Antonioli Paolo, Lampis Silvia, Chesini Irene, Vallini Giovanni, Rinalducci Sara, Zolla Lello, Righetti Pier Giorgio
Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Milan, Italy.
Appl Environ Microbiol. 2007 Nov;73(21):6854-63. doi: 10.1128/AEM.00957-07. Epub 2007 Sep 7.
Biochemical and proteomic tools have been utilized for investigating the mechanism of action of a new Stenotrophomonas maltophilia strain (SeITE02), a gammaproteobacterium capable of resistance to high concentrations of selenite [SeO(3)(2-), Se(IV)], reducing it to nontoxic elemental selenium under aerobic conditions; this strain was previously isolated from a selenite-contaminated mining soil. Biochemical analysis demonstrated that (i) nitrite reductase does not seem to take part in the process of selenite reduction by the bacterial strain SeITE02, although its involvement in this process had been hypothesized in other cases; (ii) nitrite strongly interferes with selenite removal when the two oxyanions (NO(2)(-) and SeO(3)(2-)) are simultaneously present, suggesting that the two reduction/detoxification pathways share a common enzymatic step, probably at the level of cellular transport; (iii) in vitro, selenite reduction does not take place in the membrane or periplasmic fractions but only in the cytoplasm, where maximum activity is exhibited at pH 6.0 in the presence of NADPH; and (iv) glutathione is involved in the selenite reduction mechanism, since inhibition of its synthesis leads to a considerable delay in the onset of reduction. As far as the proteomic findings are concerned, the evidence was reached that 0.2 mM selenite and 16 mM nitrite, when added to the culture medium, caused a significant modulation (ca. 10%, i.e., 96 and 85 protein zones, respectively) of the total proteins visualized in the respective two-dimensional maps. These spots were identified by mass spectrometry analysis and were found to belong to the following functional classes: nucleotide synthesis and metabolism, damaged-protein catabolism, protein and amino acid metabolism, and carbohydrate metabolism along with DNA-related proteins and proteins involved in cell division, oxidative stress, and cell wall synthesis.
生化和蛋白质组学工具已被用于研究嗜麦芽窄食单胞菌新菌株(SeITE02)的作用机制,该菌株是一种γ-变形菌,能够抵抗高浓度的亚硒酸盐[SeO(3)(2-), Se(IV)],并在有氧条件下将其还原为无毒的元素硒;该菌株先前从受亚硒酸盐污染的采矿土壤中分离得到。生化分析表明:(i)亚硝酸盐还原酶似乎不参与细菌菌株SeITE02还原亚硒酸盐的过程,尽管在其他情况下曾推测其参与该过程;(ii)当两种含氧阴离子(NO(2)(-)和SeO(3)(2-))同时存在时,亚硝酸盐强烈干扰亚硒酸盐的去除,这表明两种还原/解毒途径共享一个共同的酶促步骤,可能在细胞转运水平;(iii)在体外,亚硒酸盐还原不在膜或周质部分发生,而仅在细胞质中发生,在pH 6.0且存在NADPH的情况下表现出最大活性;(iv)谷胱甘肽参与亚硒酸盐还原机制,因为其合成受到抑制会导致还原开始出现相当大的延迟。就蛋白质组学研究结果而言,有证据表明,当向培养基中添加0.2 mM亚硒酸盐和16 mM亚硝酸盐时,各自二维图谱中可视化的总蛋白质会发生显著调节(约10%,即分别为96和85个蛋白区)。通过质谱分析鉴定了这些斑点,发现它们属于以下功能类别:核苷酸合成与代谢、受损蛋白分解代谢、蛋白质和氨基酸代谢、碳水化合物代谢以及与DNA相关的蛋白质和参与细胞分裂、氧化应激和细胞壁合成的蛋白质。