Quantitative Mass Spectrometry by SILAC in Haloferax volcanii.

The development of mass spectrometry (MS)-based proteomics methods has been critical in providing new insight about cellular processes and adaptations in all domains of life. While traditional MS-based methods are not inherently quantitative, technologies are now available to overcome this limitation. Of note, stable isotope labeling of amino acids in cell culture (SILAC) is reported as a reliable tool to label proteomes for quantitative MS-based proteomics that is accurate and flexible for multiplexing. The isotopically labeled lysine and arginine are easily incorporated into the proteome of cells auxotrophic for these amino acids. Microorganisms of the domain Archaea provide a fascinating alternative to understanding cellular adaptations and responses to environmental stresses. However, the availability of preferred SILAC-based quantitative analyses is limited. This protocol describes the use of SILAC to quantitatively analyze the proteome of Haloferax volcanii, a mesophilic halophilic archaeon that is easy to grow and requires no special equipment to maintain.

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

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 ß proteins of 20S proteasomes was hypertolerant to hypochlorite stress compared with wild type, which produces α1, α2, and ß proteins. The results of this study provide new insights into archaeal tolerance of redox active compounds such as hypochlorite.

GlpR Is a Direct Transcriptional Repressor of Fructose Metabolic Genes in Haloferax volcanii.

DeoR-type helix-turn-helix (HTH) domain proteins are transcriptional regulators of sugar and nucleoside metabolism in diverse bacteria and also occur in select archaea. In the model archaeon Haloferax volcanii, previous work implicated GlpR, a DeoR-type transcriptional regulator, in the transcriptional repression of glpR and the gene encoding the fructose-specific phosphofructokinase (pfkB) during growth on glycerol. However, the global regulon governed by GlpR remained unclear. Here, we compared transcriptomes of wild-type and ΔglpR mutant strains grown on glycerol and glucose to detect significant transcript level differences for nearly 50 new genes regulated by GlpR. By coupling computational prediction of GlpR binding sequences with in vivo and in vitro DNA binding experiments, we determined that GlpR directly controls genes encoding enzymes involved in fructose degradation, including fructose bisphosphate aldolase, a central control point in glycolysis. GlpR also directly controls other transcription factors. In contrast, other metabolic pathways appear to be under the indirect influence of GlpR. In vitro experiments demonstrated that GlpR purifies to function as a tetramer that binds the effector molecule fructose-1-phosphate (F1P). These results suggest that H. volcanii GlpR functions as a direct negative regulator of fructose degradation during growth on carbon sources other than fructose, such as glucose and glycerol, and that GlpR bears striking functional similarity to bacterial DeoR-type regulators.IMPORTANCE Many archaea are extremophiles, able to thrive in habitats of extreme salinity, pH and temperature. These biological properties are ideal for applications in biotechnology. However, limited knowledge of archaeal metabolism is a bottleneck that prevents the broad use of archaea as microbial factories for industrial products. Here, we characterize how sugar uptake and use are regulated in a species that lives in high salinity. We demonstrate that a key sugar regulatory protein in this archaeal species functions using molecular mechanisms conserved with distantly related bacterial species.

Multiplex quantitative SILAC for analysis of archaeal proteomes: a case study of oxidative stress responses.

Stable isotope labelling of amino acids in cell culture (SILAC) is a quantitative proteomic method that can illuminate new pathways used by cells to adapt to different lifestyles and niches. Archaea, while thriving in extreme environments and accounting for ∼20%-40% of the Earth's biomass, have not been analyzed with the full potential of SILAC. Here, we report SILAC for quantitative comparison of archaeal proteomes, using Haloferax volcanii as a model. A double auxotroph was generated that allowed for complete incorporation of 13 C/15 N-lysine and 13 C-arginine such that each peptide derived from trypsin digestion was labelled. This strain was found amenable to multiplex SILAC by case study of responses to oxidative stress by hypochlorite. A total of 2565 proteins was identified by LC-MS/MS analysis (q-value ≤ 0.01) that accounted for 64% of the theoretical proteome. Of these, 176 proteins were altered at least 1.5-fold (p-value < 0.05) in abundance during hypochlorite stress. Many of the differential proteins were of unknown function. Those of known function included transcription factor homologs related to oxidative stress by 3D-homology modelling and orthologous group comparisons. Thus, SILAC is found to be an ideal method for quantitative proteomics of archaea that holds promise to unravel gene function.

Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents.

Soluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PPi) to orthophosphate (Pi) are commonly used to accelerate and detect biosynthetic reactions that generate PPi as a by-product. Current PPAs are inactivated by high salt concentrations and organic solvents, which limits the extent of their use. Here we report a class A type PPA of the haloarchaeon Haloferax volcanii (HvPPA) that is thermostable and displays robust PPi-hydrolyzing activity under conditions of 25% (vol/vol) organic solvent and salt concentrations from 25 mM to 3 M. HvPPA was purified to homogeneity as a homohexamer by a rapid two-step method and was found to display non-Michaelis-Menten kinetics with a Vmax of 465 U · mg(-1) for PPi hydrolysis (optimal at 42°C and pH 8.5) and Hill coefficients that indicated cooperative binding to PPi and Mg(2+). Similarly to other class A type PPAs, HvPPA was inhibited by sodium fluoride; however, hierarchical clustering and three-dimensional (3D) homology modeling revealed HvPPA to be distinct in structure from characterized PPAs. In particular, HvPPA was highly negative in surface charge, which explained its extreme resistance to organic solvents. To demonstrate that HvPPA could drive thermodynamically unfavorable reactions to completion under conditions of reduced water activity, a novel coupled assay was developed; HvPPA hydrolyzed the PPi by-product generated in 2 M NaCl by UbaA (a "salt-loving" noncanonical E1 enzyme that adenylates ubiquitin-like proteins in the presence of ATP). Overall, we demonstrate HvPPA to be useful for hydrolyzing PPi under conditions of reduced water activity that are a hurdle to current PPA-based technologies.