Proteomic Analysis of Arsenic Resistance during Cyanide Assimilation by Pseudomonas pseudoalcaligenes CECT 5344

Abstract

Wastewater from mining and other industries usually contains arsenic and cyanide, two highly toxic pollutants, thereby creating the need to develop bioremediation strategies. Here, molecular mechanisms triggered by the simultaneous presence of cyanide and arsenite were analyzed by quantitative proteomics, complemented with qRT-PCR analysis and determination of analytes in the cyanide-assimilating bacterium Pseudomonas pseudoalcaligenes CECT 5344. Several proteins encoded by two ars gene clusters and other Ars-related proteins were up-regulated by arsenite, even during cyanide assimilation. Although some proteins encoded by the cio gene cluster responsible for cyanide-insensitive respiration decreased in the presence of arsenite, the nitrilase NitC required for cyanide assimilation was unaffected, thus allowing bacterial growth with cyanide and arsenic. Two complementary As-resistance mechanisms were developed in this bacterium, the extrusion of As(III) and its extracellular sequestration in biofilm, whose synthesis increased in the presence of arsenite, and the formation of organoarsenicals such as arseno-phosphoglycerate and methyl-As. Tetrahydrofolate metabolism was also stimulated by arsenite. In addition, the ArsH2 protein increased in the presence of arsenite or cyanide, suggesting its role in the protection from oxidative stress caused by both toxics. These results could be useful for the development of bioremediation strategies for industrial wastes co-contaminated with cyanide and arsenic.

Bacterial tolerance and detoxification of cyanide, arsenic and heavy metals: Holistic approaches applied to bioremediation of industrial complex wastes

“…In a bioreactor, the strain CECT 5344 was able to detoxify a jewellery waste containing up to 12 mM total cyanide (free and complexed to metals) (Ibáñez et al., 2017 ). The strain CECT 5344 can also grow in batch cultures with cyanide in the presence of high concentrations of arsenic or mercury (Biełło, Cabello, et al., 2023 , Biełło, Olaya‐Abril, et al., 2023 ). Other bacteria that can degrade metal‐cyanide complexes, including those with zinc, nickel, copper, silver and iron, have been also described (Dimitrova et al., 2020 ; Patil & Paknikar, 2000 ; Silva‐Avalos et al., 1990 ).…”

“…Numerous microorganisms can oxidize arsenite to arsenate, thus providing electrons for oxygen or nitrate respiration in aerobic (AioAB system) or anaerobic (ArxA system) conditions (Mazumder et al., 2020 ). Bacterial strategies for arsenic resistance include arsenic extrusion from the cytosol (Barral‐Fraga et al., 2018 ; Biełło, Cabello, et al., 2023 ; Garbinski et al., 2019 ), intracellular chelation by metal‐binding peptides (Morelli et al., 2005 ), bioaccumulation (Diba et al., 2021 ), immobilization and transformation to potentially less toxic organic forms (Yang & Rosen, 2016 ) (Figure 3 ). Arsenite is usually extruded outside the cell through efflux pumps.…”

“…Thus, accessory genes found in the ars clusters encode the glutaredoxin‐ or thioredoxin‐dependent arsenate reductase ArsC that converts arsenate into arsenite, the ATPase ArsA that improves the function of the ArsB efflux pump, the putative As(III)‐metallochaperone ArsD that transfers arsenite to ArsA, the transmembrane arsenic‐efflux protein Acr3, the S‐ adenosylmethionine dependent methyltransferase ArsM, the methylarsenite oxidase/arsenic resistance protein ArsH, the permease ArsP and the putative C‐As lyase ArsI (Andres & Bertin, 2016 ; Slyemi & Bonnefoy, 2012 ). The major facilitator superfamily (MFS) permease ArsJ that extrudes 1‐arseno‐3‐phosphoglycerate formed by glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), and the ArsT and ArsN proteins of unknown function are also encoded by ars gene clusters (Biełło, Cabello, et al., 2023 ; Chen et al., 2016 ; Huang et al., 2018 ) (Figure 3 ).…”

“…This relationship could be directly linked to the requirement of this cofactor to achieve arsenic methylation via ArsM. Additionally, this process might induce the recycling of S‐ adenosylmethione dependent proteins like NitD, which is encoded by a gene of the nit1C cluster of P. pseudoalcaligenes CECT 5344 that is essential for cyanide detoxification/assimilation in this bacterium (Biełło, Cabello, et al., 2023 ; Estepa et al., 2012 ). A quantitative proteomic analysis carried out to study the mechanisms of tolerance and resistance to arsenite, in the presence or absence of cyanide, has revealed that in this strain the mechanisms of resistance to arsenic include the extrusion of arsenite through the ArsAB efflux pump and the production of arsenical derivatives, such as arseno‐phosphoglycerate formed by an arsenic inducible glyceraldehyde‐3‐phosphate dehydrogenase, or methylarsenite formed by ArsM‐type methyltransferases, which are further exported through ArsJ, ArsP and Acr3 permeases.…”

“…In this sense, different bacteria have been described to be able to resist and degrade elevated concentrations of cyanide, metals and metalloids. In the development of a process for bioremediation of these toxic wastewaters it must be considered the possible interactions of microorganisms with metals and cyanide, the formation of different cyano‐derivatives and the catalysed and non‐catalysed reactions initiated by the reactivity of cyanide with other molecules (Biełło, Cabello, et al., 2023 , Biełło, Olaya‐Abril, et al., 2023 ; Luque‐Almagro et al., 2016 ; Newsome & Falagán, 2021 ).…”