Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, Edificio Severo Ochoa, 1a Planta, Universidad de Córdoba, 14071, Córdoba, Spain.
Environ Microbiol Rep. 2012 Jun;4(3):326-34. doi: 10.1111/j.1758-2229.2012.00337.x. Epub 2012 Mar 30.
A proteomic approach was used to identify several proteins induced by cyanide in the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344, two of them, NitB and NitG, encoded by genes that belong to the nit1C gene cluster. The predicted products of the nit1C gene cluster are a Fis-like σ(54) -dependent transcriptional activator (NitA), a nitrilase (NitC), an S-adenosylmethionine superfamily member (NitD), an N-acyltransferase superfamily member (NitE), a trifunctional polypeptide of the AIRS/GARS family (NitF), an NADH-dependent oxidoreductase (NitH) and two hypothetical proteins of unknown function (NitB and NitG). RT-PCR analysis suggested that nitBCDEFGH genes were co-transcribed, whereas the regulatory nitA gene was divergently transcribed. Real-time RT-PCR revealed that expression of the nitBCDEFGH genes was induced by cyanide and repressed by ammonium. The P. pseudoalcaligenes CECT5344 nit1C gene cluster was found to be involved in assimilation of free and organic cyanides (nitriles) as deduced for the inability to grow with cyanides showed by the NitA, NitB and NitC mutant strains. The wild-type strain CECT5344 showed a nitrilase activity that allows growth on cyanide or hydroxynitriles. The NitB and NitC mutants only presented low basal levels of nitrilase activity that were not enough to support growth on either free cyanide or aliphatic nitriles, suggesting that nitrilase NitC is specific and essential for cyanide and aliphatic nitriles assimilation.
采用蛋白质组学方法鉴定了耐碱假单胞菌 CECT5344 中被氰化物诱导的几种蛋白质，其中两种，NitB 和 NitG，由属于 nit1C 基因簇的基因编码。nit1C 基因簇的预测产物是一个类似于 Fis 的 σ(54)依赖型转录激活因子（NitA）、一种腈酶（NitC）、一种 S-腺苷甲硫氨酸超家族成员（NitD）、一种 N-酰基转移酶超家族成员（NitE）、一种 AIRS/GARS 家族的三功能多肽（NitF）、一种 NADH 依赖性氧化还原酶（NitH）和两个未知功能的假定蛋白（NitB 和 NitG）。RT-PCR 分析表明，nitBCDEFGH 基因是共转录的，而调节 nitA 基因是反向转录的。实时 RT-PCR 显示，nitBCDEFGH 基因的表达被氰化物诱导，被铵抑制。推测假单胞菌 CECT5344 的 nit1C 基因簇参与了游离和有机氰化物（腈）的同化，这是由 NitA、NitB 和 NitC 突变株无法利用氰化物生长推断出来的。野生型菌株 CECT5344 表现出腈酶活性，允许在氰化物或羟腈上生长。NitB 和 NitC 突变体仅表现出低水平的基础腈酶活性，不足以支持在游离氰化物或脂肪族腈上生长，这表明腈酶 NitC 是氰化物和脂肪族腈同化的特异性和必需的。
