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.

Nanohybrid Structures Based on Plasmonic or Fluorescent Nanoparticles and Retinal-Containing Proteins.

Rhodopsins are light-sensitive membrane proteins enabling transmembrane charge separation (proton pump) on absorption of a light quantum. Bacteriorhodopsin (BR) is a transmembrane protein from halophilic bacteria that belongs to the rhodopsin family. Potential applications of BR are considered so promising that the number of studies devoted to the use of BR itself, its mutant variants, as well as hybrid materials containing BR in various areas grows steadily. Formation of hybrid structures combining BR with nanoparticles is an essential step in promotion of BR-based devices. However, rapid progress, continuous emergence of new data, as well as challenges of analyzing the entire data require regular reviews of the achievements in this area. This review is devoted to the issues of formation of materials based on hybrids of BR with fluorescent semiconductor nanocrystals (quantum dots) and with noble metal (silver, gold) plasmonic nanoparticles. Recent data on formation of thin (mono-) and thick (multi-) layers from materials containing BR and BR/nanoparticle hybrids are presented.

Primary Transfer Step in the Light-Driven Ion Pump Bacteriorhodopsin: An Irreversible U-Turn Revealed by Dynamic Nuclear Polarization-Enhanced Magic Angle Spinning NMR.

Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the Mo intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. 15N(SB)-1H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and 13C-13C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, 15N-13C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.

[Microbial model Halobacterium salinarum in screening of synthetic analogues of antibiotic turbomycin A with anticancer activity].

The microbial test-system based on cultivation of Halobacterium salinarum developed earlier for screening inhibitors of sterol biosynthesis and proposed for screening anticancer antibiotics, proved to be efficient in revealing anticancer compounds among derivatives of tris(1-alkylindol-3-yl)methylium, synthetic analogues of antibiotic turbomycin A. Most of the methane sulfonate and chloride salts of such compounds, investigated with the help of the H. salinarum test-system, showed no activity (MIC>32 mcM), while several derivatives, containing N-butyl or N-pentyl substituents were rather active against the bacterial strain. The MICs of them against H. salinarum were 8 mcM for total and 1 mcM for partial inhibition of the bacterial growth. The results of the study correlated with the results of other investigations that revealed anticancer activity of such compounds in tumor cell cultures. Therefore, the H. salinarum test-system demonstrated its availability for screening compounds with anticancer activity.

Sodium chloride affects propidium monoazide action to distinguish viable cells.

Propidium monoazide (PMA) is a DNA-intercalating agent used to selectively detect DNA from viable cells by polymerase chain reaction (PCR). Here, we report that high concentrations (>5%) of sodium chloride (NaCl) prevents PMA from inhibiting DNA amplification from dead cells. Moreover, Halobacterium salinarum was unable to maintain cell integrity in solutions containing less than 15% NaCl, indicating that extreme halophilic microorganisms may not resist the concentration range in which PMA fully acts. We conclude that NaCl, but not pH, directly affects the efficiency of PMA treatment, limiting its use for cell viability assessment of halophiles and in hypersaline samples.

Detection of membrane protein two-dimensional crystals in living cells.

It is notoriously difficult to grow membrane protein crystals and solve membrane protein structures. Improved detection and screening of membrane protein crystals are needed. We have shown here that second-order nonlinear optical imaging of chiral crystals based on second harmonic generation can provide sensitive and selective detection of two-dimensional protein crystalline arrays with sufficiently low background to enable crystal detection within the membranes of live cells. The method was validated using bacteriorhodopsin crystals generated in live Halobacterium halobium bacteria and confirmed by electron microscopy from the isolated crystals. Additional studies of alphavirus glycoproteins indicated the presence of localized crystalline domains associated with virus budding from mammalian cells. These results suggest that in vivo crystallization may provide a means for expediting membrane protein structure determination for proteins exhibiting propensities for two-dimensional crystal formation.

Sensitive detection of protein-lipid interaction change on bacteriorhodopsin using dodecyl ß-D-maltoside.

A light-driven proton pump bacteriorhodopsin (bR) forms a two-dimensional hexagonal lattice with about 10 archaeal lipids per monomer bR on purple membrane (PM) of Halobacterium salinarum. In this study, we found that the weakening of the bR-lipid interaction on PM by addition of alcohol can be detected as the significant increase of protein solubility in a nonionic detergent, dodecyl ß-D-maltoside (DDM). The protein solubility in DDM was also increased by bR-lipid interaction change accompanied by structural change of the apoprotein after retinal removal and was about 7 times higher in the case of completely bleached membrane than that of intact PM. Interestingly, the cyclic and milliseconds order of structural change of bR under light irradiation also led to increasing the protein solubility and had a characteristic light intensity dependence with a phase transition. These results indicate that there is a photointermediate in which bR-lipid interaction has been changed by its dynamic structural change. Because partial delipidation of PM by CHAPS gave minor influence for the change of the protein solubility compared to intact PM in both dark and light conditions, it is suggested that specific interactions of bR with some lipids which remain on PM even after delipidation treatment have a key role for the change of solubility in DDM induced by alcohol binding, ligand release, and photon absorption on bR.

19F NMR analysis of the antimicrobial peptide PGLa bound to native cell membranes from bacterial protoplasts and human erythrocytes.

(19)F NMR is a unique tool to examine the structure of fluorine-labeled peptides in their native cellular environment, due to an exquisite sensitivity and lack of natural abundance background. For solid-state NMR analysis, we isolated native membranes from erythrocyte ghosts and bacterial protoplasts and prepared them as macroscopically oriented samples. They showed a high purity and quality of alignment according to (31)P NMR, and the membrane-bound antimicrobial peptide PGLa could be detected by (19)F NMR. The characteristic fingerprint splitting of its (19)F reporter group indicated that the peptide helix binds to the native membranes in a surface alignment, albeit with a higher affinity in the prokaryotic than the eukaryotic system.

The rhodopsin-like pigments of halobacteria: light-energy and signal transducers in an archaebacterium.

Three similar, small retinylidene proteins, which resemble the visual pigments of animals, are found in halobacteria: two functions as light-driven ion pumps; the third is the receptor for phototaxis and allows color discrimination.

Heat-fixation method used in an atomic force microscopy study of cell morphology.

In order to overcome the difficulties with existing methods for sample immobilization in imaging Halobacterium salinarum (H. salinarum) living in a highly salty medium by atomic force microscopy (AFM), a heat-fixation method was, for the first time, used to overcome existing problems in preparing samples for AFM. The effect on the cell morphology of the heat-fixation method was studied by MAC mode AFM, and was compared with the drop-and-dry and the polylysine-adhesion methods. It was found that the heat-fixation method can be successfully used for preparing Gram-negative and Gram-positive bacteria samples for AFM studies. Using this method, high-resolution AFM images of H. salinarum were obtained. Round protrusions on the cell surface and horn-like protrusions only at one pole of H. salinarum were observed.

Archaeal cells share common size control with bacteria despite noisier growth and division.

In nature, microorganisms exhibit different volumes spanning six orders of magnitude 1 . Despite their capability to create different sizes, a clonal population in a given environment maintains a uniform size across individual cells. Recent studies in eukaryotic and bacterial organisms showed that this homogeneity in cell size can be accomplished by growing a constant size between two cell cycle events (that is, the adder model 2-6 ). Demonstration of the adder model led to the hypothesis that this phenomenon is a consequence of convergent evolution. Given that archaeal cells share characteristics with both bacteria and eukaryotes, we investigated whether and how archaeal cells exhibit control over cell size. To this end, we developed a soft-lithography method of growing the archaeal cells to enable quantitative time-lapse imaging and single-cell analysis, which would be useful for other microorganisms. Using this method, we demonstrated that Halobacterium salinarum, a hypersaline-adapted archaeal organism, grows exponentially at the single-cell level and maintains a narrow-size distribution by adding a constant length between cell division events. Interestingly, the archaeal cells exhibited greater variability in cell division placement and exponential growth rate across individual cells in a population relative to those observed in Escherichia coli 6-9 . Here, we present a theoretical framework that explains how these larger fluctuations in archaeal cell cycle events contribute to cell size variability and control.

Transcriptome changes and cAMP oscillations in an archaeal cell cycle.

BACKGROUND: The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote. RESULTS: A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 microM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression. CONCLUSION: The analysis of cell cycle-specific transcriptome changes of H. salinarum allowed to identify a strategy of transcript level regulation that is different from all previously characterized species. The transcript levels of only 3% of all genes are regulated, a fraction that is considerably lower than has been reported for four eukaryotic species (6%-28%) and for the bacterium C. crescentus (19%). It was shown that cAMP is present in significant concentrations in an archaeon, and the phylogenetic profile of the adenylate cyclase indicates that this signaling molecule is widely distributed in archaea. The occurrence of cell cycle-dependent oscillations of the cAMP concentration in an archaeon and in several eukaryotic species indicates that cAMP level changes might be a phylogenetically old signal for cell cycle progression.

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%.

Direct observation of different surface structures on high-resolution images of native halorhodopsin.

Halorhodopsin (HR) was investigated with atomic force microscopic techniques (AFM) in aqueous solution. Two-dimensional (2D) crystals of HR were obtained by purifying an HR membrane fraction with the same buoyant density as the purple membrane (HR-PM) from the overexpressing strain Halobacterium salinarum D2. The membrane patches of HR were immobilized on mica. Images with a resolution up to 14 A were recorded. Crystals showed an orthogonal structure and the orientation of the molecules showed p42(1)2 symmetry; thus, alternate tetramers are inverted in the membrane. The crystal surface was found to display different structures depending on the imaging force used, indicating that some parts of the HR molecule are more rigid but others more compressible. From samples with single tetramers missing in the crystalline patches dimensions of the unit cell could be determined. Helix-connecting loops in single molecules of halorhodopsin were assigned. The images indicate that the large extracellular BC loop covers the whole molecule and is very flexible.

Reversible loss of crystallinity on photobleaching purple membrane in the presence of hydroxylamine.

Structural changes of purple membrane during photobleaching in the presence of hydroxylamine were monitored using atomic force microscopy (AFM). The process of bleaching was associated with the disassembly of the purple membrane crystal into smaller crystals. Imaging steps of the photobleaching progress showed that disassembly proceeds until the sample is fully bleached and its crystallinity is almost lost. As revealed from high resolution AFM topographs, the loss of crystallinity was initiated by loss of lattice forming contact between the individual bacteriorhodopsin trimers. The bacteriorhodopsin molecules, however, remained assembled into trimers during the entire photobleaching process. Regeneration of the photobleached sample into intact purple membrane resulted in the reassembly of the bacteriorhodopsin trimers into the trigonal lattice of purple membrane. The data provide novel insights into factors triggering purple membrane formation and structure.

Growth-Phase-Specific Modulation of Cell Morphology and Gene Expression by an Archaeal Histone Protein.

UNLABELLED: In all three domains of life, organisms use nonspecific DNA-binding proteins to compact and organize the genome as well as to regulate transcription on a global scale. Histone is the primary eukaryotic nucleoprotein, and its evolutionary roots can be traced to the archaea. However, not all archaea use this protein as the primary DNA-packaging component, raising questions regarding the role of histones in archaeal chromatin function. Here, quantitative phenotyping, transcriptomic, and proteomic assays were performed on deletion and overexpression mutants of the sole histone protein of the hypersaline-adapted haloarchaeal model organism Halobacterium salinarum. This protein is highly conserved among all sequenced haloarchaeal species and maintains hallmark residues required for eukaryotic histone functions. Surprisingly, despite this conservation at the sequence level, unlike in other archaea or eukaryotes, H. salinarum histone is required to regulate cell shape but is not necessary for survival. Genome-wide expression changes in histone deletion strains were global, significant but subtle in terms of fold change, bidirectional, and growth phase dependent. Mass spectrometric proteomic identification of proteins from chromatin enrichments yielded levels of histone and putative nucleoid-associated proteins similar to those of transcription factors, consistent with an open and transcriptionally active genome. Taken together, these data suggest that histone in H. salinarum plays a minor role in DNA compaction but important roles in growth-phase-dependent gene expression and regulation of cell shape. Histone function in haloarchaea more closely resembles a regulator of gene expression than a chromatin-organizing protein like canonical eukaryotic histone. IMPORTANCE: Histones comprise the major protein component of eukaryotic chromatin and are required for both genome packaging and global regulation of expression. The current paradigm maintains that archaea whose genes encode histone also use these proteins to package DNA. In contrast, here we demonstrate that the sole histone encoded in the genome of the salt-adapted archaeon Halobacterium salinarum is both unessential and unlikely to be involved in DNA compaction despite conservation of residues important for eukaryotic histones. Rather, H. salinarum histone is required for global regulation of gene expression and cell shape. These data are consistent with the hypothesis that H. salinarum histone, strongly conserved across all other known salt-adapted archaea, serves a novel role in gene regulation and cell shape maintenance. Given that archaea possess the ancestral form of eukaryotic histone, this study has important implications for understanding the evolution of histone function.

Two groups control light-induced Schiff base deprotonation and the proton affinity of Asp85 in the Arg82 his mutant of bacteriorhodopsin.

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 micros to 75 micros with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.

An agarose-in-plug bridge method to study chemotaxis in the Archaeon Halobacterium salinarum.

A simple agarose-in-plug bridge method was developed to study chemotaxis in the Archaeon Halobacterium salinarum. Preheated liquid agarose solution with chemoeffectors is pipetted in the middle of a microscope slide bridge, constructed by placing two plastic strips 16 mm apart. A coverslip is immediately placed over the agarose. The solidified agarose plug is completely encircled with the halobacterial cell suspension. Within a certain time concentrated halobacteria were seen as a ring at the edge of the agarose plug containing attractant amino acids and the control growth medium. Chemotaxis mutant Pho60 cells do not accumulate either around the attractants or around the growth medium. The kinetics of the ring formation can be readily videotaped or photographed using either phase-contrast or dark-field microscopy.

Electronic transduction of proton translocations in nanoassembled lamellae of bacteriorhodopsin.

An organic field-effect transistor (OFET) integrating bacteriorhodopsin (bR) nanoassembled lamellae is proposed for an in-depth study of the proton translocation processes occurring as the bioelectronic device is exposed either to light or to low concentrations of general anesthetic vapors. The study involves the morphological, structural, electrical, and spectroscopic characterizations necessary to assess the functional properties of the device as well as the bR biological activity once integrated into the functional biointerlayer (FBI)-OFET structure. The electronic transduction of the protons phototranslocation is shown as a current increase in the p-type channel only when the device is irradiated with photons known to trigger the bR photocycle, while Raman spectroscopy reveals an associated C═C isomer switch. Notably, higher energy photons bring the cis isomer back to its trans form, switching the proton pumping process off. The investigation is extended also to the study of a PM FBI-OFET exposed to volatile general anesthetics such as halothane. In this case an electronic current increase is seen upon exposure to low, clinically relevant, concentrations of anesthetics, while no evidence of isomer-switching is observed. The study of the direct electronic detection of the two different externally triggered proton translocation effects allows gathering insights into the underpinning of different bR molecular switching processes.