Halophilic Archaeon Halobacterium Salinarum Research Articles

A comprehensive spectral assay library to quantify the Halobacterium salinarum NRC-1 proteome by DIA/SWATH-MS

Data-Independent Acquisition (DIA) is a mass spectrometry-based method to reliably identify and reproducibly quantify large fractions of a target proteome. The peptide-centric data analysis strategy employed in DIA requires a priori generated spectral assay libraries. Such assay libraries allow to extract quantitative data in a targeted approach and have been generated for human, mouse, zebrafish, E. coli and few other organisms. However, a spectral assay library for the extreme halophilic archaeon Halobacterium salinarum NRC-1, a model organism that contributed to several notable discoveries, is not publicly available yet. Here, we report a comprehensive spectral assay library to measure 2,563 of 2,646 annotated H. salinarum NRC-1 proteins. We demonstrate the utility of this library by measuring global protein abundances over time under standard growth conditions. The H. salinarum NRC-1 library includes 21,074 distinct peptides representing 97% of the predicted proteome and provides a new, valuable resource to confidently measure and quantify any protein of this archaeon. Data and spectral assay libraries are available via ProteomeXchange (PXD042770, PXD042774) and SWATHAtlas (SAL00312-SAL00319).

Similar mutation rates but different mutation spectra in moderate and extremely halophilic archaea.

Archaea are a major part of Earth's microbiota and extremely diverse. Yet, we know very little about the process of mutation that drives such diversification. To expand beyond previous work with the moderate halophilic archaeal species Haloferax volcanii, we performed a mutation-accumulation experiment followed by whole-genome sequencing in the extremely halophilic archaeon Halobacterium salinarum. Although Hfx. volcanii and Hbt. salinarum have different salt requirements, both species have highly polyploid genomes and similar GC content. We accumulated mutations for an average of 1250 generations in 67 mutation accumulation lines of Hbt. salinarum, and revealed 84 single-base substitutions and 10 insertion-deletion mutations. The estimated base-substitution mutation rate of 3.99 × 10-10 per site per generation or 1.0 × 10-3 per genome per generation in Hbt. salinarum is similar to that reported for Hfx. volcanii (1.2 × 10-3 per genome per generation), but the genome-wide insertion-deletion rate and spectrum of mutations are somewhat dissimilar in these archaeal species. The spectra of spontaneous mutations were AT biased in both archaea, but they differed in significant ways that may be related to differences in the fidelity of DNA replication/repair mechanisms or a simple result of the different salt concentrations.

Engineering and Production of the Light-Driven Proton Pump Bacteriorhodopsin in 2D Crystals for Basic Research and Applied Technologies.

The light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum is a retinal-binding protein, which forms highly ordered and thermally stable 2D crystals in native membranes (termed purple membranes). BR and purple membranes (PMs) have been and are still being intensively studied by numerous researchers from different scientific disciplines. Furthermore, PMs are being successfully used in new, emerging technologies such as bioelectronics and bionanotechnology. Most published studies used the wild-type form of BR, because of the intrinsic difficulty to produce genetically modified versions in purple membranes homologously. However, modification and engineering is crucial for studies in basic research and, in particular, to tailor BR for specific applications in applied sciences. We present an extensive and detailed protocol ranging from the genetic modification and cultivation of H. salinarum to the isolation, and biochemical, biophysical and functional characterization of BR and purple membranes. Pitfalls and problems of the homologous expression of BR versions in H. salinarum are discussed and possible solutions presented. The protocol is intended to facilitate the access to genetically modified BR versions for researchers of different scientific disciplines, thus increasing the application of this versatile biomaterial.

Catalytic mechanism and evolutionary characteristics of thioredoxin from Halobacterium salinarum NRC-1

Thioredoxin (TRX) is an important antioxidant against oxidative stress. TRX from the extremely halophilic archaeon Halobacterium salinarum NRC-1 (HsTRX-A), which has the highest acidic residue content [(Asp + Glu)/(Arg + Lys + His) = 9.0] among known TRXs, was chosen to elucidate the catalytic mechanism and evolutionary characteristics associated with haloadaptation. X-ray crystallographic analysis revealed that the main-chain structure of HsTRX-A is similar to those of homologous TRXs; for example, the root-mean-square deviations on Cα atoms were <2.3 Å for extant archaeal TRXs and <1.5 Å for resurrected Precambrian TRXs. A unique water network was located near the active-site residues (Cys45 and Cys48) in HsTRX-A, which may enhance the proton transfer required for the reduction of substrates under a high-salt environment. The high density of negative charges on the molecular surface (3.6 × 10-3 e Å-2) should improve the solubility and haloadaptivity. Moreover, circular-dichroism measurements and enzymatic assays using a mutant HsTRX-A with deletion of the long flexible N-terminal region (Ala2-Pro17) revealed that Ala2-Pro17 improves the structural stability and the enzymatic activity of HsTRX-A under high-salt environments (>2 M NaCl). The elongation of the N-terminal region in HsTRX-A accompanies the increased hydrophilicity and acidic residue content but does not affect the structure of the active site. These observations offer insights into molecular evolution for haloadaptation and potential applications in halophilic protein-related biotechnology.

The extremely halophilic archaeon Halobacterium salinarum ETD5 from the solar saltern of Sfax (Tunisia) produces multiple halocins

The extremely halophilic archaeon Halobacterium salinarum strain ETD5 was previously isolated from the solar saltern of Sfax (Tunisia) and shown to encode and express halocin S8. The Hbt. salinarum ETD5 culture supernatant was shown here to exhibit high antimicrobial activity against several halophilic archaea and bacteria of different genera, showing a cross-domain inhibition. The antimicrobial activity was destroyed by proteases, thus pointing to halocins. A bioguided purification procedure was applied using two chromatography steps and antimicrobial assays directed against Halorubrum chaoviator ETR14. In-gel screening assay showed the presence of two antimicrobial bands of approximately 8 and 14 kDa, for which characterization was investigated by N-terminal sequencing and mass spectrometry. The full-length form of halocin S8 that contains 81 amino acids and differs from the 36 amino acid short-length halocin S8 previously described from an uncharacterized haloarchaeon S8a, was identified in the 8 kDa halocin band. A novel halocin that we termed halocin S14 was found in the 14 kDa band. It exhibits amino acid sequence identities with the N-terminally truncated region of the archaeal Mn-superoxide dismutase. These results show that Hbt. salinarum ETD5 produces multiple halocins, a feature that had not been described until now in the domain Archaea.

The Primary Antisense Transcriptome of Halobacterium salinarum NRC-1.

Antisense RNAs (asRNAs) are present in diverse organisms and play important roles in gene regulation. In this work, we mapped the primary antisense transcriptome in the halophilic archaeon Halobacterium salinarum NRC-1. By reanalyzing publicly available data, we mapped antisense transcription start sites (aTSSs) and inferred the probable 3′ ends of these transcripts. We analyzed the resulting asRNAs according to the size, location, function of genes on the opposite strand, expression levels and conservation. We show that at least 21% of the genes contain asRNAs in H. salinarum. Most of these asRNAs are expressed at low levels. They are located antisense to genes related to distinctive characteristics of H. salinarum, such as bacteriorhodopsin, gas vesicles, transposases and other important biological processes such as translation. We provide evidence to support asRNAs in type II toxin–antitoxin systems in archaea. We also analyzed public Ribosome profiling (Ribo-seq) data and found that ~10% of the asRNAs are ribosome-associated non-coding RNAs (rancRNAs), with asRNAs from transposases overrepresented. Using a comparative transcriptomics approach, we found that ~19% of the asRNAs annotated in H. salinarum belong to genes with an ortholog in Haloferax volcanii, in which an aTSS could be identified with positional equivalence. This shows that most asRNAs are not conserved between these halophilic archaea.

Archaeal Flagella as Biotemplates for Nanomaterials with New Properties.

At the end of 1980s, regions of the polypeptide chain of bacterial flagella subunits (flagellins) responsible for different properties of these protein polymers were identified by structural studies. It was found that the N- and C-terminal regions are responsible for the polymerization properties of subunits, and the central region is responsible for antigenic properties of the flagellum. Soon after that, it was proposed to use variability of the central flagellin domain for directed modification to impart new properties to the flagellum surface. Such studies of flagella and other polymeric structures of bacterial origin thrived. However bacterial polymers have some shortcomings, mainly their instability to dissociating effects. This shortcoming is absent in archaeal flagella. A limiting factor was the lack of the three-dimensional structure of archaeal flagellins. A method was developed that allowed modifying flagella of the halophilic archaeon Halobacterium salinarum in a peptide that connects positively charged ions. Later, corresponding procedures were used that allowed preparing the anode material for a lithium-ion battery whose characteristics 4-5-fold exceeded those of batteries commonly used in industrial production. We describe other advantages of archaeal flagella over bacterial analogs when used in nanotechnology.

Global transcriptional regulator TrmB family members in prokaryotes.

Members of the TrmB family act as global transcriptional regulators for the activation or repression of sugar ABC transporters and central sugar metabolic pathways, including glycolytic, gluconeogenic, and other metabolic pathways, and also as chromosomal stabilizers in archaea. As a relatively newly classified transcriptional regulator family, there is limited experimental evidence for their role in Thermococcales, halophilic archaeon Halobacterium salinarum NRC1, and crenarchaea Sulfolobus strains, despite being one of the extending protein families in archaea. Recently, the protein structures of Pyrococcus furiosus TrmB and TrmBL2 were solved, and the transcriptomic data uncovered by microarray and ChIP-Seq were published. In the present review, recent evidence of the functional roles of TrmB family members in archaea is explained and extended to bacteria.

Direct observation of rotation and steps of the archaellum in the swimming halophilic archaeon Halobacterium salinarum

Motile archaea swim using a rotary filament, the archaellum, a surface appendage that resembles bacterial flagella structurally, but is homologous to bacterial type IV pili. Little is known about the mechanism by which archaella produce motility. To gain insights into this mechanism, we characterized archaellar function in the model organism Halobacterium salinarum. Three-dimensional tracking of quantum dots enabled visualization of the left-handed corkscrewing of archaea in detail. An advanced analysis method combined with total internal reflection fluorescence microscopy, termed cross-kymography, was developed and revealed a right-handed helical structure of archaella with a rotation speed of 23 ± 5 Hz. Using these structural and kinetic parameters, we computationally reproduced the swimming and precession motion with a hydrodynamic model and estimated the archaellar motor torque to be 50 pN nm. Finally, in a tethered-cell assay, we observed intermittent pauses during rotation with ∼36° or 60° intervals, which we speculate may be a unitary step consuming a single adenosine triphosphate molecule, which supplies chemical energy of 80 pN nm when hydrolysed. From an estimate of the energy input as ten or six adenosine triphosphates per revolution, the efficiency of the motor is calculated to be ∼6-10%.

Structure of Halorhodopsin from Halobacterium salinarum in a new crystal form that imposes little restraint on the E-F loop.

Halorhodopsin from the halophilic archaeon Halobacterium salinarum is a membrane located light-driven chloride pump. Upon illumination Halorhodopsin undergoes a reversible photocycle initiated by the all-trans to 13-cis isomerization of the covalently bound retinal chromophore. The photocycle consists of several spectroscopically distinct intermediates. The structural basis of the chloride transport mechanism remains elusive, presumably because packing contacts have so far precluded protein conformational changes in the available crystals. With the intention to structurally characterize late photocycle intermediates by X-ray crystallography we crystallized Halorhodopsin in a new crystal form using the vesicle fusion method. In the new crystal form lateral contacts are mediated by helices A and G. Helices E and F that were suggested to perform large movements during the photocycle are almost unrestrained by packing contacts. This feature might permit the displacement of these helices without disrupting the crystal lattice. Therefore, this new crystal form might be an excellent system for the structural characterization of late Halorhodopsin photocycle intermediates by trapping or by time resolved experiments, especially at XFELs.

Effects of intracellular Mn on the radiation resistance of the halophilic archaeon Halobacterium salinarum

Ionizing radiation (IR) is of particular interest in biology because its exposure results in severe oxidative stress to the cell's macromolecules. Our recent work with extremophiles supports the idea that IR resistance is most likely achieved by a metabolic route, effected by manganese (Mn) antioxidants. Biochemical analysis of "super-IR resistant" mutants of H. salinarum, evolved over multiple cycles of exposure to high doses of IR, confirmed the key role for Mn antioxidants in the IR resistance of this organism. Analysis of the proteome of H. salinarum "super-IR resistant" mutants revealed increased expression for proteins involved in energy metabolism, replenishing the cell with reducing equivalents depleted by the oxidative stress inflicted by IR. Maintenance of redox homeostasis was also activated by the over-expression of coenzyme biosynthesis pathways involved in redox reactions. We propose that in H. salinarum, increased tolerance to IR is a combination of metabolic regulatory adjustments and the accumulation of Mn-antioxidant complexes.

Overexpression in a non-native halophilic host and biotechnological potential of NAD+-dependent glutamate dehydrogenase from Halobacterium salinarum strain NRC-36014

Enzymes produced by halophilic archaea are generally heat resistant and organic solvent tolerant, and accordingly important for biocatalytic applications in 'green chemistry', frequently requiring a low-water environment. NAD(+)-dependent glutamate dehydrogenase from an extremely halophilic archaeon Halobacterium salinarum strain NRC-36014 was selected to explore the biotechnological potential of this enzyme and genetically engineered derivatives. Over-expression in a halophilic host Haloferax volcanii provided a soluble, active recombinant enzyme, not achievable in mesophilic Escherichia coli, and an efficient purification procedure was developed. pH and salt dependence, thermostability, organic solvent stability and kinetic parameters were explored. The enzyme is active up to 90°C and fully stable up to 70°C. It shows good tolerance of various miscible organic solvents. High concentrations of salt may be substituted with 30% DMSO or betaine with good stability and activity. The robustness of this enzyme under a wide range of conditions offers a promising scaffold for protein engineering.

Archaeal transcriptional regulation of the prokaryotic KdpFABC complex mediating K+ uptake in H. salinarum

The genome of the halophilic archaeon Halobacterium salinarum encodes the high-affinity ATP-dependent K(+) uptake system Kdp. Previous studies have shown that the genes coding for the KdpFABC complex are arranged in a kdpFABCQ gene cluster together with an additional gene kdpQ. In bacteria, expression of the kdpFABC genes is generally regulated by the dedicated sensor kinase/response regulator pair KdpD/KdpE, which are absent in H. salinarum. Surprisingly, H. salinarum expresses the kdp genes in a manner which is strikingly similar to Escherichia coli. In this study, we show that the halobacterial kdpFABCQ genes constitute an operon and that kdpFABCQ expression is subject to a complex regulatory mechanism involving a negative transcriptional regulator and is further modulated via a so far unknown mechanism. We describe how the regulation of kdp gene expression is facilitated in H. salinarum in contrast to its bacterial counterparts. Whereas the Kdp system fulfils the same core function as an ATP-driven K(+) uptake system in both archaea and bacteria, the different mechanisms involved in the regulation of gene expression appear to have evolved separately, possibly reflecting a different physiological role of ATP-driven K(+) uptake in halophilic archaea.

A small basic protein from the brz-brb operon is involved in regulation of bop transcription in Halobacterium salinarum

BackgroundThe halophilic archaeon Halobacterium salinarum expresses bacteriorhodopsin, a retinal-protein that allows photosynthetic growth. Transcription of the bop (bacterioopsin) gene is controlled by two transcription factors, Bat and Brz that induce bop when cells are grown anaerobically and under light.ResultsA new gene was identified that is transcribed together with the brz gene that encodes a small basic protein designated as Brb (bacteriorhodopsin-regulating basic protein). The translation activity of the start codon of the brb gene was confirmed by BgaH reporter assays. In vivo site-directed mutagenesis of the brb gene showed that the Brb protein cooperates with Brz in the regulation of bop expression. Using a GFP reporter assay, it was demonstrated that Brb cooperates with both Brz and Bat proteins to activate bop transcription under phototrophic growth conditions.ConclusionsThe activation of the bop promoter was shown to be dependent not only on two major factors, Bat and Brz, but is also tuned by the small basic protein, Brb.

Bacterioopsin-Mediated Regulation of Bacterioruberin Biosynthesis in Halobacterium salinarum

Integral membrane protein complexes consisting of proteins and small molecules that act as cofactors have important functions in all organisms. To form functional complexes, cofactor biosynthesis must be coordinated with the production of corresponding apoproteins. To examine this coordination, we study bacteriorhodopsin (BR), a light-induced proton pump in the halophilic archaeon Halobacterium salinarum. This complex consists of a retinal cofactor and bacterioopsin (BO), the BR apoprotein. To examine possible novel regulatory mechanisms linking BO and retinal biosynthesis, we deleted bop, the gene that encodes BO. bop deletion resulted in a dramatic increase of bacterioruberins, carotenoid molecules that share biosynthetic precursors with retinal. Additional studies revealed that bacterioruberins accumulate in the absence of BO regardless of the presence of retinal or BR, suggesting that BO inhibits bacterioruberin biosynthesis to increase the availability of carotenoid precursors for retinal biosynthesis. To further examine this potential regulatory mechanism, we characterized an enzyme, encoded by the lye gene, that catalyzes bacterioruberin biosynthesis. BO-mediated inhibition of bacterioruberin synthesis appears to be specific to the H. salinarum lye-encoded enzyme, as expression of a lye homolog from Haloferax volcanii, a related archaeon that synthesizes bacterioruberins but lacks opsins, resulted in bacterioruberin synthesis that was not reduced in the presence of BO. Our results provide evidence for a novel regulatory mechanism in which biosynthesis of a cofactor is promoted by apoprotein-mediated inhibition of an alternate biochemical pathway. Specifically, BO accumulation promotes retinal production by inhibiting bacterioruberin biosynthesis.

Interaction of hexa-His tag with acidic amino acids results in facilitated refolding of halophilic nucleoside diphosphate kinase

We have previously reported that amino-terminal extension sequence containing hexa-His facilitated refolding and assembly of hexameric nucleoside diphosphate kinase from extremely halophilic archaeon Halobacterium salinarum (NDK). In this study, we made various mutations in both the tag sequence and within NDK molecule. SerNDK, in which hexa-His was replaced with hexa-Ser, showed no facilitated folding. In addition, HisD58GD63G, in which both Asp58 and Asp63 in NDK were replaced with Gly, also showed no refolding enhancement. These results suggest that hexa-His in His-tag interact cooperatively with either Asp58 or Asp63 or both. Furthermore, G114D mutant, which formed a dimer in low salt solution, was strongly stabilized by His-tag to form a stable hexamer.

MALDI-TOF/MS analysis of archaebacterial lipids in lyophilized membranes dry-mixed with 9-aminoacridine

A method of direct lipid analysis by MALDI mass spectrometry in intact membranes, without prior extraction/separation steps, is described. The purple membrane isolated from the extremely halophilic archaeon Halobacterium salinarum was selected as model membrane. Lyophilized purple membrane were grinded with 9-aminoacridine (9-AA) as dry matrix, and the powder mixture was crushed in a mechanical die press to form a thin pellet. Small pieces of the pellet were then attached to the MALDI target and directly analyzed. In parallel, individual archaebacterial phospholipids and glycolipids, together with the total lipid extract of the purple membrane, were analyzed by MALDI-TOF/MS using 9-AA as the matrix in solution. Results show that 9-AA represents a suitable matrix for the conventional MALDI-TOF/MS analysis of lipid extracts from archaeal microorganisms, as well as for fast and reliable direct dry lipid analysis of lyophilized archaebacterial membranes. This method might be of general application, offering the advantage of quickly gaining information about lipid components without disrupting or altering the membrane matrix.

MutS and MutL Are Dispensable for Maintenance of the Genomic Mutation Rate in the Halophilic Archaeon Halobacterium salinarum NRC-1

BackgroundThe genome of the halophilic archaeon Halobacterium salinarum NRC-1 encodes for homologs of MutS and MutL, which are key proteins of a DNA mismatch repair pathway conserved in Bacteria and Eukarya. Mismatch repair is essential for retaining the fidelity of genetic information and defects in this pathway result in the deleterious accumulation of mutations and in hereditary diseases in humans.Methodology/Principal FindingsWe calculated the spontaneous genomic mutation rate of H. salinarum NRC-1 using fluctuation tests targeting genes of the uracil monophosphate biosynthesis pathway. We found that H. salinarum NRC-1 has a low incidence of mutation suggesting the presence of active mechanisms to control spontaneous mutations during replication. The spectrum of mutational changes found in H. salinarum NRC-1, and in other archaea, appears to be unique to this domain of life and might be a consequence of their adaption to extreme environmental conditions. In-frame targeted gene deletions of H. salinarum NRC-1 mismatch repair genes and phenotypic characterization of the mutants demonstrated that the mutS and mutL genes are not required for maintenance of the observed mutation rate.Conclusions/SignificanceWe established that H. salinarum NRC-1 mutS and mutL genes are redundant to an alternative system that limits spontaneous mutation in this organism. This finding leads to the puzzling question of what mechanism is responsible for maintenance of the low genomic mutation rates observed in the Archaea, which for the most part do not have MutS and MutL homologs.

Gene expression and characterization of a novel GH family 18 chitinase from extremely halophilic archaeon Halobacterium salinarum NRC-1

An open reading frame encoding a chitinase homolog (ChiN1) was found in the genome of extremely halophilic archaeon Halobacterium salinarum NRC-1. ChiN1 is a multidomain enzyme consisting of a chitin-binding domain, a polycystic kidney disease domain and a catalytic domain belonging to glycoside hydrolase family 18. chiN1 gene was successfully expressed in extremely halophilic archaeon Haloarcula japonica TR-1 by employing the promoter sequence of its cell surface glycoprotein gene. A large amount of recombinant ChiN1 was secreted into the culture supernatant. The Ha. japonica-produced ChiN1 was purified and characterized. The optimal pH and temperature of ChiN1 are pH 4.5 and 55°C, respectively. ChiN1 was most active at 1.0 M NaCl and stable over a wide range of NaCl concentration from 1.0 to 4.5 M. This is the first report on a chitinase from extremely halophilic archaeon.

Life‐style changes of a halophilic archaeon analyzed by quantitative proteomics

Quantitative proteomics based on isotopic labeling has become the method of choice to accurately determine changes in protein abundance in highly complex mixtures. Isotope-coded protein labeling (ICPL), which is based on the nicotinoylation of proteins at lysine residues and free N-termini was used as a simple, reliable and fast method for the comparative analysis of three different cellular states of the halophilic archaeon Halobacterium salinarum through pairwise comparison. The labeled proteins were subjected to SDS-PAGE, in-gel digested and the proteolytic peptides were separated by LC and analyzed by MALDI-TOF/TOF MS. Automated quantitation was performed by comparing the MS peptide signals of (12)C and (13)C nicotinoylated isotopic peptide pairs. The transitions between (i) aerobic growth in complex versus synthetic medium and (ii) aerobic versus anaerobic/phototrophic growth, both in complex medium, provide a wide span in nutrient and energy supply for the cell and thus allowed optimal studies of proteome changes. In these two studies, 559 and 643 proteins, respectively, could be quantified allowing a detailed analysis of the adaptation of H. salinarum to changes of its living conditions. The subtle cellular response to a wide variation of nutrient and energy supply demonstrates a fine tuning of the cellular protein inventory.