Molecular Diversity of New Thermococcales Isolates from a Single Area of Hydrothermal Deep-Sea Vents as Revealed by Randomly Amplified Polymorphic DNA Fingerprinting and 16S rRNA Gene Sequence Analysis

Members of the Thermococcales are anaerobic Archaea belonging to the kingdom Euryarchaea that are studied in many laboratories as model organisms for hyperthermophiles. We describe here a molecular analysis of 86 new Thermococcales isolates collected from six different chimneys of a single hydrothermal field located in the 13°N 104°W segment of the East Pacific ridge at a depth of 2,330 m. These isolates were sorted by randomly amplified polymorphic DNA (RAPD) fingerprinting into nine groups, and nine unique RAPD profiles were obtained. One RAPD group corresponds to new isolates of Thermococcus hydrothermalis, whereas all other groups and isolates with unique profiles are different from the 22 reference strains included in this study. Analysis of 16S rRNA gene sequences of representatives of each RAPD group and unique profiles showed that one group corresponds to Pyrococcus strains, whereas all the other isolates are Thermococcus strains. We estimated that our collection may contain at least 11 new species. These putative species, isolated from a single area of hydrothermal deep-sea vents, are dispersed in the 16S rRNA tree among the reference strains previously isolated from diverse hot environments (terrestrial, shallow water, hydrothermal vents) located around the world, suggesting that there is a high degree of dispersal of Thermococcales. About one-half of our isolates contain extrachromosomal elements that could be used to search for novel replication proteins and to develop genetic tools for hyperthermophiles.     

Investigation of structure and antigenic capacities of Thermococcales cell envelopes and reclassification of "Caldococcus litoralis" Z-1301 as Thermococcus litoralis Z-1301

Fourteen strains of hyperthermophilic organotrophic anaerobic marine Archaea were isolated from shallow water and deep-sea hot vents, and four of them were characterized. These isolates, eight previously published strains, and six type strains of species of the order Thermococcales were selected for the study of cell wall components by means of thin sectioning or freeze-etching electron microscopy. The cell envelopes of most isolates were shown to consist of regularly arrayed surface protein layers, either single or double, with hexagonal lattice (p6) symmetry, as the exclusive constituents outside the cytoplasmic membrane. The S-layers studied differed in center-to-center spacing and molecular mass of the constituent protein subunits. Polyclonal antisera raised against the cells of 10 species were found to be species-specific and allowed 12 new isolates from shallow water hot vents to be identified as representatives of the species Thermococcus litoralis, Thermococcus stetteri, Thermococcus chitonophagus, and Thermococcus pacificus. Of the 7 deep-sea isolates, only 1 was identified as a T. litoralis strain. Thus, hyperthermophilic marine organotrophic isolates obtained from deep-sea hot vents showed greater diversity with regard to their S-layer proteins than shallow water isolates.     

Starvation survivability of Thermococcus strains isolated from Japanese oil reservoirs

The starvation survivability of seven Thermococcus strains isolated from four Japanese oil reservoirs was compared with that of Thermococcus strains from marine hydrothermal fields. 16S rDNA analyses showed the isolates to be closely related to Thermococcus litoralis. Growth of the isolates was dependent on amino acids, which were present at low concentrations in the oil reservoirs. At 80 degrees C in the formation water, strain CKU-1 from the oil reservoir showed a higher starvation survivability than strain KS-1 from the marine hydrothermal field. Crude oil did not affect the starvation survivability of strain CKU-1, but it reduced that of strain KS-1. These results indicate that strain CKU-1 could survive longer than stain KS-1 under the conditions of an oil reservoir. At 90 degrees C in artificial seawater without organic nutrients, the half-lives of the isolates were between 7.7 and 25.1 days. However, those of the strains from marine hydrothermal fields, except Thermococcus litoralis and Thermococcus chitonophagus, were less than 1.0 day. The higher starvation survivability is probably important for the hyperthermophiles to continue to exist in a hot subterranean oil reservoir where the supply of nutrients seems to be limited.     

Design of 16S rRNA-targeted oligonucleotide probes for detecting cultured and uncultured archaeal lineages in high-temperature environments

In order to facilitate the evaluation of archaeal community diversity and distribution in high-temperature environments, 14 16S rRNA oligonucleotide probes were designed. Adequate hybridization and wash conditions of the probes encompassing most known hyperthermophilic Archaea, members of the orders Thermococcales, Desulfurococcales and Sulfolobales, of the families Methanocaldococcaceae, Pyrodictiaceae and Thermoproteaceae, of the genera Archaeoglobus, Methanopyrus and Ignicoccus, and of the as yet uncultured lineages Korarchaeota, Crenarchaeota marine group I, deep-sea hydrothermal vent euryarchaeotic group 2 (DHVE 2), and deep-sea hydrothermal vent euryarchaeotic group 8 (DHVE 8) were determined by dot-blot hybridization from target and non-target reference organisms and environmental clones. The oligonucleotide probes were also used to evaluate the archaeal community composition in nine deep-sea hydrothermal vent samples. All probes, except those targeting members of Sulfolobales, Thermoproteaceae, Pyrodictiaceae and Korarchaeota, gave positive hybridization signals when hybridized against 16S rDNA amplification products obtained from hydrothermal DNA extracts. The results confirmed the widespread occurrence of Thermococcales, Desulfurococcales, Methanocaldococcaceae and Archaeoglobus in deep-sea hydrothermal vents, and extended the known ecological habitats of uncultured lineages. Despite their wide coverage, the probes were unable to resolve the archaeal communities associated with hydrothermally influenced sediments, suggesting that these samples may contain novel lineages. This suite of oligonucleotide probes may represent an efficient tool for rapid qualitative and quantitative characterization of archaeal communities. Their application would help to provide new insights in the future into the composition, distribution and abundance of Archaea in high-temperature environments.     

Diversity among three novel groups of hyperthermophilic deep-sea Thermococcus species from three sites in the northeastern Pacific Ocean

Eight new strains of deep-sea hyperthermophilic sulfur reducers were isolated from hydrothermal vent fields at 9 degrees 50'N East Pacific Rise (EPR) and at the Cleft and CoAxial segments along the Juan de Fuca Ridge (JdFR). 16S rRNA gene sequence analysis showed that each strain belongs to the genus Thermococcus. Restriction fragment length polymorphism patterns of the 16S/23S rRNA intergenic spacer region revealed that these isolates fell into three groups: those from the EPR, those from fluid and rock sources on the JdFR, and those isolated from Paralvinella spp. polychaete vent worms from the JdFR. The optimum-temperature specific growth rates and the temperature ranges for growth were significantly higher and broader for those strains isolated from worms relative to those isolated from low-temperature diffuse hydrothermal fluids. Furthermore, the worm-derived isolates generally produced a larger array of proteases and amylases based on zymogram analyses. The zymogram patterns also changed with growth temperature suggesting that these organisms alter their lytic protein suites in response to changes in temperature. This study suggests that there is significant phenotypic diversity in Thermococcus that is not apparent from their highly conserved 16S rRNA nucleotide sequences.     

Two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins

Thermococcales (phylum Euryarchaeota) are model organisms for physiological and molecular studies of hyperthermophiles. Here we describe three new plasmids from Thermococcales that could provide new tools and model systems for genetic and molecular studies in Archaea. The plasmids pTN2 from Thermococcus nautilus sp. 30-1 and pP12-1 from Pyrococcus sp. 12-1 belong to the same family. They have similar size (∼12 kb) and share six genes, including homologues of genes encoded by the virus PAV1 from Pyrococcus abyssi. The plasmid pT26-2 from Thermococcus sp. 26-2 (21.5 kb), that corresponds to another plasmid family, encodes many proteins having homologues in virus-like elements integrated in several genomes of Thermococcales and Methanococcales. Our analyses confirm that viruses and plasmids are evolutionary related and co-evolve with their hosts. Whereas all plasmids previously isolated from Thermococcales replicate by the rolling circle mechanism, the three plasmids described here probably replicate by the theta mechanism. The plasmids pTN2 and pP12-1 encode a putative helicase of the SFI superfamily and a new family of DNA polymerase, whose activity was demonstrated in vitro, whereas pT26-2 encodes a putative new type of helicase. This strengthens the idea that plasmids and viruses are a reservoir of novel protein families involved in DNA replication.     

Novel bacterial and archaeal lineages from an in situ growth chamber deployed at a Mid-Atlantic Ridge hydrothermal vent

The phylogenetic diversity was determined for a microbial community obtained from an in situ growth chamber placed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge (23 degrees 22' N, 44 degrees 57' W). The chamber was deployed for 5 days, and the temperature within the chamber gradually decreased from 70 to 20 degrees C. Upon retrieval of the chamber, the DNA was extracted and the small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for the Archaea or Bacteria domain and cloned. Unique rDNA sequences were identified by restriction fragment length polymorphisms, and 38 different archaeal and bacterial phylotypes were identified from the 85 clones screened. The majority of the archaeal sequences were affiliated with the Thermococcales (71%) and Archaeoglobales (22%) orders. A sequence belonging to the Thermoplasmales confirms that thermoacidophiles may have escaped enrichment culturing attempts of deep-sea hydrothermal vent samples. Additional sequences that represented deeply rooted lineages in the low-temperature eurarchaeal (marine group II) and crenarchaeal clades were obtained. The majority of the bacterial sequences obtained were restricted to the Aquificales (18%), the epsilon subclass of the Proteobacteria (epsilon-Proteobacteria) (40%), and the genus Desulfurobacterium (25%). Most of the clones (28%) were confined to a monophyletic clade within the epsilon-Proteobacteria with no known close relatives. The prevalence of clones related to thermophilic microbes that use hydrogen as an electron donor and sulfur compounds (S(0), SO(4), thiosulfate) indicates the importance of hydrogen oxidation and sulfur metabolism at deep-sea hydrothermal vents. The presence of sequences that are related to sequences from hyperthermophiles, moderate thermophiles, and mesophiles suggests that the diversity obtained from this analysis may reflect the microbial succession that occurred in response to the shift in temperature and possible associated changes in the chemistry of the hydrothermal fluid.     

Investigation of structure and antigenic capacities of Thermococcales cell envelopes and reclassification of "Caldococcus litoralis" Z-1301 as Thermococcus litoralis Z-1301

Fourteen strains of hyperthermophilic organotrophic anaerobic marine Archaea were isolated from shallow water and deep-sea hot vents, and four of them were characterized. These isolates, eight previously published strains, and six type strains of species of the order Thermococcales were selected for the study of cell wall components by means of thin sectioning or freeze-etching electron microscopy. The cell envelopes of most isolates were shown to consist of regularly arrayed surface protein layers, either single or double, with hexagonal lattice (p6) symmetry, as the exclusive constituents outside the cytoplasmic membrane. The S-layers studied differed in center-to-center spacing and molecular mass of the constituent protein subunits. Polyclonal antisera raised against the cells of 10 species were found to be species-specific and allowed 12 new isolates from shallow water hot vents to be identified as representatives of the species Thermococcus litoralis, Thermococcus stetteri, Thermococcus chitonophagus, and Thermococcus pacificus. Of the 7 deep-sea isolates, only 1 was identified as a T. litoralis strain. Thus, hyperthermophilic marine organotrophic isolates obtained from deep-sea hot vents showed greater diversity with regard to their S-layer proteins than shallow water isolates. Received: February 5, 1999 / Accepted: May 11, 1999     

Isolation and characterization of Thermococcus sibiricus sp. nov. from a Western Siberia high-temperature oil reservoir

Anaerobic organotrophic hyperthermophilic Archaea were isolated from five of eight samples from oil wells of the Samotlor oil reservoir (depth, 1,799-2,287 m; temperature, 60 degrees-84 degrees C). Three strains were isolated in pure cultures and characterized phylogenetically on the basis of comparison of the 16S rRNA gene sequences. All strains belonged to a new species of the genus Thermococcus, with Thermococcus litoralis, Thermococcus aggregans, Thermococcus fumicolans, and Thermococcus alcaliphilus being the nearest relatives (range of sequence similarity, 97.2%-98.8%). Strain MM 739 was studied in detail. The new isolate grew on peptides but not on carbohydrates. Elemental sulfur had a stimulatory effect on growth. The temperature range for growth was between 40 degrees and 88 degrees C, with the optimum at 78 degrees C; the pH range was 5.8 to 9.0, with the optimum around 7.3; and the salinity range was 0.5% to 7.0%, with the optimum at 1.8%-2.0%. The doubling time at optimal growth conditions was about 43 min. The G+C content of the DNA was 38.4 mol%. The DNA-DNA relatedness between strain MM 739 and T. litoralis was 27%; between strain MM 739 and T. aggregans, it was 22%. Based on the phenotypic and genomic differences with known Thermococcus species, the new species Thermococcus sibiricus is proposed. The isolation of a hyperthermophilic archaeum from a deep subsurface environment, significantly remote from shallow or abyssal marine hot vents, indicates the existence of a subterranean biosphere inhabited by indigenous hyperthermophilic biota.     

Compatible solutes of the hyperthermophile Palaeococcus ferrophilus: osmoadaptation and thermoadaptation in the order thermococcales

The effect of salinity and growth temperature on the accumulation of intracellular organic solutes was examined in the hyperthermophilic archaeon Palaeococcus ferrophilus. The genus Palaeococcus represents a deep-branching lineage of the order Thermococcales, which diverged before Thermococcus and Pyrococcus. Palaeococcus ferrophilus accumulated mannosylglycerate, glutamate, and aspartate as major compatible solutes. Unlike members of the genera Pyrococcus and Thermococcus, Palaeococcus ferrophilus did not accumulate di-myo-inositol phosphate, a canonical solute of hyperthermophiles. The level of mannosylglycerate increased in response to both heat and salt stress; glutamate increased at supraoptimal growth temperatures, whereas aspartate increased at supraoptimal salt concentration. Proline, alanine, and trehalose were also found in lesser amounts, but their levels did not respond significantly to any of the stresses imposed. Additionally, the genes involved in the synthesis of mannosylglycerate in Palaeococcus ferrophilus and Thermococcus litoralis were identified. In both organisms the synthesis proceeds via the two-step pathway comprising mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The mpgS and mpgP genes of Palaeococcus ferrophilus were expressed in Escherichia coli and the proteins were characterized. MPGS had maximal activity at 90 degrees C and pH near 7.0, and was strictly dependent on Mg2+. MPGP had optimal activity at 90 degrees C and pH 6.0 and was barely dependent on Mg2+. The half-life values for inactivation of MPGS and MPGP at 83 degrees C were 18 and 25 min, respectively. A comparative discussion of the osmo- and thermoadaptation strategies in these three genera of the Thermococcales is presented.     

Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids.

Three hyperthermophilic sulfur-dependent heterotrophs were isolated from a shallow submarine hydrothermal system at an inlet of Kodakara-jima island, Kagoshima, Japan. The isolates grew at 60 to 97 degrees C, with the optimum temperatures at 85 to 90 degrees C. Sensitivity to rifampin and the existence of ether lipids indicated that the isolates are hyperthermophilic archaea. Partial sequencing of the genes coding for 16S rRNA showed that the three isolates are closely related to the genus Thermococcus. They grew on proteinaceous mixtures, such as yeast extract, Casamino Acids, and purified proteins (e.g., casein and gelatin), but not on carbohydrates or organic acids as sole carbon and energy sources. Nine amino acids were essential for growth of isolate KS-1 (Thr, Leu, Ile, Val, Met, Phe, His, Tyr, and Arg). Isolate KS-2 required Lys in addition to the nine amino acids, and KS-8 required Lys instead of Tyr. In comparative studies, it was shown that Thermococcus celer DSM 2476 required 10 amino acids (Thr, Leu, Ile, Val, Met, Phe, Tyr, Trp, Lys, and Arg) while Pyrococcus furiosus DSM 3638 required only Ile and Val. The hyperthermophilic fermentative eubacterium Thermotoga neapolitana DSM 4359 did not require any amino acids for growth.     

Identification of extracellular enzyme producing thermophilic bacilli from Balcova (Agamemnon) geothermal site by ITS rDNA RFLP

AIMS: Molecular characterization of extracellular enzyme producing thermophilic bacilli from Balcova geothermal site. METHODS AND RESULTS: Three types of geothermal samples were collected: mud, re-injection water, and samples from uncontrolled hydrothermal vents. Isolates grown at 55 degrees C in culture media prepared in sterilized re-injection water, were screened for extracellular enzyme activity by using eight different substrates: casein, carboxymethylcellulose, pectin, polygalacturonic acid (PGA), soluble starch, Tween 20 and 80, and xylan. In total, 109 thermoaerophilic isolates were selected. All of the isolates could hydrolyse Tween 20 (100%) but not Tween 80. Soluble starch was hydrolysed by 96%, casein by 55%, xylan and carboxymethylcellulose by 9%, and pectin and PGA by 2% of the isolates. The isolates were grouped into 14 different homology groups by the restriction pattern analysis of 16S-internal transcribed spacer (ITS) rDNA RFLP. Each of the RFLP groups was also studied by 16S rRNA gene partial sequence analysis. Plasmid DNA profiles revealed that 15 of the isolated strains contained small plasmid DNA molecules ranging in size from 12 000 to 35 000 bp. CONCLUSIONS: Combined analysis of 16S-ITS rDNA RFLP and 16S rRNA gene partial sequence results indicated the presence of novel or existing species of Anoxybacillus (nine species) and Geobacillus (three species). SIGNIFICANCE AND IMPACT OF THE STUDY: In this study 16S-ITS rDNA RFLP was applied for the first time to differentiate thermophilic bacilli. It was also the first study on thermophilic bacilli of Balcova geothermal site.     

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA inter-genic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S–23S IGS RFLP pat-terns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. legumino-sarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Precipitation of greigite and pyrite induced by Thermococcales: an advantage to live in Fe- and S-rich environments?

Thermococcales, a major order of archaea inhabiting the iron- and sulfur-rich anaerobic parts of hydrothermal deep-sea vents, have been shown to rapidly produce abundant quantities of pyrite FeS₂ in iron–sulfur-rich fluids at 85°C, suggesting that they may contribute to the formation of ‘low temperature’ FeS₂ in their ecosystem. We show that this process operates in Thermococcus kodakarensis only when zero-valent sulfur is directly available as intracellular sulfur vesicles. Whether in the presence or absence of zero-valent sulfur, significant amounts of Fe₃S₄ greigite nanocrystals are formed extracellularly. We also show that mineralization of iron sulfides induces massive cell mortality but that concomitantly with the formation of greigite and/or pyrite, a new generation of cells can grow. This phenomenon is observed for Fe concentrations of 5 mM but not higher suggesting that above a threshold in the iron pulse all cells are lysed. We hypothesize that iron sulfides precipitation on former cell materials might induce the release of nutrients in the mineralization medium further used by a fraction of surviving non-mineralized cells allowing production of new alive cells. This suggests that biologically induced mineralization of iron-sulfides could be part of a survival strategy employed by Thermococcales to cope with mineralizing high-temperature hydrothermal environments.     

Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1

A hyperthermophilic archaeal strain, KOD1, isolated from a solfatara on Kodakara Island, Japan, has previously been reported as Pyrococcus sp. KOD1. However, a detailed phylogenetic tree, made possible by the recent accumulation of 16S rRNA sequences of various species in the order Thermococcales, indicated that strain KOD1 is a member of the genus Thermococcus. We performed DNA-DNA hybridization tests against species that displayed high similarity in terms of 16S ribosomal DNA sequences, including Thermococcus peptonophilus and Thermococcus stetteri. Hybridization results and differences in growth characteristics and substrate utilization differentiated strain KOD1 from T. peptonophilus and T. stetteri at the species level. Our results indicate that strain KOD1 represents a new species of Thermococcus, which we designate as Thermococcus kodakaraensis KOD1 sp. nov.     

Genetic diversity of root-nodulating bacteria isolated from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA intergenic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S-23S IGS RFLP patterns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. leguminosarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Towards the ecology of hyperthermophiles: biotopes, new isolation strategies and novel metabolic properties

Ecological studies have shown that water-containing terrestrial, subterranean and submarine high-temperature environments harbor a great diversity of hyperthermophilic prokaryotes, growing fastest at temperatures of 80 degrees C or above. The investigations included cultivation, isolation and detailed analysis of these hyperthermophiles as well as in situ 16S rRNA gene sequence analysis and in situ hybridization studies. For a safe and fast isolation of novel hyperthermophiles from mixed cultures, a new, plating-independent isolation technique was developed, based on the use of a laser microscope ('optical tweezers'). This method, combined with 16S rRNA gene sequence analysis and whole-cell hybridization using fluorescently labelled oligonucleotide probes, even allows the recovery of pure cultures of phylogenetically predicted organisms harboring novel 16S rRNA gene sequences. In their natural habitats, hyperthermophiles form complex food webs, consisting of primary producers and consumers of organic material. Their metabolic potential includes various types of aerobic and anaerobic respiration and different modes of fermentation. In hydrothermal and geothermal environments, hyperthermophiles have important ecological functions in biogeochemical processes. Members of the Sulfolobales are able to mobilize heavy metals from sulfidic ores like pyrite or chalcopyrite. Biomineralization processes of hyperthermophiles include the formation of magnetite from iron or the precipitation of arsenate as realgar, a reaction performed by a novel hyperthermophile that was isolated from Pisciarelli Solfatara, Naples, Italy.     

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

ABSTRACT: BACKGROUND: Haemophilus parasuis is the causative agent of Glasser's disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. RESULTS: The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson's index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. CONCLUSIONS: The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.     

RAPD analysis of Yersinia enterocolitica

A total of 87 isolates of Yersinia enterocolitica were examined with randomly amplified polymorphic DNA (RAPD) by use of three different primers. Based on the RAPD profiles, the strains could be divided into three major groups: (1) the pathogenic American serotypes, O: 8, O: 13ab, O: 20 and O: 21; (2) the pathogenic European serotypes, O: 3, O: 5,27 and O: 9; and (3) the nonpathogenic serotypes. Five tested strains of the American serotype O: 4 gave unique profiles with YCPEL, but did not give reproducible profiles with the other primers. The European serotypes could be further subdivided into a group consisting of strains of O: 3 and O: 5,27 and a group of strains of O: 9. RAPD profiling provides an easy approachable method to divide isolates of Y. enterocolitica into pathogenic and nonpathogenic strains and further to differentiate between the pathogenic isolates.     

Purification, characterization, and application of a novel dye-linked L-proline dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus

The distribution of dye-linked L-amino acid dehydrogenases was investigated in several hyperthermophiles, and the activity of dye-linked L-proline dehydrogenase (dye-L-proDH, L-proline:acceptor oxidoreductase) was found in the crude extract of some Thermococcales strains. The enzyme was purified to homogeneity from a hyperthermophilic archaeon, Thermococcus profundus DSM 9503, which exhibited the highest specific activity in the crude extract. The molecular mass of the enzyme was about 160 kDa, and the enzyme consisted of heterotetrameric subunits (alpha(2) beta(2)) with two different molecular masses of about 50 and 40 kDa. The N-terminal amino acid sequences of the alpha-subunit (50-kDa subunit) and the beta-subunit (40-kDa subunit) were MRLTEHPILDFSERRGRKVTIHF and XRSEAKTVIIGGGIIGLSIAYNLAK, respectively. Dye-L-proDH was extraordinarily stable among the dye-linked dehydrogenases under various conditions: the enzyme retained its full activity upon incubation at 70 degrees C for 10 min, and ca. 40% of the activity still remained after heating at 80 degrees C for 120 min. The enzyme did not lose the activity upon incubation over a wide range of pHs from 4.0 to 10.0 at 50 degrees C for 10 min. The enzyme exclusively catalyzed L-proline dehydrogenation using 2,6-dichloroindophenol (Cl2Ind) as an electron acceptor. The Michaelis constants for L-proline and Cl2Ind were determined to be 2.05 and 0.073 mM, respectively. The reaction product was identified as Delta(1)-pyrroline-5-carboxylate by thin-layer chromatography. The prosthetic group of the enzyme was identified as flavin adenine dinucleotide by high-pressure liquid chromatography. In addition, the simple and specific determination of L-proline at concentrations from 0.10 to 2.5 mM using the stable dye-L-proDH was achieved.