Effect of Mutations in GvpJ and GvpM on Gas Vesicle Formation of Halobacterium salinarum

The two haloarchaeal proteins, GvpM and GvpJ, are homologous to GvpA, the major gas vesicle structural protein. All three are hydrophobic and essential for gas vesicle formation. The effect of mutations in GvpJ and GvpM was studied in Haloferax volcanii transformants by complementing the respective mutated gene with the remaining gvp genes and inspecting the cells for the presence of gas vesicles (Vac+). In case of GvpJ, 56 of 66 substitutions analyzed yielded Vac– ΔJ + Jmut transformants, indicating that GvpJ is very sensitive to alterations, whereas ten of the 38 GvpM variants resulted in Vac– ΔM + Mmut transformants. The variants were also tested by split-GFP for their ability to interact with their partner protein GvpL. Some of the alterations leading to a Vac– phenotype affected the J/L or M/L interaction. Also, the interactions J/A and J/M were studied using fragments to exclude an unspecific aggregation of these hydrophobic proteins. Both fragments of GvpJ interacted with the M1–25 and M60–84 fragments of GvpM, and fragment J1–56 of GvpJ interacted with the N-terminal fragment A1–22 of GvpA. A comparison of the results on the three homologous proteins indicates that despite their relatedness, GvpA, GvpJ, and GvpM have unique features and cannot substitute each other.

Identifying Components of a Halobacterium salinarum N-Glycosylation Pathway

Whereas N-glycosylation is a seemingly universal process in Archaea, pathways of N-glycosylation have only been experimentally verified in a mere handful of species. Toward expanding the number of delineated archaeal N-glycosylation pathways, the involvement of the putative Halobacterium salinarum glycosyltransferases VNG1067G, VNG1066C, and VNG1062G in the assembly of an N-linked tetrasaccharide decorating glycoproteins in this species was addressed. Following deletion of each encoding gene, the impact on N-glycosylation of the S-layer glycoprotein and archaellins, major glycoproteins in this organism, was assessed by mass spectrometry. Likewise, the pool of dolichol phosphate, the lipid upon which this glycan is assembled, was also considered in each deletion strain. Finally, the impacts of such deletions were characterized in a series of biochemical, structural and physiological assays. The results revealed that VNG1067G, VNG1066C, and VNG1062G, renamed Agl25, Agl26, and Agl27 according to the nomenclature used for archaeal N-glycosylation pathway components, are responsible for adding the second, third and fourth sugars of the N-linked tetrasaccharide decorating Hbt. salinarum glycoproteins. Moreover, this study demonstrated how compromised N-glycosylation affects various facets of Hbt. salinarum cell behavior, including the transcription of archaellin-encoding genes.

Rhodopsins at a glance

ABSTRACT Rhodopsins are photoreceptive membrane proteins consisting of a common heptahelical transmembrane architecture that contains a retinal chromophore. Rhodopsin was first discovered in the animal retina in 1876, but a different type of rhodopsin, bacteriorhodopsin, was reported to be present in the cell membrane of an extreme halophilic archaeon, Halobacterium salinarum, 95 years later. Although these findings were made by physiological observation of pigmented tissue and cell bodies, recent progress in genomic and metagenomic analyses has revealed that there are more than 10,000 microbial rhodopsins and 9000 animal rhodopsins with large diversity and tremendous new functionality. In this Cell Science at a Glance article and accompanying poster, we provide an overview of the diversity of functions, structures, color discrimination mechanisms and optogenetic applications of these two rhodopsin families, and will also highlight the third distinctive rhodopsin family, heliorhodopsin.

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, its precise function remains unclear. Here we use quantitative phenotyping, genetics, and functional genomic 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.

Enhanced hydrogen production of Rhodobacter sphaeroides promoted by extracellular H+ of Halobacterium salinarum

Purpose This study utilized the principle that the bacteriorhodopsin (BR) produced by Halobacterium salinarum could increase the hydrogen production of Rhodobacter sphaeroides. H. salinarum are co-cultured with R. sphaeroides to determine the impact of purple membrane fragments (PM) on R. sphaeroides and improve its hydrogen production capacity. Methods In this study, low-salinity in 14 % NaCl domesticates H salinarum. Then, 0–160 nmol of different concentration gradient groups of bacteriorhodopsin (BR) and R. sphaeroides was co-cultivated, and the hydrogen production and pH are measured; then, R. sphaeroides and immobilized BR of different concentrations are used to produce hydrogen to detect the amount of hydrogen. Two-chamber microbial hydrogen production system with proton exchange membrane-assisted proton flow was established, and the system was operated. As additional electricity added under 0.3 V, the hydrogen production rate increased with voltages in the coupled system. Results H salinarum can still grow well after low salt in 14% NaCl domestication. When the BR concentration is 80 nmol, the highest hydrogen production reached 217 mL per hour. Both immobilized PC (packed cells) and immobilized PM (purple membrane) of H. salinarum could promote hydrogen production of R. sphaeroides to some extent. The highest production of hydrogen was obtained by the coupled system with 40 nmol BR of immobilized PC, which increased from 127 to 232 mL, and the maximum H2 production rate was 18.2 mL−1 h−1 L culture. In the 192 h experiment time, when the potential is 0.3 V, the hydrogen production amount can reach 920 mL, which is 50.3% higher than the control group. Conclusions The stability of the system greatly improved after PC was immobilized, and the time for hydrogen production of R. sphaeroides significantly extended on same condition. As additional electricity added under 0.3 V, the hydrogen production rate increased with voltages in the coupled system. These results are helpful to build a hydrogen production-coupled system by nitrogenase of R. sphaeroides and proton pump of H. salinarum. Graphical abstract

Femtochemistry of Rhodopsins

The review considers the spectral kinetic data obtained by us by femtosecond absorption laser spectroscopy for the photochromic reaction of retinal isomerization in animal rhodopsin (type II), namely, bovine visual rhodopsin and microbial rhodopsins (type I), such as Exiguobacterium sibiricum rhodopsin and Halobacterium salinarum bacteriorhodopsin. It is shown that the elementary act of the photoreaction of retinal isomerization in type I and type II rhodopsins can be interpreted as a transition through a conical intersection with retention of the coherence of the vibrational wave packets generated during excitation. The coherent nature of the reaction is most pronounced in visual rhodopsin as a result of the barrier-free movement along the excited surface of potential energy, which also leads to an extremely high rate of retinal isomerization compared to microbial rhodopsins. Differences in the dynamics of photochemical reactions of type I and type II rhodopsins can be related to both differences in the initial isomeric forms of their chromophores (all-trans and 11-cis retinal, respectively), as well as with the effect of the protein environment on the chromophore. Despite the practically identical values of the quantum yields of the direct photoreaction of visual rhodopsin and bacteriorhodopsin, the reverse photoreaction of visual rhodopsin is much less effective (φ = 0.15) than in the case of bacteriorhodopsin (φ = 0.81). It can be assumed that the photobiological mechanism for converting light into an information process in the evolutionarily younger visual rhodopsins (type II rhodopsins) should be more reliable than the mechanism for converting light into a photoenergetic process in the evolutionarily more ancient microbial rhodopsins (type I rhodopsins). The low value of the quantum yield of the reverse reaction of visual rhodopsin can be considered as an increase in the reliability of the forward reaction, which triggers the process of phototransduction.