Departamento de Bioquímica y Biología Molecular, Edificio Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, Córdoba, Spain.
Servicio Central de Apoyo a la Investigación (SCAI), Unidad de Proteómica, Campus de Rabanales, Córdoba, Spain.
PLoS One. 2019 Feb 8;14(2):e0212032. doi: 10.1371/journal.pone.0212032. eCollection 2019.
The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 uses free cyanide and several metal-cyanide complexes as the sole nitrogen source and tolerates high concentrations of metals like copper, zinc and iron, which are present in the jewelry wastewaters. To understand deeply the regulatory mechanisms involved in the transcriptional regulation of cyanide-containing wastewaters detoxification by P. pseudoalcaligenes CECT5344, RNA-Seq has been performed from cells cultured with a cyanide-containing jewelry wastewater, sodium cyanide or ammonium chloride as the sole nitrogen source. Small RNAs (sRNAs) that may have potential regulatory functions under cyanotrophic conditions were identified. In total 20 sRNAs were identified to be differentially expressed when compared the jewelry residue versus ammonium as nitrogen source, 16 of which could be amplified successfully by RT-PCR. As predicted targets of these 16 sRNAs were several components of the nit1C gene cluster encoding the nitrilase NitC essential for cyanide assimilation, the cioAB gene cluster that codes for the cyanide-insensitive cytochrome bd-type terminal oxidase, the medium length-polyhydroxyalkanoates (ml-PHAs) gene cluster, and gene clusters related with a global nitrogen limitation response like those coding for glutamine synthase and urease. Other targets were non-clustered genes (or their products) involved in metal resistance and iron acquisition, such as metal extrusion systems and the ferric uptake regulatory (Fur) protein, and a GntR-like regulatory family member probably involved in the regulation of the cyanide assimilation process in the strain CECT5344. Induction of genes targeted by sRNAs in the jewelry residue was demonstrated by qRT-PCR.
耐碱假单胞菌 Pseudomonas pseudoalcaligenes CECT5344 可将游离氰化物和几种金属氰化物络合物用作唯一氮源，并耐受高浓度的金属，如铜、锌和铁，这些金属存在于珠宝废水中。为了深入了解 Pseudomonas pseudoalcaligenes CECT5344 对含氰废水解毒的转录调控机制，我们对以含氰珠宝废水、氰化钠或氯化铵作为唯一氮源培养的细胞进行了 RNA-Seq 分析。鉴定了在氰同化条件下可能具有潜在调控功能的小 RNA（sRNA）。与以珠宝残渣作为氮源相比，当以珠宝残渣作为氮源时，共鉴定出 20 个差异表达的 sRNA，其中 16 个可通过 RT-PCR 成功扩增。由于这些 16 个 sRNA 的预测靶标是编码氰化物同化所必需的腈酶 NitC 的 nit1C 基因簇、编码对氰化物不敏感的细胞色素 bd 型末端氧化酶 cioAB 基因簇、中长链聚羟基烷酸（ml-PHAs）基因簇，以及编码与全局氮限制反应相关的基因簇，如编码谷氨酰胺合成酶和脲酶的基因簇。其他靶标是非聚类基因（或其产物），如金属抗性和铁获取相关基因，如金属外排系统和铁摄取调节（Fur）蛋白，以及一个可能参与调控菌株 CECT5344 氰化物同化过程的 GntR 样调控家族成员。通过 qRT-PCR 证明了 sRNA 靶向珠宝残渣中基因的诱导。
