Phylogenetic analyses of some extremely halophilic archaea isolated from Dead Sea water, determined on the basis of their 16S rRNA sequences.

Twenty-two extremely halophilic aerobic archaeal strains were isolated from enrichments prepared from Dead Sea water samples collected 57 years ago. The isolates were phenotypically clustered into five different groups, and a representative from each group was chosen for further study. Almost the entire sequences of the 16S rRNA genes of these representatives, and of Haloarcula hispanica ATCC 33960, were determined to establish their phylogenetic positions. The sequences of these strains were compared to previously published sequences of 27 reference halophilic archaea (members of the family Halobacteriaceae) and two other archaea, Methanobacterium formicicum DSM 1312 and Methanospirillum hungatei DSM 864. Phylogenetic analysis using approximately 1,400 base comparisons of 16S rRNA-encoding gene sequences demonstrated that the five isolates clustered closely to species belonging to three different genera--Haloferax, Halobacterium, and Haloarcula. Strains E1 and E8 were closely related and identified as members of the species Haloferax volcanii, and strain E12 was closely related and identified as a member of the species Halobacterium salinarum. However, strains E2 and E11 clustered in the Haloarcula branch with Haloarcula hispanica as the closest relative at 98.9 and 98.8% similarity, respectively. Strains E2 and E11 could represent two new species of the genus Haloarcula. However, because strains of these two new species were isolated from a single source, they will not be named until additional strains are isolated from other sources and fully characterized.     

A potential anti-oxidant protein in a ferrous iron-oxidizing Sulfolobus species

Abstract Eight species of halophilic Archaea were tested for the presence of isocitrate lyase activity. High activities (up to 100 nmol min⁻¹ mg protein⁻¹) were detected in Haloferax mediterranei and Haloferax volcanii when grown in medium containing acetate as the principal carbon source. Little activity was found in representatives of the genera Halobacterium and Haloarcula . Isocitrate lyase from Haloferax mediterranei required high potassium chloride concentrations, optimal activity being found at 1.5–3 M potassium chloride and pH 7.0. Replacement of potassium chloride by sodium chloride resulted in much lower activities. Sulfhydryl compounds (cysteine, glutathione) were not stimulatory. In other properties (stimulation by magnesium ions, sensitivity to different inhibitors) the enzyme resembled isocitrate lyases from representatives of the Bacteria and Eucarya.     

Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

The outer surface of the archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer protein was cloned and sequenced. The mature polypeptide is composed of 794 amino acids and is preceded by a typical signal sequence of 34 amino acid residues. A highly hydrophobic stretch of 20 amino acids at the C-terminal end probably serves as a transmembrane domain. Clusters of threonine residues are located adjacent to this membrane anchor. The S-layer protein is a glycoprotein containing both N- and O-glycosidic bonds. Glucosyl-(1----2)-galactose disaccharides are linked to threonine residues. The primary structure and the glycosylation pattern of the S-layer glycoproteins from Haloferax volcanii and from Halobacterium halobium were compared and found to exhibit distinct differences, despite the fact that three-dimensional reconstructions from electron micrographs revealed no structural differences at least to the 2.5-nm level attained so far (M. Kessel, I. Wildhaber, S. Cohe, and W. Baumeister, EMBO J. 7:1549-1554, 1988).     

Cloning and sequencing of ftsZ homolog from extremely halophilic archaeon Haloarcula japonica strain TR-1.

The gene encoding FtZ was cloned from triangular disc-shaped extremely halophilic archaeon Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the possible ftsZ gene revealed that the structural gene consisted of an open reading frame of 1,182 nucleotides encoding 394 amino acids. The deduced amino acid sequence of the Ha. japonica FtsZ showed high identities with those Halobacterium salinarom, Haloferax volcanii and Haloferax mediterranei FtsZs.     

Ketocarotenoids in halobacteria: 3-hydroxy-echinenone and trans-astaxanthin

HPLC testing of the carotenoid content of the halobacteria Halobacterium salinarium, Haloarcula hispanica and Haloferax mediterranei showed that all contained high amounts of ketocarotenoids. Halobacterium salinarium produced 2400 μg of total carotenoids per gram of dried bacteria, including 265 μg of trans -astaxanthin (11%), and 588 μg of 3-hydroxy-echinenone (24%). The biotechnological properties of Halobacterium salinarium as a natural pigment source are also presented. The results are compared with those from the yeast Phaffia rhodozyma currently used in the industry as a source of trans -astaxanthin.     

Drastic differences in glycosylation of related S-layer glycoproteins from moderate and extreme halophiles.

The outer surface of the moderate halophilic archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer glycoprotein. The polypeptide (794 amino acid residues) contains 7 N-glycosylation sites. Four of these sites were isolated as glycopeptides and the structure of one of the corresponding saccharides was determined. Oligosaccharides consisting of beta-1,4-linked glucose residues are attached to the protein via the linkage unit asparaginyl-glucose. In the related glycoprotein from the extreme halophile Halobacterium halobium, the glucose residues are replaced by sulfated glucuronic acid residues, causing a drastic increase in surface charge density. This is discussed in terms of a recent model explaining the stability of halophilic proteins.     

Utilization of protein profiles for the characterization of halophilic bacteria

Proteins from 24 halophilic bacteria, including Haloarcula marismortui, Haloarcula vallismortis, Haloferax mediteranei, Haloferax gibbonsii, Halobacterium salinarium, as well as unknown isolates from Enid, Oklahoma; Jefferson Island, Louisiana; and the Salado Formation-New Mexico, were analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their protein profiles were compared, and the bacteria were grouped according to the Statistical Analysis System (SAS Institute, Cary, North Carolina) on an IBM 4316 computer. The groupings made from protein profiles showed agreement with groupings made from DNA reassociation data. The various known halophiles were easily separated into the three main halobacterial genera. The data show that one-dimensional SDS-PAGE can be easily used to rapidly screen large numbers of unknown strains to group them into related clusters. This technique offers a way to reduce the total number of halophilic isolates being studied in large taxonomic research programs.     

Taxonomic analysis of extremely halophilic archaea isolated from 56-years-old dead sea brine samples

A taxonomic study comprising both phenotypic and genotypic characterization, has been carried out on a total of 158 extremely halophilic aerobic archaeal strains. These strains were isolated from enrichments prepared from Dead Sea water samples dating from 1936 that were collected by B. E. Volcani for the demonstration of microbial life in the Dead Sea. The isolates were examined for 126 morphological, physiological, biochemical and nutritional tests. Numerical analysis of the data, by using the S(J) coefficient and UPGMA clustering method, showed that the isolates clustered into six phenons. Twenty-two out of the 158 strains used in this study were characterized previously (ARAHAL et al., 1996) and were placed into five phenotypic groups. The genotypic study included both the determination of the guanineplus-cytosine content of the DNA and DNA-DNA hybridization studies. For this purpose, representative strains from the six phenons were chosen. These groups were found to represent some members of three different genera - Haloarcula (phenons A, B, and C), Haloferax (phenons D and E) and Halobacterium (phenon F) - of the family Halobacteriaceae, some of them never reported to occur in the Dead Sea, such as Haloarcula hispanica, while Haloferax volcanii (phenons D and E) was described in the Dead Sea by studies carried out several decades later than Volcani's work.     

嗜盐古菌R1中与真核同源基因rad25启动子的序列分析和启动活性研究

在盐生盐杆菌(Halobacterium halobium)R1中分析了真核同源基因rad25的转录,分析了rad25同源基因启动子片段的序列特征,用β_半乳糖苷酶基因(bgaH)为报告基因,利用启动子探针检测技术验证了rad25同源基因启动子片段在嗜盐古生菌WFD11中的启动功能;缺失分析进一步确认了rad25同源基因启动子具有嗜盐古菌启动子典型特征。从启动子和转录水平上证明盐生盐杆菌R1中真核同源基因rad25存在生物学功能,推测其可能在核苷酸切除修复(NER)起作用。     

Genetic analysis of a novel plasmid pZMX101 from Halorubrum saccharovorum: determination of the minimal replicon and comparison with the related haloarchaeal plasmid pSCM201

The DNA sequence of a novel haloarchaeal plasmid pZMX101 (3918 bp) from Halorubrum saccharovorum was determined and six ORFs were predicted. The largest ORF encodes a putative replication initiation protein RepA, which shares 40% sequence similarity with the Rep201 of a theta-replication plasmid pSCM201 recently isolated from Haloarcula, suggesting that pZMX101 might replicate via a theta-type mechanism. Using pZMX101 as the only haloarchaeal replicon, a shuttle vector pZMX108 was constructed and successfully transformed into Haloferax volcanii DS70. Based on this in vivo system, the minimal replicon (1978 bp) of pZMX101 was determined. It is composed of the repA gene plus c. 400-bp upstream and 300-bp downstream sequences. Significantly, the putative replication origin of pZMX101 and that of pSCM201 contain different types of sequence motifs, and these two plasmids exhibit distinct host preference for Haloferax and Haloarcula, respectively.     

Molecular cloning of A1-ATPase gene from extremely halophilic archaeon Haloarcula japonica strain TR-1.

The genes encoding A1-ATPase A- and B-subunits were cloned from Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the A1-ATPase gene revealed that the A- and B-subunits consisted of 586 and 473 amino acids, respectively. The deduced amino acid sequences of the A- and B-subunits of Ha. japonica showed high identities with those of Halobacterium salinarum and Haloferax volcanii. The consensus ATP-binding motif was found in the A-subunit.     

PCR and blot hybridization for rapid identification of Haloferax species

Based on the amplification of a 16S rDNA, a PCR assay for the identification of species of Haloferax to genus level was performed. Two variable regions of the 16S rDNA in Haloferax spp. were selected as genus-specific primers for the PCR assay and hybridization probe. Five genera of halophilic Archaea and Escherichia coli were examined as outside groups. Using this approach, all strains of Haloferax spp. were positive. In contrast, all species belonging to the most closely related genera, including Natrinema, Halorubrum, Halobacterium, and Haloarcula, were negative. In addition, the mass bloom of halophilic Archaea that develops in the El-Mallahet saltern of Alexandria City was positive using the same approach. This assay, which does not require pure cultures of microorganisms, is a specific and rapid method for identifying Haloferax spp. in hypersaline environments.     

From replication to cultivation: hot news from Haloarchaea

Haloarchaea have developed into model organisms that are utilized to study many biological processes. Examples are the mechanisms of chromosome maintenance, gene expression and its regulation, protein export and degradation, and motility and sensing. In addition to the analysis of model species like Halobacterium salinarum and Haloferax volcanii, natural communities have been characterized. Halophilic Archaea were found in low-salt environments and are thus more widespread than previously thought.     

Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning

We report the presence of long stretches of tandem repeats in the genome of the halophilic Archaea Haloferax mediterranei and Haloferax volcanii A 30 bp sequence with dyad symmetry (including 5 bp inverted repeats) was repeated in tandem, interspersed with 33–39 bp unique sequences. This structure extends for long stretches — 1.4kb at one location in H. mediterranei chromosome and about 3kb in the H. volcanii chromosome. The tandem repeats (designated TREPs) show a similar distribution in both organisms, appearing once or twice in the H. volcanii and H. mediterranei chromosomes, and once in the largest, probably essential megaplasmid of each organism but not in the smaller replicons. Sequencing of the structures in both H. volcanii replicons revealed an extremely high sequence conservation in both replicons within the species, as well as in the different organisms. Homologous sequences have also been found in other more distantly related halophilic members of the Archaea. Transformation of H. volcanii with a recombinant plasmid containing a 1.1 kb fragment of the TREPs produced significant alterations in the host cells, particularly in terms of cell viability. The introduction of extra copies of TREPs within the vector significantly alters the distribution of the genome among the daughter cells, as observed by DAPI staining. Although the precise biological role cannot be completely ascertained, all the data conform with the tandem repeats being involved in replicon partitioning in halobacteria.     

Construction of a novel shuttle vector based on an RCR-plasmid from a haloalkaliphilic archaeon and transformation into other haloarchaea

The pNB101 is the first plasmid to be isolated from an haloalkaliphilic archaea. With insertion of the ColE1 replicon of Escherichia coli, as well as two antibiotic resistance genes at its unique Hin dIII site, a novel shuttle vector between haloarchaea and E. coli was developed. This vector, named pNB102, was successfully transformed into two non-alkaliphilic haloarchaea, Halobacterium salinarum SNOB and Haloarcula hispanica ATCC33960. The presence and stability of pNB102 in the transformants were confirmed by PCR identification, Southern blotting and restriction endonuclease digestion. Results also indicated that the presence of restriction-modification (R-M) systems in some Halobacterium species prevented this transformation. It is the first report that the replicon of pNB101 has such a wide host range, and has taken the first step for construction of the vector/host system in haloalkaliphilic archaea.     

Absorption of 14C-dicarboxylic acids by bacteria of the family Halobacteriaceae

Cultures of the family Halobacteriaceae belonging to the species Halobacterium halobium, H. cutirubrum, H. vallismortis and Halococcus morrhuae were shown to be capable of assimilating 14C-succinate. Halobacterium salinarium lacked this ability. The transport systems of C4-dicarboxylates differed in Halobacterium halobium 996 and H. vallismortis 1398, on the one hand, and Halococcus morrhuae 1235, on the other. The differences involve the kinetic parameters and stereospecificity of transport systems, the ability to take up different labelled C4-dicarboxylates, the pH-dependence of transport, and the action of CCCP, a protonophorous uncoupling agent. Halobacteria are capable of labelled succinate uptake at a lower NaCl content in the incubation medium than it is necessary for their growth. The optimal temperature for 14C-succinate uptake by halobacteria is higher than the optimal temperature of their growth. For all of the studied cultures, the transport system of dicarboxylate was shown to differ from that of E. coli common for C4-dicarboxylates and aspartate.     

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.     

Comparative analysis of ribonuclease P RNA structure in Archaea.

Although the structure of the catalytic RNA component of ribonuclease P has been well characterized in Bacteria, it has been little studied in other organisms, such as the Archaea. We have determined the sequences encoding RNase P RNA in eight euryarchaeal species: Halococcus morrhuae, Natronobacterium gregoryi, Halobacterium cutirubrum, Halobacteriurn trapanicum, Methanobacterium thermoautotrophicum strains deltaH and Marburg, Methanothermus fervidus and Thermococcus celer strain AL-1. On the basis of these and previously available sequences from Sulfolobus acidocaldarius, Haloferax volcanii and Methanosarcina barkeri the secondary structure of RNase P RNA in Archaea has been analyzed by phylogenetic comparative analysis. The archaeal RNAs are similar in both primary and secondary structure to bacterial RNase P RNAs, but unlike their bacterial counterparts these archaeal RNase P RNAs are not by themselves catalytically proficient in vitro.     

The cell surface glycoprotein layer of the extreme halophile Halobacterium salinarum and its relation to Haloferax volcanii: cryo-electron tomography of freeze-substituted cells and projection studies of negatively stained envelopes

We have studied the surface layer (S-layer) of Halobacterium salinarum (formerly Halobacterium halobium), an extreme halophile requiring high concentrations of sodium, by electron microscopy of (a) isolated, negatively stained, flattened envelopes and (b) cryo-fixation of intact cells in their high-salt growth medium followed by freeze substitution and tomography of thin sections. From the negatively stained isolated envelopes we have calculated a two-dimensional, projection map that is strikingly similar to that of Haloferax volcanii, an extreme halophile requiring high concentrations of magnesium; both projection maps show the hexagonal arrangement of the morphological units with an identical center-to-center spacing of 150 A; each of the morphological units of the two species has six subunits with a similar density distribution and apparent domain organization. In contrast to the two-dimensional map, the tomographic reconstruction of Halob. salinarum does not agree in a straightforward way with the three-dimensional, electron crystallographic map of negatively stained Halof. volcanii envelopes, although the main features of the lattice and the morphological units are evident. The tomographic reconstruction of sections from epoxy-embedded material suffers from directional compression due to sectioning stress and continuous dimensional changes and mass loss due to electron irradiation. This communication consists, therefore, of three parts: (a) a comparison of the projection maps of negatively stained envelopes of Halof. volcanii and Halob. salinarum; (b) a comparison of the three-dimensional maps obtained by electron crystallography (Halof. volcanii) and low-dose cryo-tomography (Halob. salinarum); and (c) a methodological study of mass loss and dimensional changes of plastic-embedded material under low-dose conditions at room and liquid nitrogen temperatures.