Magnesium orthophosphate,a new form of reserve phosphates in the halophilic archaeon Halobacterium salinarium

The accumulation and utilization of reserve phosphates in the extremely halophilic archaeon Halobacterium salinarium were studied. The growth of H. salinarium was found to depend on the initial concentration of inorganic phosphate (Pi) in the culture medium and its content in the inoculum. Growing cells consumed 85-95% of Pi from the medium. Unlike the reserve phosphates of many other microorganisms, which are mainly polyphosphates, the reserve phosphates of H. salinarium cells contain no more than 15% polyphosphates, the rest being magnesium orthophosphate. The excessive consumption of Pi from the medium changed cell morphology and caused the death of part of the cell population. The cells that remained viable could grow in a Pi-deficient medium, utilizing about 70% of reserve magnesium phosphate as the phosphorus source.     

Comparative genomic analysis of the Haloferax volcanii DS2 and Halobacterium salinarium GRB contig maps reveals extensive rearrangement.

Anonymous probes from the genome of Halobacterium salinarium GRB and 12 gene probes were hybridized to the cosmid clones representing the chromosome and plasmids of Halobacterium salinarium GRB and Haloferax volcanii DS2. The order of and pairwise distances between 35 loci uniquely cross-hybridizing to both chromosomes were analyzed in a search for conservation. No conservation between the genomes could be detected at the 15-kbp resolution used in this study. We found distinct sets of low-copy-number repeated sequences in the chromosome and plasmids of Halobacterium salinarium GRB, indicating some degree of partitioning between these replicons. We propose alternative courses for the evolution of the haloarchaeal genome: (i) that the majority of genomic differences that exist between genera came about at the inception of this group or (ii) that the differences have accumulated over the lifetime of the lineage. The strengths and limitations of investigating these models through comparative genomic studies are discussed.     

Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium

Dundas, Ian E. D. (University of Illinois, Urbana), and H. Orin Halvorson. Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium. J. Bacteriol. 91:113-119. 1966.-Arginine was shown to be essential for growth of Halobacterium salinarium strain 1 in a chemically defined medium. Citrulline was the only compound which could substitute for arginine without affecting growth. Resting cells of H. salinarium converted arginine to citrulline and citrulline to ornithine. Cells grown in an arginine-free medium with C(14)-ureido-labeled citrulline incorporated the isotope mainly into the arginine of their proteins. The enzymes arginine desimidase and ornithine transcarbamylase were found and studied in cell-free extracts of H. salinarium. Experiments indicated that arginine was degraded in H. salinarium by arginine desimidase to citrulline, and that citrulline was further degraded by ornithine transcarbamylase to carbamyl phosphate and ornithine. Synthesis of arginine from citrulline seems to occur via the formation of argininosuccinic acid.     

Isolation and characterization of an extremely halophilic archaeobacterium from traditionally fermented Thai fish sauce (nam pla)

An extremely halophilic archaeobacterium (halobacterium), strain ORE, was isolated from traditionally fermented Thai fish sauce (nam pla), which has a concentration of 4·4–5·1 M NaCl. Polar liquid analysis and DNA hybridization revealed that it was a representative of the species Halobacterium salinarium. In common with many other strains of H. salinarium this organism produces salt-stable extracellular proteases which are likely to be important in the fermentation process.     

Isolation of an ftsZ homolog from the archaebacterium Halobacterium salinarium: implications for the evolution of FtsZ and tubulin.

We have isolated a homolog of the cell division gene ftsZ from the extremely halophilic archaebacterium Halobacterium salinarium. The predicted protein of 39 kDa is divergent relative to eubacterial homologs, with 32% identity to Escherichia coli FtsZ. No other eubacterial cell division gene homologs were found adjacent to H. salinarium ftsZ. Expression of the ftsZ gene region in H. salinarium induced significant morphological changes leading to the loss of rod shape. Phylogenetic analysis demonstrated that the H. salinarium FtsZ protein is more related to tubulins than are the FtsZ proteins of eubacteria, supporting the hypothesis that FtsZ may have evolved into eukaryotic tubulin.     

Chemotaxis and phototaxis require a CheA histidine kinase in the archaeon Halobacterium salinarium.

Histidine kinases are part of the two-component signal transduction system responsible for eubacterial responses to diverse environmental signals. They have recently been detected in eukaryotes but their existence in the kingdom Archaea remains uncertain. Here we report the sequence and function of a histidine kinase (CheAH.s.) from Halobacterium salinarium, the first such transmitter in Archaea. The protein CheAH.s. (668 residues) has significant sequence identity with the CheA proteins known from eubacterial signal transduction (e.g. 34% identity with CheA from Bacillus subtilis). Antibodies were raised against CheAH.s. as expressed in Escherichia coli and were used in Western blotting to demonstrate the expression of cheAH.s. in H. salinarium. As has been observed for other halophilic proteins, CheAH.s. has a deviant electrophoretic migration, with an apparent molecular weight of 103 kDa on SDS-PAGE compared with a calculated molecular weight of 72 kDa. Deletion of a part of the cheAH.s. gene leads to loss of both chemotactic and phototactic responses in H. salinarium as measured by swarm plate assays, motion analysis and tethering experiments. This indicates that CheAH.s. plays a crucial role in chemical and light signal integration, presumably interacting with at least two phototransducers and a number of chemoreceptors.     

Nucleic acid studies on halophilic archaebacteria

DNA-16S rRNA hybridization studies of archaebacterial halophiles revealed nine major groups. High (greater than 45%) DNA-DNA homologies were found only within DNA-rRNA groups. The DNA-DNA homology between the type strains of Halobacterium halobium, Hb. salinarium and Hb. cutirubrum was greater than 70%. The implications for the taxonomy of the extreme halophiles are discussed.     

Distribution of glycerol dehydrogenase and glycerol kinase activity in halophilic archaea

Abstract A constitutive NAD⁺-dependent glycerol dehydrogenase activity was detected in Halobacterium salinarium and Halobacterium cutirubrum . Optimal activity was found at 3 M KCl and pH 8–10. No glycerol dehydrogenase activity could be demonstrated in representatives of the genera Haloferax and Haloarcula , even when grown in the presence of glycerol, or in Halobacterium saccharovorum and Halobacterium sodomense . Glycerol kinase activity was shown to be present constitutively in all halophilic archaea examined. The finding that glycerol dehydrogenase is found only in part of the halophilic arachaea makes dihydroxyacetone an improbable candidate as the precursor for the glycerol moiety of halobacterial lipids.     

Inorganic polyphosphates and phosphohydrolases from Halobacterium salinarium

Halobacterium salinarium grown in a liquid medium consumed up to 75% of phosphates originally present in the growth medium and accumulated up to 100 mumol Pi/g wet biomass by the time it entered the growth retardation phase. The content of acid-soluble oligophosphates in the biomass was maximum at the early stage of active growth and drastically decreased when cells reached the growth-retardation phase. The total content of alkali-soluble and acid-insoluble polyphosphates changed very little throughout the cultivation period (five days). The polyphosphate content of H. salinarium cells was close to that of yeasts and eubacteria. The pyrophosphatase, polyphosphatase, and nonspecific phosphatase activities of H. salinarium cells were several times lower than those of the majority of eubacteria. The specific activity of pyrophosphatase, the most active hydrolase of H. salinarium, gradually increased during cultivation, reaching 540 mU/mg protein by the end of the cultivation period. Half of the total pyrophosphatase activity of this halobacterium was localized in the cytosol. The molecular weight of pyrophosphatase, evaluated by gel filtration, was 86 kDa. The effective Km of this enzyme with respect to pyrophosphate was 115 microM.     

The flagellar bundle of Halobacterium salinarium is inserted into a distinct polar cap structure.

Flagellated envelopes of Halobacterium salinarium cells were prepared by lysis with taurodeoxycholate. After solubilization of the envelopes with Triton X-100 at high ionic strength, flagella and round patches from which numerous flagella emerged were isolated by gel filtration chromatography. We conclude that the flagellar bundle of H. salinarium is inserted into a differentiated polar cap structure.     

Archaebacterial class I and class II aldolases from extreme halophiles

Both, class I (Schiff-base forming) and class II (metal requiring) fructose biphosphate aldolases were found to be distributed among halophilic archaebacteria. The aldolase activity fromHalobacterium halobium, H. salinarium, H. cutirubrum, H. mediterranei andH. volcanii exhibited properties of a bacterial class II aldolase as it was metal-dependent for activity and therefore inhibited by EDTA. In contrast, aldolase fromH. saccharovorum, Halobacterium R-113, H. vallismortis andHalobacterium CH-1 formed a Schiff-base intermediate with the substrate and therefore resembled to eukaryotic class I type. The type of aldolase did not vary by changes in the growth medium.     

Immunological Relationships Among Some Species of Extremely Halophilic Bacteria

Rabbits were immunized with four strains of halobacteria, Halobacterium halobium NRL, H. halobium R-1, H. salinarium NRL-9, and H. cutirubrum NRL-10, that had been fixed with formaldehyde. The antisera obtained detected the presence of an antigen common to the Halobacterium genus and, after absorption, detected three distinct antigenic groups within the Halobacterium genus. A fourth group was agglutinated only by unabsorbed sera.     

Characterization of plasmids in halobacteria.

Extrachromosomal, covalently closed circular deoxyribonucleic acid has been isolated from different species of halobacteria. Three strains of Halobacterium halobium and one of Halobacterium cutirubrum, all of which synthesize purple membrane (Pum+) and bacterioruberin (Rub+), contain plasmids of different size which share extensive sequence homologies. One strain of Halobacterium salinarium, another one of Halobacterium capanicum, and two new Halobacterium isolates from Tunisia, which are also Pum+ Rub+, do not harbor covalently closed circular deoxyribonucleic acid but contain sequences, presumably integrated into the chromosome, which are similar if not identical to those of pHH1, i.e., the plasmid originally isolated from H. halobium. Three other halophilic strains, Halobacterium trapanicum, Halobacterium volcanii, and a new isolate from Israel, do not carry pHH1-like sequences. These strains are, by morphological and physiological criteria, different from the others examined and harbor plasmids unrelated to pHH1.     

Formation of insoluble magnesium phosphates during growth of the archaea Halorubrum distributum and Halobacterium salinarium and the bacterium Brevibacterium antiquum

Stationary phase cells of the halophilic archaea Halobacterium salinarium and Halorubrum distributum, growing at 3-4 M NaCl, and of the halotolerant bacterium Brevibacterium antiquum, growing with and without 2.6 NaCl, took up approximately 90% of the phosphate from the culture media containing 2.3 and 11.5 mM phosphate. The uptake was blocked by the uncoupler FCCP. In B. antiquum, EDTA inhibited the phosphate uptake. The content of polyphosphates in the cells was significantly lower than the content of orthophosphate. At a high phosphate concentration, up to 80% of the phosphate taken up from the culture medium was accumulated as Mg(2)PO(4)OH x 4H(2)O in H. salinarium and H. distributum and as NH(4)MgPO(4) x 6H(2)O in B. antiquum. Consolidation of the cytoplasm and enlargement of the nucleoid zone were observed in the cells during phosphate accumulation. At phosphate surplus, part of the H. salinarium and H. distributum cell population was lysed. The cells of B. antiquum were not lysed and phosphate crystals were observed in the cytoplasm.     

Indole-3-acetic acid and 2-(indol-3-ylmethyl)indol-3-yl acetic acid in the thermophilic archaebacterium Sulfolobus acidocaldarius.

Indole-3-acetic acid (IAA) and 2-(indol-3-ylmethyl)indol-3-yl acetic acid were identified in lipid extracts of Sulfolobus acidocaldarius; they occurred at concentrations of 0.57 and 0.59 mumol/g (dry weight), respectively. The amount of IAA found in these cells is more than a thousand times greater than that found in a typical extract of a plant in which IAA serves as a plant growth hormone. Neither of these compounds was detected in the other archaebacteria that were analyzed; these included Sulfolobus sulfataricus, Halobacterium salinarium, and several strains of methanogenic bacteria. This is the first report of the natural occurrence of 2-(indol-3-ylmethyl)indol-3-yl acetic acid.     

DNA-uptake in the naturally competent cyanobacterium, Synechocystis sp. PCC 6803

Abstract Eight species of halophilic Archaea were tested for the presence of the enzymes of the methylglyoxal bypass. Methylglyoxal synthase was found in extracts of all species tested, with the exception of Halobacterium salinarium and Halobacterium cutirubrum . The enzyme of Haloferax volcanii was most active at pH 7 in the absence of salt, and in the presence of 3 M NaCl or KCl activity was half of that without salt, and was inhibited by phosphate. Glyoxalase I was detected in all species tested. Optimal activity of H. volcanii glyoxalase I was found at pH 7 and 3 M KCl; in the absence of salt, activity was strongly reduced. Glutathione could be replaced by γ-glutamylcysteine as the acceptor of the D-lactoyl group. The work shows that the methylglyoxal bypass may be operative in representatives of the archaeal kingdom.     

Conservation of chromosomal arrangement among three strains of the genetically unstable archaeon Halobacterium salinarium.

Phenotypic variants of Halobacterium salinarium NRC-1 arise at a frequency of 10(-2). These result from transpositions of halobacterial insertion sequences and rearrangements mediated by halobacterial insertion sequences. We have tested the hypothesis that such mutations are confined to only a portion of the genome by comparing the chromosomal restriction map of H. salinarium NRC-1 and that of the derivative S9, which was made in 1969. The two chromosomes were mapped by using two-dimensional pulsed-field gel electrophoresis and the restriction enzymes AflII, AseI, and DraI. A comparison of the two deduced maps showed a domain of about 210 kbp to be subject to many rearrangements, including an inversion in S9 relative to NRC-1. However, the rest of the chromosome was conserved among NRC-1, S9, and an independent Halobacterium isolate, GRB, previously mapped by St. Jean et al. (A. St. Jean, B. A. Trieselmann, and R. L. Charlebois, Nucleic Acids Res. 22:1476-1483, 1994). This concurs with data from eubacteria suggesting strong selective forces maintaining gene order even in the face of rearrangement events occurring at a high frequency.     

Combined effect of temperature and salt concentration/water activity on the growth rate of Halobacterium spp.

Growth responses of the halophilic bacteria, Halobacterium sp. strain HB9 and Halobacterium salinarium strain CM42/12, to temperature and water activity/sodium chloride concentration were described by the square root model and T _min (the theoretical minimum temperature for growth) was fixed. Little change in growth rate was observed in response to added NaCl at water activities below which cell lysis was avoided. Hence, growth of halobacteria on products such as salted, dried fish at water activities below 0.85 may be based on the square root temperature response without the need to incorporate a water activity term.     

Isolation and characterization of a calmodulin-like protein from Halobacterium salinarium.

The first evidence for a calmodulin-like protein in an archaeon, Halobacterium salinarium, is reported here. The calmodulin-like protein, with a molecular mass of 24 kDa and an estimated pI of 4.8, stimulated cyclic nucleotide phosphodiesterase in a calcium-dependent manner. This stimulation could be suppressed by calmodulin inhibitors. The Ca(2+)-binding ability was verified by 45Ca autoradiography.