新疆两盐湖可培养嗜盐古菌多样性研究

从新疆地区艾比盐湖和艾丁盐湖卤水及泥土样品中分离到86株嗜盐古菌。16S rRNA基因序列分析结果表明,分离自艾比湖的嗜盐古菌分别属于Haloarcula、Halobacterium、Halorubrum、Haloterrigena、Natrinema和Natronorubrum6个属的11个分类单元,而分离自艾丁湖的嗜盐古菌分别属于Haloarcula、Halobiforma、Halorubrum、Haloterrigena、Natrialba、Natrinema6个属的8个分类单元,这一结果表明艾比湖可培养嗜盐古菌生物多样性稍高于艾丁湖。基于16S rRNA基因序列的系统发育分析表明代表菌株ABH15应为Natronorubrum属的中性嗜盐古菌新种,代表菌株ABH07、ABH12、ABH17、ABH19、ABH51和AD30可能是Halobacterium、Halorubrum、Haloterrigena、Haloarcula的新成员。     

Diversity of halophilic archaea from six hypersaline environments in Turkey

The diversity of archaeal strains from six hypersaline environments in Turkey was analyzed by comparing their phenotypic characteristics and 16S rDNA sequences. Thirty-three isolates were characterized in terms of their phenotypic properties including morphological and biochemical characteristics, susceptibility to different antibiotics, and total lipid and plasmid contents, and finally compared by 16S rDNA gene sequences. The results showed that all isolates belong to the family Halobacteriaceae. Phylogenetic analyses using approximately 1,388 bp comparisions of 16S rDNA sequences demonstrated that all isolates clustered closely to species belonging to 9 genera, namely Halorubrum (8 isolates), Natrinema (5 isolates), Haloarcula (4 isolates), Natronococcus (4 isolates), Natrialba (4 isolates), Haloferax (3 isolates), Haloterrigena (3 isolates), Halalkalicoccus (1 isolate), and Halomicrobium (1 isolate). The results revealed a high diversity among the isolated halophilic strains and indicated that some of these strains constitute new taxa of extremely halophilic archaea.     

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.     

Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons.

We have stably transformed both Haloarcula vallismortis and Haloarcula hispanica with the halobacterium-Escherichia coli shuttle vectors pWL102 (based on the Haloferax volcanii pHV2 replicon) and pUBP2 (based on the Halobacterium halobium pHH1 replicon). Haloferax volcanii, Halobacterium halobium, and Haloarcula vailismortis are equally distant from one another and span the phylogenetic depth of the halophilic Archaea; thus, these vectors may be generally useful for the halophiles. Both Haloarcula vallismortis and Haloarcula hispanica exhibit previously unreported complex life cycles and are therefore significant as genetically approachable models of cellular differentiation within the Archaea.     

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.     

Diversity of Archaea in hypersaline environments characterized by molecular-phylogenetic and cultivation studies

The diversity of Archaea from three different hypersaline environments was analyzed and compared by polymerase chain reaction (PCR)-based molecular phylogenetic techniques and cultivation approaches. The samples originated from a crystallization pond of a solar saltern in Spain (FC); an alkaline lake in Nevada, USA, (EMF); and a small pond from a slag heap of a potassium mine in Germany (DIE). Except for two 16S rDNA sequences that were related to crenarchaeota from soil and did not apparently belong to the indigenous halophilic community, all sequences recovered from environmental DNA or cultivated strains grouped within the Halobacteriaceae. Mostly 16S rDNA sequences related to the genera Halorubrum and Haloarcula were detected in sample FC, and organisms belonging to these genera were also recovered by cultivation. In contrast, sequences related to five different groups of halophilic archaea were amplified from sample DIE (including novel lineages with only uncultivated phylotypes), but the organisms that were cultivated from this sample fell into different groups (i.e., Natronococcus, Halorubrum, or unaffiliated) and did not overlap with those predicted using the culture-independent approach. With respect to the highly alkaline sample, EMF, four groups were predicted from the environmental 16S rDNA sequences, two of which ( Natronomonas and Haloarcula) were also recovered through cultivation together with Natronococcus isolates. In summary, we found that halophilic archaea dominate the archaeal populations in these three hypersaline environments and show that culturability of the organisms predicted by molecular surveys might strongly depend on the habitat chosen. While a number of novel halophilic archaea have been isolated, we have not been able to cultivate representatives of the new lineages that were detected in this and several other environmental studies.     

Ureases of extreme halophiles of the genus Haloarcula with a unique structure of gene cluster

We searched for urease activities in 71 strains of extreme halophiles by a urea-phenol red-agar plate method. Positive strains were further investigated by measuring the ammonia released from urea in cell-free extracts. Only 4 strains of the genus Haloarcula, Har. aidinensis, Har. hispanica, Har. japonica, and Har. marismortui were finally shown as the urease producers. A partially purified urease from Har. hispanica was a typical halophilic enzyme in that it showed maximum activity at 18-23% NaCl and lost the activity irreversibly in the absence of NaCl. Partial genes (1596 bp) of the urease encoding from upstream of the beta subunit down to the N-terminal 139 amino acids of the alpha subunit, were PCR amplified from the four strains, as well as from five urease-negative Haloarcula strains. Strains of other genera, which were urease-negative, did not yield PCR products. The deduced amino acid sequences of the beta subunit and partial alpha subunit were similar to each other (92-100% similarities) and to those from other organisms. Analysis of the draft genome sequence of Har. marismortui, however, suggested that the order of the genes encoding the three subunits (with the total number of amino acids of 834) and four accessory proteins was beta-alpha-gamma-UreG-UreD-UreE-UreF. This order is quite unique, since in other microorganisms the order is gamma-beta-alpha-UreE-UreF-UreG-UreD in most cases. No open reading frames were detected in the PCR-amplified upstream of the beta subunit, suggesting that all Haloarcula species have the same unique structure of the urease gene cluster.     

岱山盐场可培养嗜盐菌的多样性及其产酶活性筛选

从岱山盐场采集样品,利用选择性培养基分离培养嗜盐菌,对盐田环境中可培养嗜盐菌的多样性及产酶活性进行研究.共分离得到181株嗜盐菌菌株,通过真细菌和古生菌两对通用引物扩增其16S rRNA 基因,并采用限制性内切酶Hinf I进行ARDRA(amplified rDNA restriction analysis)多态性分析,共分为21个不同的操作分类单元(operation taxonomy units, OTUs),其中嗜盐细菌有12个OTUs,嗜盐古菌有9个OTUs.选取具有不同酶切图谱的代表菌株进行克隆测序,BLAST 比对及系统发育分析将嗜盐细菌归于7个属,其中嗜盐单胞菌属(Halomonas)的菌株数占优势,是嗜盐细菌总数的46.8%;嗜盐古菌归于4个属,盐盒菌属(Haloarcula)的菌株数占优势,是嗜盐古菌总数的49.1%.对分离菌株的产酶活性进行检测表明,岱山盐田环境蕴含丰富的产淀粉酶、蛋白酶和脂肪酶等生物活性酶的嗜盐菌, 其中盐盒菌属产酶菌株数最丰富.研究结果表明,岱山盐田环境中具有较为丰富的嗜盐菌多样性,是筛选产酶菌株的重要资源库.     

内蒙古锡林浩特地区嗜盐古菌多样性的研究

从内蒙古锡林浩特地区3个不同的盐湖中共分离到165株古菌,通过ARDRA分析后得到不同的类群,从各个类群中随机选取1~2个代表菌株进行16S rDNA序列测定和系统发育的分析。结果表明分离的菌株分布在Halorubrum,Natronococcus,Natronorubrum,Haloterrigena,Halorhabdus,Halobiforma,Haloarcula,Haloferax8个属和另外两个分支中,表现了锡林浩特地区嗜盐古菌的多样性。部分菌株的16S rDNA序列同源性低于97%,可能是潜在的新属或新种,代表了该地区嗜盐古菌的独特类型。     

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.     

Characterization of the dominant halophilic archaea in a bacterial bloom in the dead sea

Abstract A mass bloom of halophilic archaea developed in the Dead Sea in the summer of 1992, with peak densities of more than 3 × 10⁷ cells/ml, imparting a red coloration to the water. Microscopical examination showed a numerical dominance of pleomorphic, flat cells. Attempts to identify the dominant type of halophilic archaea by means of growth experiments, both on agar plates and by dilution in liquid media, were unsuccessful, as viable counts obtained were two or more orders of magnitude lower than the total microscopic counts. Analysis of the polar lipids in the Dead Sea biomass during the bloom showed one major glycolipid to be present in the extracts, corresponding with the sulfated diglycosyl diether lipid (S-DGD-1) characteristic of the genus Haloferax . No indications were found for the presence of significant amounts of other glycolipids that indicate the presence of large numbers of Dead Sea archaea such as Halobacterium sodomense or Haloarcula marismortui , or Halobacterium species such as H. halobium, H. salinarium and H. saccharovorum . Thus, the numerically dominant organisms in the bloom is probably a difficult to culture, not yet isolated, representative of the genus Haloferax .     

Metabolism of halophilic archaea

In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

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.     

连云港台北和盐城三圩盐田土壤嗜盐菌多样性研究

盐田土壤嗜盐微生物对盐田生态系统的良性循环和盐的生产至关重要。本文对江苏连云港台北盐田土壤和盐城三圩盐田土壤的嗜盐细菌和古菌的多样性进行了研究, 结果表明两地盐土嗜盐细菌和古菌的分布具有相似性和独特性。采用培养法从两地盐土中共分离到17株嗜盐细菌, 其中Halomonas为两地盐土共有的嗜盐细菌, 而Halobacillus和Pontibacillus仅在三圩盐土中发现。通过非培养的16S rDNA 基因文库法从两地盐土中发现了13种嗜盐古菌, 台北盐土有Halobacterium 和 Haloplanus, 三圩盐土有Halobacterium, Natronobacterium, Halogeometricum 和 Haloarcula。10个嗜盐古菌的16S rDNA和GenBank已知序列的同源性为92%~97%, 可能为这些属中的新种。该研究为盐田环境嗜盐微生物资源的开发和利用奠定了基础。     

连云港台北和盐城三圩盐田土壤嗜盐菌多样性研究

盐田土壤嗜盐微生物对盐田生态系统的良性循环和盐的生产至关重要.本文对江苏连云港台北盐田土壤和盐城三圩盐田土壤的嗜盐细菌和古菌的多样性进行了研究,结果表明两地盐土嗜盐细菌和古菌的分布具有相似性和独特性.采用培养法从两地盐土中共分离到17株嗜盐细菌,其中Halomonas为两地盐土共有的嗜盐细菌,而Halobacillus和Pontibacillus仅在三圩盐土中发现.通过非培养的16S rDNA基因文库法从两地盐土中发现了13种嗜盐古菌,台北盐土有Halobacterium和Haloplanus,三圩盐土有Halobacterium, Natronobacterium, Halogeometricum和Haloarcula. 10个嗜盐古菌的16S rDNA和GenBank已知序列的同源性为92%～97%.可能为这些属中的新该研究为盐田环境嗜盐微生物资源的开发和利用奠定了基础.     

Natrinema salaciae sp. nov., a halophilic archaeon isolated from the deep, hypersaline anoxic Lake Medee in the Eastern Mediterranean Sea

Two halophilic archaea, strains MDB25(T) and MDB20, were isolated from a sample of the brine from Lake Medee, at a depth of 3050m, in the Mediterranean Sea. Cells of the organisms were Gram-negative, non-motile and pleomorphic, and colonies were red pigmented. Strains MDB25(T) and MDB20 showed optimum growth at 45°C, in 2.6-3.4M NaCl and at pH 7.0-8.0. The major polar lipids of the two strains were phosphatidylglycerol (PG1 and PG2), phosphatidylglycerol phosphate methyl ester (PGP-Me) and mannose-2,6-dissulfate (1→2)-glucose glycerol diether (S(2)-DGD). Menaquinone MK-8 and MK-8(H(2)) were the major respiratory quinones. The DNA G+C content of strain MDB25(T) was 63.0%. The strains were facultatively anaerobic but grew better under aerobic conditions, nitrate served as electron acceptor. Analysis of the almost complete 16S rRNA gene sequence indicated that the strains MDB25(T) and MDB20 represented a member of the genus Natrinema in the family Halobacteriaceae. Both strains formed a distinct cluster and were most closely related to Natrinema ejinorense JCM 13890(T) and Haloterrigena longa JCM 13562(T) (98.0% and 97.9% sequence similarity, respectively). Based on 16S rRNA gene sequence analysis, DNA-DNA hybridization results, physiological and biochemical characteristics we describe a new species represented by strain MDB25(T) (=DSM 25055(T) =JCM 17869(T)) for which we propose the name Natrinema salaciae sp. nov.     

Haloarcula marismortui (Volcani) sp. nov., nom. rev., an extremely halophilic bacterium from the Dead Sea

An extremely halophilic red archaebacterium isolated from the Dead Sea (Ginzburg et al., J. Gen. Physiol. 55: 187-207, 1970) belongs to the genus Haloarcula and differs sufficiently from the previously described species of the genus to be designated a new species; we propose the name Haloarcula marismortui (Volcani) sp. nov., nom. rev. because of the close resemblance of this organism to "Halobacterium marismortui," which was first described by Volcani in 1940. The type strain is strain ATCC 43049.     

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.     

Halophilic archaea isolated from man-made young (200 years) salt lakes in Slănic, Prahova, Romania

We isolated a number of extremely halophilic archaea from four salt lakes (Red Bath, Green Bath, Shepherd Bath and Bride Cave) located in Slanic Prahova, Romania. The characterization of 12 representative isolates by polyphasic approach revealed that 11 strains were members of the genus Haloferax and only one was a member of the genus Haloarcula. The 11 Haloferax isolates possessed sulfated diglycosylarchaeol-1 as the major membrane glycolipid, and G+C contents of total DNA were 63.4–65.8 mol%. The predominant isolation of Haloferax species from the lakes may suggest that the underground salt deposit possesses Haloferax species as the major biota of ancient origin. To the best of the authors’ knowledge, this is the first paper on the survey of halophilic archaea of man-made young salt lakes.