Molecular Factors of Hypochlorite Tolerance in the Hypersaline Archaeon Haloferax volcanii.

Miguel Gomez,Lana J Mcmillan,Harri Savilahti,Alexander Pillai,Saija Kiljunen,Swathi Dantuluri,Whinkie Leung,Zyan Gani,Julie A Maupin-Furlow,Sungmin Hwang

doi:10.3390/genes9110562

Miguel Gomez, Lana J Mcmillan + Show 8 more

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https://doi.org/10.3390/genes9110562

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Abstract

Halophilic archaea thrive in hypersaline conditions associated with desiccation, ultraviolet (UV) irradiation and redox active compounds, and thus are naturally tolerant to a variety of stresses. Here, we identified mutations that promote enhanced tolerance of halophilic archaea to redox-active compounds using Haloferax volcanii as a model organism. The strains were isolated from a library of random transposon mutants for growth on high doses of sodium hypochlorite (NaOCl), an agent that forms hypochlorous acid (HOCl) and other redox acid compounds common to aqueous environments of high concentrations of chloride. The transposon insertion site in each of twenty isolated clones was mapped using the following: (i) inverse nested two-step PCR (INT-PCR) and (ii) semi-random two-step PCR (ST-PCR). Genes that were found to be disrupted in hypertolerant strains were associated with lysine deacetylation, proteasomes, transporters, polyamine biosynthesis, electron transfer, and other cellular processes. Further analysis revealed a ΔpsmA1 (α1) markerless deletion strain that produces only the α2 and β proteins of 20S proteasomes was hypertolerant to hypochlorite stress compared with wild type, which produces α1, α2, and β proteins. The results of this study provide new insights into archaeal tolerance of redox active compounds such as hypochlorite.

Highlights

Reactive oxygen species (ROS) and other redox-active compounds can overwhelm the antioxidant mechanisms of a cell and cause damage to most biomolecules including proteins, nucleic acids, lipids, and carbohydrates [1,2]
Our prior work demonstrated that H. volcanii responds to hypochlorite stress in a manner that can be quantified at the proteome level by stable isotope labeling in cell culture (SILAC) coupled with tandem mass spectrometry analysis (LC-MS/MS) [15]. To further understand these responses on a global scale, we report the development of an approach to select for H. volcanii mutants that are tolerant of extreme doses of NaOCl on a defined medium
We developed an assay to select and isolate H. volcanii mutant strains from a random transposon library that displayed enhanced tolerance to hypochlorite stress when grown on glycerol

Summary

Introduction

Reactive oxygen species (ROS) and other redox-active compounds can overwhelm the antioxidant mechanisms of a cell and cause damage to most biomolecules including proteins, nucleic acids, lipids, and carbohydrates [1,2]. Oxidation can lead to mutations in DNA by generating single- and double-stranded breaks in the backbone, crosslinks (interstrand and intrastrand), and adducts of bases and sugars [3]. Protein oxidation is disruptive, as it leads to protein misfolding, aggregation, breaks in the protein backbone, modified amino acid residues, and loss of catalytic function causing bottlenecks in metabolism [5]. Hypochlorous acid (HOCl) and its derivatives are redox-active compounds commonly encountered in environments of high concentrations of chloride [7,8]. Sodium hypochlorite (NaOCl) forms sodium hydroxide (NaOH) and the strong oxidant HOCl, which can dissociate into hydroxide (OH− ) and hypochlorite (OCl− ) anions [2]

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