Laboratory of Environmental Microbiology and Ecology (LEnME), Department of Life Science, National Institute of Technology, Rourkela, Odisha, 769 008, India.
Environ Sci Pollut Res Int. 2019 Oct;26(28):28763-28774. doi: 10.1007/s11356-019-05916-2. Epub 2019 Aug 2.
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.
浮游细菌中已经精确研究了重金属耐受能力的遗传基础和生化方面；然而，在自然界中，细菌主要以附着在表面的群落形式生长，称为生物膜。生物膜的一个显著特征是与浮游细菌的抗性相比，其对重金属的抗性增加。一种被认为有助于增加这种抗性的机制是增强金属抗性基因的表达。编码金属硫蛋白的 bmtA 基因是许多细菌 spp 中发现的一种金属抗性基因。在本研究中，探索了形成生物膜的海洋细菌铜绿假单胞菌 N6P6 对铅 (Pb) 的修复潜力。形成生物膜的海洋细菌铜绿假单胞菌 N6P6 拥有 bmtA 基因，并对许多重金属，即 Pb、Cd、Hg、Cr 和 Zn 表现出抗性。在 48 小时龄生物膜培养物中，金属硫蛋白编码基因 bmtA 的表达显著升高（11.4 倍），其次是铜绿假单胞菌 N6P6 的 24 小时龄生物膜培养物（4.7 倍）（P<0.05）。然而，在铜绿假单胞菌 N6P6 的浮游培养物的情况下，在 24 小时龄培养物中观察到 bmtA 基因的最高表达。bmtA 的表达也随着 Pb 浓度的增加而显著增加，最高可达 800 ppm。CSLM 分析表明，在 Pb（亚致死浓度）添加培养基中生长的铜绿假单胞菌 N6P6 生物膜的生物膜相关脂类和多糖（PS）的原始积分密度显著降低（P<0.05），而 EPS 相关蛋白的原始积分密度没有观察到显著降低。通过在大肠杆菌 BL21(DE3)中过表达来自铜绿假单胞菌 N6P6 的 bmtA 基因，表征了 bmtA 基因作为 Pb(II)-抗性决定因素的作用。ESI-MS 和 SDS-PAGE 分析验证了从 Pb(II)诱导的含有 bmtA 基因的重组大肠杆菌 BL21(DE3)中分离出的 11.5 kDa MT 蛋白的存在。
