Photoenergy transduction in halobacterium halobium

The structure and function of a rhodopsin-like pigment bacteriorhodopsin, discovered and isolated from the membranes of the halophile Halobacterium halobium, were studied. Intermediatesm appearing in the cyclic photoreaction that drives the proton translocation, were spectroscopically characterized. The charge translocation in membrane monolayers and mulitlayers placed between electrodes were kinetically resolved. A model was developed for the proton translocation process, in which the isomerization of the retinal Schiff base decreases its pK to drive the proton off and simultaneaouly changes the connectivity from the cytoplasmic surface to the external surface. The stoichiometry of proton pumping in intact cells and the effect of the light generated electrochemical potential on the kinetics of the photoreaction cycle and the synthesis of ATP were investigated

Excited-state reaction dynamics of bacteriorhodopsin studied by femtosecond spectroscopy

The photodynamics of bacteriorhodopsin were studied by transient absorption and gain measurements after excitation with femtosecond pulses at 620 nm. With probing pulses at longer wavelengths (λ > 770 nm) the previously reported formation of the J intermediate (with a time constant of 500±100 fs) was confirmed. With probing pulses around 700 nm, a faster process with a relaxation time of 200±70 fs was observed. The data analysis strongly suggests that this kinetic constant describes the reactive motion of the polyatomic molecule on its excited-state potential energy surface, i.e. one observes directly the incipient isomerization of the retinal molecule. The minimum of the S1 potential energy surface reached in 200 fs lies approximately 13300 cm−1 above the ground state of bacteriorhodopsin and from this minimum the intermediate J is formed with a time constant of 500 fs

Morphological and Structural Aspects of the Extremely Halophilic Archaeon Haloquadratum walsbyi

Ultrathin square cell Haloquadratum walsbyi from the Archaea domain are the most abundant microorganisms in the hypersaline water of coastal salterns and continental salt lakes. In this work, we explore the cell surface of these microorganisms using amplitude-modulation atomic-force microscopy in nearly physiological conditions. We demonstrate the presence of a regular corrugation with a periodicity of 16–20 nm attributed to the surface layer (S-layer) protein lattice, striped domains asymmetrically distributed on the cell faces and peculiar bulges correlated with the presence of intracellular granules. Besides, subsequent images of cell evolution during the drying process indicate the presence of an external capsule that might correspond to the giant protein halomucin, predicted by the genome but never before observed by other microscopy studies

Optical Silencing of C. elegans Cells with Arch Proton Pump

BACKGROUND: Optogenetic techniques using light-driven ion channels or ion pumps for controlling excitable cells have greatly facilitated the investigation of nervous systems in vivo. A model organism, C. elegans, with its small transparent body and well-characterized neural circuits, is especially suitable for optogenetic analyses. METHODOLOGY/PRINCIPAL FINDINGS: We describe the application of archaerhodopsin-3 (Arch), a recently reported optical neuronal silencer, to C. elegans. Arch::GFP expressed either in all neurons or body wall muscles of the entire body by means of transgenes were localized, at least partially, to the cell membrane without adverse effects, and caused locomotory paralysis of worms when illuminated by green light (550 nm). Pan-neuronal expression of Arch endowed worms with quick and sustained responsiveness to such light. Worms reliably responded to repeated periods of illumination and non-illumination, and remained paralyzed under continuous illumination for 30 seconds. Worms expressing Arch in different subsets of motor neurons exhibited distinct defects in the locomotory behavior under green light: selective silencing of A-type motor neurons affected backward movement while silencing of B-type motor neurons affected forward movement more severely. Our experiments using a heat-shock-mediated induction system also indicate that Arch becomes fully functional only 12 hours after induction and remains functional for more than 24 hour. CONCLUSIONS/SGNIFICANCE: Arch can be used for silencing neurons and muscles, and may be a useful alternative to currently widely used halorhodopsin (NpHR) in optogenetic studies of C. elegans

Microarray Analysis in the Archaeon Halobacterium salinarum Strain R1

Background: Phototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea. Methodology/Principal Findings: We developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis. Conclusion/Significance: This is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis

Haloquadratum walsbyi yields a versatile, NAD+/NADP+ dual affinity, thermostable, alcohol dehydrogenase (HwADH)

This study presents the first example of an alcohol dehydrogenase (ADH) from the halophilic archaeum Haloquadratum walsbyi (HwADH). A hexahistidine-tagged recombinant HwADH was heterologously overexpressed in Haloferax volcanii. HwADH was purified in one step and was found to be thermophilic with optimal activity at 65 °C. HwADH was active in the presence of 10 % (v/v) organic solvent. The enzyme displayed dual cofactor specificity and a broad substrate scope, maximum activity was detected with benzyl alcohol and 2-phenyl-1- propanol. HwADH accepted aromatic ketones, acetophenone and phenylacetone as substrates. The enzyme also accepted cyclohexanol and aromatic secondary alcohols, 1- phenylethanol and 4-phenyl-2-butanol. H. walsbyi may offer an excellent alternative to other archaeal sources to expand the toolbox of halophilic biocatalysts