Perturbed N-glycosylation of Halobacterium salinarum archaellum filaments leads to filament bundling and compromised cell motility.

The swimming device of archaea-the archaellum-presents asparagine (N)-linked glycans. While N-glycosylation serves numerous roles in archaea, including enabling their survival in extreme environments, how this post-translational modification contributes to cell motility remains under-explored. Here, we report the cryo-EM structure of archaellum filaments from the haloarchaeon Halobacterium salinarum, where archaellins, the building blocks of the archaellum, are N-glycosylated, and the N-glycosylation pathway is well-resolved. We further determined structures of archaellum filaments from two N-glycosylation mutant strains that generate truncated glycans and analyzed their motility. While cells from the parent strain exhibited unidirectional motility, the N-glycosylation mutant strain cells swam in ever-changing directions within a limited area. Although these mutant strain cells presented archaellum filaments that were highly similar in architecture to those of the parent strain, N-linked glycan truncation greatly affected interactions between archaellum filaments, leading to dramatic clustering of both isolated and cell-attached filaments. We propose that the N-linked tetrasaccharides decorating archaellins act as physical spacers that minimize the archaellum filament aggregation that limits cell motility.

Ultrafast terahertz Stark spectroscopy reveals the excited-state dipole moments of retinal in bacteriorhodopsin.

The photoinduced all-trans to 13-cis isomerization of the retinal Schiff base represents the ultrafast first step in the reaction cycle of bacteriorhodopsin (BR). Extensive experimental and theoretical work has addressed excited-state dynamics and isomerization via a conical intersection with the ground state. In conflicting molecular pictures, the excited state potential energy surface has been modeled as a pure S[Formula: see text] state that intersects with the ground state, or in a 3-state picture involving the S[Formula: see text] and S[Formula: see text] states. Here, the photoexcited system passes two crossing regions to return to the ground state. The electric dipole moment of the Schiff base in the S[Formula: see text] and S[Formula: see text] state differs strongly and, thus, its measurement allows for assessing the character of the excited-state potential. We apply the method of ultrafast terahertz (THz) Stark spectroscopy to measure electric dipole changes of wild-type BR and a BR D85T mutant upon electronic excitation. A fully reversible transient broadening and spectral shift of electronic absorption is induced by a picosecond THz field of several megavolts/cm and mapped by a 120-fs optical probe pulse. For both BR variants, we derive a moderate electric dipole change of 5 [Formula: see text] 1 Debye, which is markedly smaller than predicted for a neat S[Formula: see text]-character of the excited state. In contrast, S[Formula: see text]-admixture and temporal averaging of excited-state dynamics over the probe pulse duration gives a dipole change in line with experiment. Our results support a picture of electronic and nuclear dynamics governed by the interaction of S[Formula: see text] and S[Formula: see text] states in a 3-state model.

Effects of stand-alone polar residue on membrane protein stability and structure.

Helical membrane proteins generally have a hydrophobic nature, with apolar side chains comprising the majority of the transmembrane (TM) helices. However, whenever polar side chains are present in the TM domain, they often exert a crucial role in structural interactions with other polar residues, such as TM helix associations and oligomerization. Moreover, polar residues in the TM region also often participate in protein functions, such as the Schiff base bonding between Lys residues and retinal in rhodopsin-like membrane proteins. Although many studies have focused on these functional polar residues, our understanding of stand-alone polar residues that are energetically unfavored in TM helixes is limited. Here, we adopted bacteriorhodopsin (bR) as a model system and systematically mutated 17 of its apolar Leu or Phe residues to polar Asn. Stability measurements of the resulting mutants revealed that all of these polar substitutions reduced bR stability to various extents, and the extent of destabilization of each mutant bR is also correlated to different structural factors, such as the relative accessible surface area and membrane depth of the mutation site. Structural analyses of these Asn residues revealed that they form sidechain-to-backbone hydrogen bonds that alleviate the unfavorable energetics in hydrophobic and apolar surroundings. Our results indicate that membrane proteins are able to accommodate certain stand-alone polar residues in the TM region without disrupting overall structures.

Detection of Lipid-Bound Bacteriorhodopsin Trimer Complex Directly from Purple Membrane by Native Mass Spectrometry.

Native mass spectrometry (MS) was used to detect the membrane protein, bacteriorhodopsin (bR), in its 27 kDa monomeric form and trimeric assemblies directly from lipid-containing purple membranes (PMs) from the halophilic archaeon, Halobacterium salinarum. Trimer bR ion populations bound to lipid molecules were detected with n-octyl ß-d-glucopyranoside as the solubilizing detergent; the use of octyl tetraethylene glycol monooctyl ether or n-dodecyl-ß-d-maltopyranoside resulted in only detection of monomeric bR. The archaeal lipids phosphotidylglycerolphosphate methyl ester and 3-HSO3-Galp-ß1,6-Manp-α1,2-Glcp-α1,1-sn-2,3-diphytanylglycerol were the only lipids in the PMs found to bind to bR, consistent with previous high-resolution structural studies. Removal of the lipids from the sample resulted in the detection of only the bR monomer, highlighting the importance of specific lipids for stabilizing the bR trimer. To the best of our knowledge, this is the first report of the detection of the bR trimer with resolved lipid-bound species by MS.

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).

Key determinants for signaling in the sensory rhodopsin II/transducer complex are different between Halobacterium salinarum and Natronomonas pharaonis.

The cell membrane of Halobacterium salinarum contains a retinal-binding photoreceptor, sensory rhodopsin II (HsSRII), coupled with its cognate transducer (HsHtrII), allowing repellent phototaxis behavior for shorter wavelength light. Previous studies on SRII from Natronomonas pharaonis (NpSRII) pointed out the importance of the hydrogen bonding interaction between Thr204NpSRII and Tyr174NpSRII in signal transfer from SRII to HtrII. Here, we investigated the effect on phototactic function by replacing residues in HsSRII corresponding to Thr204NpSRII and Tyr174NpSRII . Whereas replacement of either residue altered the photocycle kinetics, introduction of any mutations at Ser201HsSRII and Tyr171HsSRII did not eliminate negative phototaxis function. These observations imply the possibility of the presence of an unidentified molecular mechanism for photophobic signal transduction differing from NpSRII-NpHtrII.

Halobacterium salinarum: Life with more than a grain of salt.

Halobacterium salinarum is a halophilic (salt-loving) archaeon that grows in salt concentrations near or at saturation. Although isolated from salted fish a century ago, it was the 1971 discovery of bacteriorhodopsin, the light-driven proton pump, that raised interest in Hbt. salinarum across a range of disciplines, including biophysics, chemistry, molecular evolution and biotechnology. Hbt. salinarum have since contributed to numerous discoveries, such as advances in membrane protein structure determination and the first example of a non-eukaryal glycoprotein. Work on Hbt. salinarum, one of the species used to define Archaea, has also elucidated molecular workings in the third domain. Finally, Hbt. salinarum presents creative solutions to the challenges of life in high salt.

Agl28 and Agl29 are key components of a Halobacterium salinarum N-glycosylation pathway.

Although Halobacterim salinarum provided the first example of N-glycosylation outside the Eukarya, only recently has attention focused on delineating the pathway responsible for the assembly of the N-linked tetrasaccharide decorating selected proteins in this haloarchaeon. In the present report, the roles of VNG1053G and VNG1054G, two proteins encoded by genes clustered together with a set of genes demonstrated to encode N-glycosylation pathway components, were considered. Relying on both bioinformatics and gene deletion and subsequent mass spectrometry analysis of known N-glycosylated proteins, VNG1053G was determined to be the glycosyltransferase responsible for addition of the linking glucose, while VNG1054G was deemed to be the flippase that translocates the lipid-bound tetrasaccharide across the plasma membrane to face the cell exterior, or to contribute to such activity. As observed with Hbt. salinarum lacking other components of the N-glycosylation machinery, both cell growth and motility were compromised in the absence of VNG1053G or VNG1054G. Thus, given their demonstrated roles in Hbt. salinarum N-glycosylation, VNG1053G and VNG1054G were re-annotated as Agl28 and Agl29, according to the nomenclature used to define archaeal N-glycosylation pathway components.

Combined effect of the head groups and alkyl chains of archaea lipids when interacting with bacteriorhodopsin.

Bacteriorhodopsin (bR), a transmembrane protein with seven α-helices, is highly expressed in the purple membrane (PM) of archaea such as Halobacterium salinarum. It is well known that bR forms two-dimensional crystals with acidic lipids such as phosphatidylglycerol phosphate methyl ester (PGP-Me)-a major component of PM lipids bearing unique chemical structures-methyl-branched alkyl chains, ether linkages, and divalent anionic head groups with two phosphodiester groups. Therefore, we aimed to determine which functional groups of PGP-Me are essential for the boundary lipids of bR and how these functionalities interact with bR. To this end, we compared various well-known phospholipids (PLs) that carry one of the structural features of PGP-Me, and evaluated the affinity of PLs to bR using the centerband-only analysis of rotor-unsynchronized spin echo (COARSE) method in solid-state NMR measurements and thermal shift assays. The results clearly showed that the branched methyl groups of alkyl chains and double negative charges in the head groups are important for PL interactions with bR. We then examined the effect of phospholipids on the monomer-trimer exchange of bR using circular dichroism (CD) spectra. The results indicated that the divalent negative charge in a head group stabilizes the trimer structure, while the branched methyl chains significantly enhance the PLs' affinity for bR, thus dispersing bR trimers in the PM even at high concentrations. Finally, we investigated the effects of PL on the proton-pumping activity of bR based on the decay rate constant of the M intermediate of a bR photocycle. The findings showed that bR activities decreased to 20% in 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), and in 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers as compared to that in PM. Meanwhile, 1,2-Diphytanoyl-sn-glycero-3-phosphate (DPhPA) bilayers bearing both negative charges and branched methyl groups preserved over 80% of the activity. These results strongly suggest that the head groups and alkyl chains of phospholipids are essential for boundary lipids and greatly influence the biological function of bR.

Extremely halophilic strains of Halobacterium salinarum as a potential starter culture for fish sauce fermentation.

Proteinase-producing halophilic archaea were isolated from Thai fish sauce collected from industrial fermentation tanks at various periods of fermentation. Five isolates namely, J-1-S4, J-1-S13, J-1-S22, 2 m-40-15-R2, and P-1-S8, were identified as Halobacterium salinarum with slightly different colony and morphological characteristics among isolates. Starters of five isolates were prepared and added to the anchovy mixed with 25% solar salt and fermented for 180 days at 30-35°C. Halophilic bacteria/archaea counts of inoculated samples were decreased and undetected after 120 days of fermentation. At 180 days of fermentation, α-amino group contents of inoculated fish sauce samples (856-1010 mM) and total nitrogen content (2.2%-2.5%) were higher than the control without archaea inoculation (p < 0.05). All samples contained low amounts of biogenic amines, suggesting that all starters were not biogenic amine formers. The major volatile compound found in samples inoculated with H. salinarum P-1-S8 and H. salinarum 2 m-40-15-R2 was 3-methylbutanal, which contributes to the meaty note. Dimethyl disulfide, a compound that contributes to fecal note, was detected in all inoculated samples in a lower amount than in the commercial fish sauce (p < 0.05). Thus, H. salinarum accelerated protein hydrolysis and produced desirable volatile compounds during fish sauce fermentation in a strain-specific manner.

An N-linked tetrasaccharide from Halobacterium salinarum presents a novel modification, sulfation of iduronic acid at the O-3 position.

Halobacterium salinarum, a halophilic archaeon that grows at near-saturating salt concentrations, provided the first example of N-glycosylation outside Eukarya. Yet, almost 50 years later, numerous aspects of such post-translational protein processing in this microorganism remain to be determined, including the architecture of glycoprotein-bound glycans. In the present report, nuclear magnetic resonance spectroscopy was used to define a tetrasaccharide N-linked to both archaellins, building blocks of the archaeal swimming device (the archaellum), and the S-layer glycoprotein that comprises the protein shell surrounding the Hbt. salinarum cell as ß-GlcA(2S)-(1 â†’ 4)-α-IdoA(3S)-(1 â†’ 4)-ß-GlcA-(1 â†’ 4)-ß-Glc-Asn. The structure of this tetrasaccharide fills gaps remaining from previous studies, including confirmation of the first known inclusion of iduronic acid in an archaeal N-linked glycan. At the same time, the sulfation of this iduronic acid at the O-3 position has not, to the best of our knowledge, been previously seen. As such, this may represent yet another unique facet of N-glycosylation in Archaea.

A conserved Trp residue in HwBR contributes to its unique tolerance toward acidic environments.

Bacteriorhodopsin (BR) is a light-driven outward proton pump found mainly in halophilic archaea. A BR from an archaeon Haloquadratum walsbyi (HwBR) was found to pump protons under more acidic conditions compared with most known BR proteins. The atomic structural study on HwBR unveiled that a pair of hydrogen bonds between the BC and FG loop in its periplasmic region may be a factor in such improved pumping capability. Here, we further investigated the retinal-binding pocket of HwBR and found that Trp94 contributes to the higher acid tolerance. Through single mutations in a BR from Halobacterium salinarum and HwBR, we examined the conserved tryptophan residues in the retinal-binding pocket. Among these residues of HwBR, mutagenesis at Trp94 facing the periplasmic region caused the most significant disruption to optical stability and proton-pumping capability under acidic conditions. The other tryptophan residues of HwBR exerted little impact on both maximum absorption wavelength and pH-dependent proton pumping. Our findings suggest that the residues from Trp94 to the hydrogen bonds at the BC loop confer both optical stability and functionality on the overall protein in low-pH environments.

A functional promoter from the archaeon Halobacterium salinarum is also transcriptionally active in E. coli.

BACKGROUND: Archaea form a third domain of life that is distinct from Bacteria and Eukarya. So far, many scholars have elucidated considerable details about the typical promoter architectures of the three domains of life. However, a functional promoter from the archaeon Halobacterium salinarum has never been studied in Escherichia coli. RESULTS: This paper found that the promoter of Halobacterium salinarum showed a promoter function in Escherichia coli. This Escherichia coli promoter structure contains - 10 box, -10 box extension and - 29 elements, however, no -35 box. The - 29 element is exercised by the TATA box in archaea. And we isolated the RM10 fragment that possessed the fusion characteristics of bacteria and archaea, which was overlapped with functionality of TATA box and - 29 elements. CONCLUSIONS: The - 29 element reflects the evolutionary relationship between the archaeal promoter and the bacterial promoter. The result possibly indicated that there may be a certain internal connection between archaea and bacteria. We hypothesized that it provided a new viewpoint of the evolutionary relationship of archaea and other organisms.

An archaeal histone-like protein regulates gene expression in response to salt stress.

Histones, ubiquitous in eukaryotes as DNA-packing proteins, find their evolutionary origins in archaea. Unlike the characterized histone proteins of a number of methanogenic and themophilic archaea, previous research indicated that HpyA, the sole histone encoded in the model halophile Halobacterium salinarum, is not involved in DNA packaging. Instead, it was found to have widespread but subtle effects on gene expression and to maintain wild type cell morphology. However, the precise function of halophilic histone-like proteins remain unclear. Here we use quantitative phenotyping, genetics, and functional genomics to investigate HpyA function. These experiments revealed that HpyA is important for growth and rod-shaped morphology in reduced salinity. HpyA preferentially binds DNA at discrete genomic sites under low salt to regulate expression of ion uptake, particularly iron. HpyA also globally but indirectly activates other ion uptake and nucleotide biosynthesis pathways in a salt-dependent manner. Taken together, these results demonstrate an alternative function for an archaeal histone-like protein as a transcriptional regulator, with its function tuned to the physiological stressors of the hypersaline environment.

Halobacterium salinarum and Haloferax volcanii Comparative Transcriptomics Reveals Conserved Transcriptional Processing Sites.

Post-transcriptional processing of messenger RNA is an important regulatory strategy that allows relatively fast responses to changes in environmental conditions. In halophile systems biology, the protein perspective of this problem (i.e., ribonucleases which implement the cleavages) is generally more studied than the RNA perspective (i.e., processing sites). In the present in silico work, we mapped genome-wide transcriptional processing sites (TPS) in two halophilic model organisms, Halobacterium salinarum NRC-1 and Haloferax volcanii DS2. TPS were established by reanalysis of publicly available differential RNA-seq (dRNA-seq) data, searching for non-primary (monophosphorylated RNAs) enrichment. We found 2093 TPS in 43% of H. salinarum genes and 3515 TPS in 49% of H. volcanii chromosomal genes. Of the 244 conserved TPS sites found, the majority were located around start and stop codons of orthologous genes. Specific genes are highlighted when discussing antisense, ribosome and insertion sequence associated TPS. Examples include the cell division gene ftsZ2, whose differential processing signal along growth was detected and correlated with post-transcriptional regulation, and biogenesis of sense overlapping transcripts associated with IS200/IS605. We hereby present the comparative, transcriptomics-based processing site maps with a companion browsing interface.

Structure and RNA-Binding Properties of Lsm Protein from Halobacterium salinarum.

The structure and the RNA-binding properties of the Lsm protein from Halobacterium salinarum have been determined. A distinctive feature of this protein is the presence of a short L4 loop connecting the ß3 and ß4 strands. Since bacterial Lsm proteins (also called Hfq proteins) have a short L4 loop and form hexamers, whereas archaeal Lsm proteins (SmAP) have a long L4 loop and form heptamers, it has been suggested that the length of the L4 loop may affect the quaternary structure of Lsm proteins. Moreover, the L4 loop covers the region of SmAP corresponding to one of the RNA-binding sites in Hfq, and thus can affect the RNA-binding properties of the protein. Our results show that the SmAP from H. salinarum forms heptamers and possesses the same RNA-binding properties as homologous proteins with the long L4 loop. Therefore, the length of the L4 does not govern the number of monomers in the protein particles and does not affect the RNA-binding properties of Lsm proteins.

Revisiting N-glycosylation in Halobacterium salinarum: Characterizing a dolichol phosphate- and glycoprotein-bound tetrasaccharide.

Although Halobacterium salinarum provided the first example of N-glycosylation outside the Eukarya, much regarding such post-translational modification in this halophilic archaea remains either unclear or unknown. The composition of an N-linked glycan decorating both the S-layer glycoprotein and archaellins offers one such example. Originally described some 40 years ago, reports from that time on have presented conflicted findings regarding the composition of this glycan, as well as differences between the protein-bound glycan and that version of the glycan attached to the lipid upon which it is assembled. To clarify these points, liquid chromatography-electrospray ionization mass spectrometry was employed here to revisit the composition of this glycan both when attached to selected asparagine residues of target proteins and when bound to the lipid dolichol phosphate upon which the glycan is assembled. Such efforts revealed the N-linked glycan as corresponding to a tetrasaccharide comprising a hexose, a sulfated hexuronic acid, a hexuronic acid and a second sulfated hexuronic acid. When attached to dolichol phosphate but not to proteins, the same tetrasaccharide is methylated on the final sugar. Moreover, in the absence of the oligosaccharyltransferase AglB, there is an accumulation of the dolichol phosphate-linked methylated and disulfated tetrasaccharide. Knowing the composition of this glycan at both the lipid- and protein-bound stages, together with the availability of gene deletion approaches for manipulating Hbt. salinarum, will allow delineation of the N-glycosylation pathway in this organism.

Membrane protein crystallography in the era of modern structural biology.

The aim of structural biology has been always the study of biological macromolecules structures and their mechanistic behaviour at molecular level. To achieve its goal, multiple biophysical methods and approaches have become part of the structural biology toolbox. Considered as one of the pillars of structural biology, X-ray crystallography has been the most successful method for solving three-dimensional protein structures at atomic level to date. It is however limited by the success in obtaining well-ordered protein crystals that diffract at high resolution. This is especially true for challenging targets such as membrane proteins (MPs). Understanding structure-function relationships of MPs at the biochemical level is vital for medicine and drug discovery as they play critical roles in many cellular processes. Though difficult, structure determination of MPs by X-ray crystallography has significantly improved in the last two decades, mainly due to many relevant technological and methodological developments. Today, numerous MP crystal structures have been solved, revealing many of their mechanisms of action. Yet the field of structural biology has also been through significant technological breakthroughs in recent years, particularly in the fields of single particle electron microscopy (cryo-EM) and X-ray free electron lasers (XFELs). Here we summarise the most important advancements in the field of MP crystallography and the significance of these developments in the present era of modern structural biology.

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 Šfor extant archaeal TRXs and <1.5 Šfor 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.

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins.