importance of marine phytoplankton, zinc availability in the oceans is likely to have an impact on both carbon and phosphorus cycles. Zinc concentrations in seawater vary over several orders of magnitude, and in the open oceans adopt a nutrient-like profile. Most studies on zinc handling by cyanobacteria have focused on freshwater strains and zinc toxicity; much less information is available on marine strains and zinc limitation. Several systems for zinc homeostasis have been characterized in the freshwater species Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803, but little is known about zinc requirements or zinc handling by marine species. Comparative metallo-genomics has begun to explore not only the putative zinc proteome, but also specific protein families predicted to have an involvement in zinc homeostasis, including sensors for excess and limitation (SmtB and its homologs as well as Zur), uptake systems (ZnuABC), putative intracellular zinc chaperones (COG0523) and metallothioneins (BmtA), and efflux pumps (ZiaA and its homologs).