Iron-uptake in the Euryarchaeon Halobacterium salinarum

Iron-uptake is well studied in a plethora of pro- and eukaryotic organisms with the exception of Archaea, which thrive mainly in extreme environments. In this study, the mechanism of iron transport in the extremely halophilic Euryarchaeon Halobacterium salinarum strain JW 5 was analyzed. Under low-iron growth conditions no siderophores were detectable in culture supernatants. However, various xenosiderophores support growth of H. salinarum. In [⁵⁵Fe]–[¹⁴C] double-label experiments, H. salinarum displays uptake of iron but not of the chelator citrate. Uptake of iron was inhibited by cyanide and at higher concentrations by Ga. Furthermore, a K_M for iron uptake in cells of 2.36 μM and a V_max of approximately 67 pmol Fe/min/mg protein was determined. [⁵⁵Fe]-uptake kinetics were measured in the absence and presence of Ga. Uptake of iron was inhibited merely at very high Ga concentrations. The results indicate an energy dependent iron uptake process in H. salinarum and suggest reduction of the metal at the membrane level.     

Improved production of bacteriorhodopsin from Halobacterium salinarum through direct amino acid supplement in the basal medium

Extremophiles - Enhanced production and growth of Halobacterium salinarum are achieved by direct supplement of essential amino acids in the modified nutrient culture medium. As arginine (R) and...     

An efficient system for the synthesis of bacteriorhodopsin in Halobacterium halobium

The mechanism by which bacteriorhodopsin (BR) transports protons across the cell membrane of Halobacterium halobium is actively studied in many laboratories. Currently available systems for the synthesis of mutant proteins obtained by site-directed mutagenesis of the gene encoding BR (bop) require reconstitution of the denatured polypeptide after its synthesis Escherichia coli or yeast; this approach is technically difficult and labor intensive, and raises questions about possible differences between in vivo and in vitro folding. Using a newly described transformation system and a halobacterial plasmid vector, we show that it is possible to reintroduce the bop gene into BR- strains of H. halobium. The bop-carrying plasmid expresses native BR in amounts similar to those obtained in several wild type strains. This system allows facile site-directed mutagenesis in halophilic archaebacteria.     

Investigations of iron uptake in Halobacterium salinarum

The iron transport in the extremely halophilic Euryarchaeon Halobacterium salinarum JW5 was investigated. Experiments to detect endogenous siderophores from H. salinarum failed, but it was able to utilize exogenous siderophores. Measurement of the uptake of (55)Fe and [(14)C]citrate gave evidence only for the accumulation of iron. Two additional membrane proteins could be detected in iron-starved cells, one in iron-repleted membranes and one that is up-regulated there. Respiratory rates of iron-starved membranes after the addition of succinate and NADH differed considerably from iron-repleted ones. Furthermore, both types of membrane exhibited different degrees of inhibition by cyanide.     

Synthesis of spyropyran derivatives of retinal and study of their interaction with bacterioopsin from Halobacterium salinarum

The synthesis of retinal analogue series that contain a spyropyran moiety instead of a trimethylcyclohexene ring was proposed. The process of the retinal analogue interaction with bacterioopsin from apomembranes of Halobacterium salinarum and the spectral properties of the newly formed pigments were studied. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

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.     

Archaeal promoter-directed expression of the Halobacterium salinarum catalase-peroxidase gene

The Halobacterium salinarum catalase-peroxidase gene was subcloned into shuttle vectors pWL102 and pWL202 and expressed under the control of different archaeal promoters. When Hbt. salinarum was transformed with the catalase-peroxidase gene under the control of its own promoter, catalase-peroxidase activity increased twofold. Catalase-peroxidase activity increased threefold when Hbt. salinarum was transformed with the catalase-peroxidase gene under the control of a tRNA promoter. This bifunctional enzyme in Hbt. salinarum was not induced by environmental stresses such as H₂O₂, intense light, darkness, high temperature, low temperature, redox inhibitors, heavy metals, or ions. Received: May 5, 2000 / Accepted: August 28, 2000     

Biofilms of Halobacterium salinarum as a tool for phenanthrene bioremediation

Abstract

The use of hyperhalophilic microorganisms is emerging as a sustainable alternative to clean hydrocarbon-polluted hypersaline water bodies. In line with this practice, this work reports on the ability of the archaeon Halobacterium salinarum to develop biofilms on a solid surface conditioned by the presence of phenanthrene crystals, which results in the removal of the contaminating compound. The cell surface hydrophobicity does not change during the removal process and this organism is shown to constitutively produce a surfactant molecule with specific action on aromatic hydrocarbons, both indicating that phenanthrene removal might proceed through a non-contact mechanism. A new approach is presented to follow the process in situ through epifluorescence microscopy by monitoring phenanthrene auto-fluorescence.     

A major role for nonenzymatic antioxidant processes in the radioresistance of Halobacterium salinarum

Oxidative stress occurs when the generation of reactive oxygen species (ROS) exceeds the capacity of the cell's endogenous systems to neutralize them. Our analyses of the cellular damage and oxidative stress responses of the archaeon Halobacterium salinarum exposed to ionizing radiation (IR) revealed a critical role played by nonenzymatic antioxidant processes in the resistance of H. salinarum to IR. ROS-scavenging enzymes were essential for resistance to chemical oxidants, yet those enzymes were not necessary for H. salinarum's resistance to IR. We found that protein-free cell extracts from H. salinarum provided a high level of protection for protein activity against IR in vitro but did not protect DNA significantly. Compared with cell extracts of radiation-sensitive bacteria, H. salinarum extracts were enriched in manganese, amino acids, and peptides, supporting an essential role in ROS scavenging for those small molecules in vivo. With regard to chemical oxidants, we showed that the damage caused by gamma irradiation was mechanistically different than that produced by hydrogen peroxide or by the superoxide-generating redox-cycling drug paraquat. The data presented support the idea that IR resistance is most likely achieved by a "metabolic route," with a combination of tightly coordinated physiological processes.     

Regulation of bacteriorhodopsin synthesis by growth rate in continuous cultures of Halobacterium halobium

The independent effects of oxygen tension and growth rate on bacteriorhodopsin synthesis in Halobacterium halobium have been studied in chemostat cultures. Bacteriorhodopsin synthesis occurs only at low growth rates and is stimulated by low oxygen tension. Fast growth rates override the stimulatory effects of oxygen tension, with the result that bacteriorhodopsin can scarcely be detected. Illumination of cultures maintained at low growth rate and low oxygen tension significantly increases the steady state cell yield. This finding suggests that under these conditions the purple membrane proton pump is coupled to energy transduction.     

The DpsA-homologue of the archaeon Halobacterium salinarum is a ferritin

An iron-rich protein, DpsA(Hsal), was isolated from the archaeon Halobacterium salinarum sharing a sequence identity of 35% with the starvation-induced DNA-binding protein, DpsA, of Synechecoccus sp. PCC7942. It consists of 20-kDa subunits forming a dodecameric structure. The protein exhibits a ferric iron loading of up to 100 Fe ions per mole of holoprotein. CD spectra and secondary structure calculations are consistent with an alpha-helical contribution of 60%. The UV/VIS spectrum provides no evidence for the presence of heme groups. This protein exhibits features of a non-heme type bacterial ferritin (Ftn) although it shares only little sequence homology with Ftn. Molecular modelling disclosed a high structural similarity to E. coli Dps.     

Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation

The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence. The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure. A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5. Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight. Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF). There were 94 proteins that were found in multiple spots, indicating post-translational modification. An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map. Thus about 40% of the cytosolic proteome was identified. In addition to the 2784 protein-coding genes, the H. salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons. Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments. The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments. The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids. In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes. About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology. The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).     

Systems Analysis of Bioenergetics and Growth of the Extreme Halophile Halobacterium salinarum

Halobacterium salinarum is a bioenergetically flexible, halophilic microorganism that can generate energy by respiration, photosynthesis, and the fermentation of arginine. In a previous study, using a genome-scale metabolic model, we have shown that the archaeon unexpectedly degrades essential amino acids under aerobic conditions, a behavior that can lead to the termination of growth earlier than necessary. Here, we further integratively investigate energy generation, nutrient utilization, and biomass production using an extended methodology that accounts for dynamically changing transport patterns, including those that arise from interactions among the supplied metabolites. Moreover, we widen the scope of our analysis to include phototrophic conditions to explore the interplay between different bioenergetic modes. Surprisingly, we found that cells also degrade essential amino acids even during phototropy, when energy should already be abundant. We also found that under both conditions considerable amounts of nutrients that were taken up were neither incorporated into the biomass nor used as respiratory substrates, implying the considerable production and accumulation of several metabolites in the medium. Some of these are likely the products of forms of overflow metabolism. In addition, our results also show that arginine fermentation, contrary to what is typically assumed, occurs simultaneously with respiration and photosynthesis and can contribute energy in levels that are comparable to the primary bioenergetic modes, if not more. These findings portray a picture that the organism takes an approach toward growth that favors the here and now, even at the cost of longer-term concerns. We believe that the seemingly “greedy” behavior exhibited actually consists of adaptations by the organism to its natural environments, where nutrients are not only irregularly available but may altogether be absent for extended periods that may span several years. Such a setting probably predisposed the cells to grow as much as possible when the conditions become favorable.     

Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum

Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCl). Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations. In this study, the protein expression profile of H. salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE). As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips. The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry. Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins. Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application. IMPDH gene (1.6 kb fragment) expected to exist in H. salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector. Recombinant IMPDH purified from H. salinarum has a higher activity in the presence of salt than in the absence of salt.     

Differential transport properties of D-leucine and L-leucine in the archaeon, Halobacterium salinarum

The transport of D-leucine was compared with that of L-leucine in Halobacterium salinarum. When a high-outside/low-inside Na+ gradient was imposed, D-leucine as well as L-leucine accumulated in envelope vesicles, supporting the hypothesis that D-leucine is transported via a symport system along with Na+. Kinetic analyses, including inhibition experiments, indicated that both enantiomers are transported via a common carrier. However, a Hill plot indicated a single binding site for Na+ during L-leucine transport, but dual binding sites for Na+ during D-leucine transport. Furthermore, D-leucine transport was dependent on electrical membrane potential, suggesting that a transporter bound with D-leucine is positively charged. L-leucine transport was slightly, if at all, dependent on membrane potential, suggesting that a transporter bound with L-leucine is electrically neutral. These results indicate that the leucine carrier in Halobacterium salinarum translocates two moles of Na+ per mole of D-leucine, and one mole of Na+ per mole of L-leucine.     

High-pressure tolerance in Halobacterium salinarum NRC-1 and other non-piezophilic prokaryotes

In this study, we examined the high-pressure survival of a range of prokaryotes not found in high-pressure environments to determine the effects of adaptations to osmotic and oxidative stresses on piezo-resistance. The pressure survivals of Halobacterium salinarum NRC-1, Deinococcus radiodurans R1, and Chromohalobacter salexigens were compared to that of Escherichia coli MG1655. C. salexigens, which uses the compatible solute ectoine as an osmolyte, was as piezo-sensitive as E. coli MG1655, suggesting that ectoine is not a piezolyte. D. radiodurans R1 and H. salinarum NRC-1, both resistant to oxidative stress, were found to be highly piezo-resistant. H. salinarum NRC-1 showed nearly full survival after pressurization up to 400 MPa; a survival 3.5 log units higher than E. coli MG1655. This piezo-resistance was maintained in H. salinarum NRC-1 for pressurizations up to 1 h. We hypothesize that the high-pressure resistance of H. salinarum NRC-1 is due to a combination of factors including cell envelope structure and the presence of intracellular salts.