Olaya-Abril Alfonso, Pérez María Dolores, Cabello Purificación, Martignetti Diego, Sáez Lara Paloma, Luque-Almagro Víctor Manuel, Moreno-Vivián Conrado, Roldán María Dolores
Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Córdoba, Spain.
Departamento de Botánica, Ecología y Fisiología Vegetal, Edificio Celestino Mutis, Campus de Rabanales, Universidad de Córdoba, Córdoba, Spain.
Front Microbiol. 2020 Jan 23;11:28. doi: 10.3389/fmicb.2020.00028. eCollection 2020.
Cyanide is a toxic compound widely used in mining and jewelry industries, as well as in the synthesis of many different chemicals. Cyanide toxicity derives from its high affinity for metals, which causes inhibition of relevant metalloenzymes. However, some cyanide-degrading microorganisms like the alkaliphilic bacterium CECT5344 may detoxify hazardous industrial wastewaters that contain elevated cyanide and metal concentrations. Considering that iron availability is strongly reduced in the presence of cyanide, mechanisms for iron homeostasis should be required for cyanide biodegradation. Previous omic studies revealed that in the presence of a cyanide-containing jewelry residue the strain CECT5344 overproduced the dihydrodipicolinate synthase DapA1, a protein involved in lysine metabolism that also participates in the synthesis of dipicolinates, which are excellent metal chelators. In this work, a mutant of CECT5344 has been generated and characterized. This mutant showed reduced growth and cyanide consumption in media with the cyanide-containing wastewater. Intracellular levels of metals like iron, copper and zinc were increased in the mutant, especially in cells grown with the jewelry residue. In addition, a differential quantitative proteomic analysis by LC-MS/MS was carried out between the wild-type and the mutant strains in media with jewelry residue. The mutation in the gene altered the expression of several proteins related to urea cycle and metabolism of arginine and other amino acids. Additionally, the mutant showed increased levels of the global nitrogen regulator PII and the glutamine synthetase. This proteomic study has also highlighted that the DapA1 protein is relevant for cyanide resistance, oxidative stress and iron homeostasis response, which is mediated by the ferric uptake regulator Fur. DapA1 is required to produce dipicolinates that could act as iron chelators, conferring protection against oxidative stress and allowing the regeneration of Fe-S centers to reactivate cyanide-damaged metalloproteins.
氰化物是一种有毒化合物，广泛应用于采矿业和珠宝行业，以及许多不同化学品的合成中。氰化物毒性源于其对金属的高亲和力，这会导致相关金属酶的抑制。然而，一些氰化物降解微生物，如嗜碱细菌CECT5344，可以对含有高浓度氰化物和金属的危险工业废水进行解毒。考虑到在氰化物存在的情况下铁的可用性会大大降低，氰化物生物降解应该需要铁稳态机制。先前的组学研究表明，在含有氰化物的珠宝残渣存在的情况下，菌株CECT5344过量产生二氢吡啶二羧酸合酶DapA1，这是一种参与赖氨酸代谢的蛋白质，也参与二吡啶甲酸盐的合成，二吡啶甲酸盐是优秀的金属螯合剂。在这项工作中，已经构建并表征了CECT5344的一个突变体。该突变体在含有氰化物废水的培养基中生长和氰化物消耗减少。突变体中细胞内铁、铜和锌等金属的水平增加，特别是在用珠宝残渣培养的细胞中。此外，在含有珠宝残渣的培养基中，对野生型和突变体菌株进行了液相色谱-串联质谱的差异定量蛋白质组学分析。该基因突变改变了几种与尿素循环以及精氨酸和其他氨基酸代谢相关的蛋白质的表达。此外，突变体中全局氮调节因子PII和谷氨酰胺合成酶的水平增加。这项蛋白质组学研究还强调，DapA1蛋白与抗氰化物、氧化应激和铁稳态反应相关，这是由铁摄取调节因子Fur介导的。需要DapA1来产生可以作为铁螯合剂的二吡啶甲酸盐，提供对氧化应激的保护，并允许Fe-S中心再生以重新激活被氰化物破坏的金属蛋白。
