Análisis transcriptómico y proteómico de pseudomonas pseudoalcaligenes cect5344 en respuesta a cianuro

Abstract

Cyanide is a natural toxic compound that displays a very high affinity for metals, inhibiting metaloenzymes which are essential for life. This affinity of cyanide for metals may also be responsible of a decrease in the biodisponibility of essential metals for specific biological processes. A large number of microorganisms are able to synthesize (cyanogenics), tolerate or even assimilate cyanide (cyanotrophics). Plants are the main source of cyanide in nature. Some microorganisms, including cyanogenics, have developed mechanisms of resistance forward cyanide, mainly based on the synthesis of a cyanide insensitive alternative oxidase. In addition to a cyanide resistance system, cyanide assimilation requires a degradation pathway in order to use cyanide as a nitrogen source. Cyanotrophic microorganisms have a significant biotechnological potential because they can be applied in bioremediation of industrial cyanide-containing wastewaters, including jewelry and mining residues. Pseudomonas pseudoalcaligenes CECT5344 is a cyanotrophic bacterium isolated from the Guadalquivir River (Córdoba). This is an alkaliphilic bacterium, with an optimun pH of growth of 9,5-10. This fact allows to establish a very alkaline pH value, higher than the pKa 9,2 of the CN-/HCN pair, thus avoiding HCN losses. Several studies have elucidated the main mechanisms of cyanide resistance, as well as the cyanide assimilation pathway in P. pseudoalcaligenes CECT5344. In this bacterium cyanide chemically reacts with oxaloacetate producing a cyanohydrin that it is further metabolised to ammonia by the nitrilase NitC. Although cyanide resistance and assimilation have been studied in detail in this bacterium, the global response to cyanide of the strain CECT5344 is unknown. Sequencing and analysis of the whole genome of P. pseudoalcaligenes CECT5344 has facilitated to carry out this work that includes transcriptomic and proteomic studies in response to cyanide. Specifically, we have studied iron bioavailability related with cyanide, and the response of P. pseudoalcaligenes CECT5344 to iron starvation at proteomic level has been established. Proteins induced by iron starvation were not present in the proteome of cyanide-asimilating cells, discarding the hypothesis that cyanide causes iron stress in the strain CECT5344. DNA microarrays were performed in order to study the transcriptome of this cyanotrophic strain in response to cyanide, as well as to cyanide-containing wastewater and nitrogen starvation conditions. A functional analysis of the results allowed the identification of specific biological processes affected by cyanide, including the cyanide insensitive oxidase (CIO) for respiration in the presence of cyanide and several nitrilases (one of then, the nitrilase NitC). Specificaly induced by the jewelry residue were genes involved in tolerance to metals like regulatory genes and metal extrusion related genes. Some of the cellular components induced by cyanide, and possibly involved in cyanide resistance, were studied by mutational analysis. Mutant strains deficient in DapA (dihydrodipicolinate synthase) or AhpC (alkyl hydroperoxide reductase) presented a phenotype more sensitive to cyanide, thus confirming the participation of these proteins in cyanide metabolism in P. pseudoalcaligenes CECT5344. The DapA protein could participate in the reorganization of the iron-sulphur clusters, a target for cyanide, while AhpC could be involved in the resistance to oxidative stress generated by cyanide.