Identification of Resistance Genes and Response to Arsenic in Rhodococcus aetherivorans BCP1.

Andrea Firrincieli,Giusi Favoino,Enrica Allevato,Maurizio Petruccioli,Rosita Marabottini,Silvia Rita Stazi,Raymond J Turner,Davide Zannoni,Alessandro Presentato,Martina Cappelletti,Antoine Harfouche,Giuseppe Scarascia Mugnozza

doi:10.3389/fmicb.2019.00888

Abstract

Arsenic (As) ranks among the priority metal(loid)s that are of public health concern. In the environment, arsenic is present in different forms, organic or inorganic, featured by various toxicity levels. Bacteria have developed different strategies to deal with this toxicity involving different resistance genetic determinants. Bacterial strains of Rhodococcus genus, and more in general Actinobacteria phylum, have the ability to cope with high concentrations of toxic metalloids, although little is known on the molecular and genetic bases of these metabolic features. Here we show that Rhodococcus aetherivorans BCP1, an extremophilic actinobacterial strain able to tolerate high concentrations of organic solvents and toxic metalloids, can grow in the presence of high concentrations of As(V) (up to 240 mM) under aerobic growth conditions using glucose as sole carbon and energy source. Notably, BCP1 cells improved their growth performance as well as their capacity of reducing As(V) into As(III) when the concentration of As(V) is within 30–100 mM As(V). Genomic analysis of BCP1 compared to other actinobacterial strains revealed the presence of three gene clusters responsible for organic and inorganic arsenic resistance. In particular, two adjacent and divergently oriented ars gene clusters include three arsenate reductase genes (arsC1/2/3) involved in resistance mechanisms against As(V). A sequence similarity network (SSN) and phylogenetic analysis of these arsenate reductase genes indicated that two of them (ArsC2/3) are functionally related to thioredoxin (Trx)/thioredoxin reductase (TrxR)-dependent class and one of them (ArsC1) to the mycothiol (MSH)/mycoredoxin (Mrx)-dependent class. A targeted transcriptomic analysis performed by RT-qPCR indicated that the arsenate reductase genes as well as other genes included in the ars gene cluster (possible regulator gene, arsR, and arsenite extrusion genes, arsA, acr3, and arsD) are transcriptionally induced when BCP1 cells were exposed to As(V) supplied at two different sub-lethal concentrations. This work provides for the first time insights into the arsenic resistance mechanisms of a Rhodococcus strain, revealing some of the unique metabolic requirements for the environmental persistence of this bacterial genus and its possible use in bioremediation procedures of toxic metal contaminated sites.

Highlights

Arsenic is the most prevalent environmental toxic compound and ranks first on the U.S Environmental Protection Agency’s Superfund List1
Arsenate speciation was assessed by exposing BCP1 cells at subinhibitory concentrations of arsenate As(V), i.e., 6 and 33 mM in order to determine the capability of BCP1 to reduce As(V) into arsenite As(III) and to reveal if this process was dependent on the initial concentration of As(V)
This study demonstrates that BCP1 strain is resistant to arsenic, with Minimal Inhibitory Concentration (MIC) of 8 mM for arsenite As(III) and 240 mM for As(V) (Figure 1), these values being similar to those reported in Corynebacterium glutamicum and in a few arsenic resistant Rhodococcus and other Actinobacteria strains isolated from contaminated sites (Mateos et al, 2006; Jain et al, 2012; Ghosh and Sar, 2013; Shagol et al, 2014; Maizel et al, 2018; RetamalMorales et al, 2018)

Summary

Introduction

Arsenic is the most prevalent environmental toxic compound and ranks first on the U.S Environmental Protection Agency’s Superfund List. Three distinct functional classes of bacterial arsenite reductases (encoded by arsC genes) belong to the low-molecular weight phosphotyrosine phosphatase (LMWP) protein family and are involved in resistance mechanisms and detoxification, being their catalytic activity linked to specific reducing systems: (i) the thioredoxin (Trx)/thioredoxin reductase (TrxR)-dependent class, (ii) the glutathione (GSH)/glutaredoxin (Grx)-coupled class, (iii) and the mycothiol (MSH)/mycoredoxin (Mrx)-dependent class (Páez-Espino et al, 2009). Until now, the latter reducing mechanism has only been described in Actinobacteria (Ordóñez et al, 2009) and among these, only Corynebacterium glutamicum has been studied in detail, despite the high abundance of diverse Actinobacteria genera in arsenic contaminated sites and their high resistance to toxic metals (Bankar and Nagaraja, 2018)

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Discussion

Conclusion