The isolation and use of iron-oxidizing,moderately thermophilic acidophiles from the Collie coal mine for the generation of ferric iron leaching solution

Moderately thermophilic, iron-oxidizing acidophiles were enriched from coal collected from an open-cut mine in Collie, Western Australia. Iron-oxidizers were enriched in fluidized-bed reactors (FBR) at 60 degrees C and 70 degrees C; and iron-oxidation rates were determined. Ferrous iron oxidation by the microbiota in the original coal material was inhibited above 63;C. In addition to four iron-oxidizers, closely related to Sulfobacillus spp that had been earlier isolated from the 60 degrees C FBR, one heterotroph closely related to Alicyclobacillus spp was isolated. The Alicyclobacillus sp. isolated from the Collie coal mine tolerated a lower pH than known Alicyclobacillus spp and therefore may represent a new species. The optimum temperature for growth of the iron-oxidizing strains was approximately 50 degrees C and their maximum temperatures were approximately 60 degrees C. The FBR was adjusted to operate at 50 degrees C and was inoculated with all of the isolated iron-oxidizing strains. At 60 degrees C, an iron-oxidation rate of 0.5 g Fe(2+) l(-1) x h(-1) was obtained. At 50 degrees C, the iron-oxidation rate was only 0.3 g Fe(2+) l(-1) x h(-1). These rates compare favourably with the iron-oxidation rate of Acidianus brierleyi in shake-flasks, but are considerably lower than mesophilic iron-oxidation rates.     

Microbial diversity in nonsulfur, sulfur and iron geothermal steam vents

Fumaroles, commonly called steam vents, are ubiquitous features of geothermal habitats. Recent studies have discovered microorganisms in condensed fumarole steam, but fumarole deposits have proven refractory to DNA isolation. In this study, we report the development of novel DNA isolation approaches for fumarole deposit microbial community analysis. Deposit samples were collected from steam vents and caves in Hawaii Volcanoes National Park, Yellowstone National Park and Lassen Volcanic National Park. Samples were analyzed by X-ray microanalysis and classified as nonsulfur, sulfur or iron-dominated steam deposits. We experienced considerable difficulty in obtaining high-yield, high-quality DNA for cloning: only half of all the samples ultimately yielded sequences. Analysis of archaeal 16S rRNA gene sequences showed that sulfur steam deposits were dominated by Sulfolobus and Acidianus, while nonsulfur deposits contained mainly unknown Crenarchaeota. Several of these novel Crenarchaeota lineages were related to chemoautotrophic ammonia oxidizers, indicating that fumaroles represent a putative habitat for ammonia-oxidizing Archaea. We also generated archaeal and bacterial enrichment cultures from the majority of the deposits and isolated members of the Sulfolobales. Our results provide the first evidence of Archaea in geothermal steam deposits and show that fumaroles harbor diverse and novel microbial lineages.     

Low incidence of Vibrio vulnificus among Vibrio isolates from sea water and shellfish of the western Mediterranean coast

A specific search for Vibrio vulnificus in natural marine samples from the Spanish Mediterranean Sea was carried out by nested PCR and cultural approaches using thiosulphate-citrate-bile salts-sucrose agar (TCBS) and cellobiose-polymixin B-colistin agar (CPC), incubated at 40 degrees C, as selective media. Presumptive colonies were identified by PCR using specific primers against 23S rRNA sequences. This species was isolated from sea water and edible bivalves, mainly after preenrichment in alkaline peptone water (APW) at 40 degrees C followed by CPC agar. None of the V. vulnificus isolates identified corresponded to serovar E. Dominant Vibrio species on directly inoculated TCBS plates incubated at 25 degrees C were V. splendidus below 20 degrees C and V. harveyi and V. mediterranei above that temperature. Low percentages of several pathogenic vibrios were recorded but V. vulnificus was never recovered at this incubation temperature. The incidence of this species in the samples studied was lower than that described for other geographical areas, probably due to the high salinity values of the Mediterranean Sea.     

Detection of Putatively Thermophilic Anaerobic Methanotrophs in Diffuse Hydrothermal Vent Fluids

The anaerobic oxidation of methane (AOM) is carried out by a globally distributed group of uncultivated Euryarchaeota, the anaerobic methanotrophic arachaea (ANME). In this work, we used G+C analysis of 16S rRNA genes to identify a putatively thermophilic ANME group and applied newly designed primers to study its distribution in low-temperature diffuse vent fluids from deep-sea hydrothermal vents. We found that the G+C content of the 16S rRNA genes (P_GC) is significantly higher in the ANME-1GBa group than in other ANME groups. Based on the positive correlation between the P_GC and optimal growth temperatures (T_opt) of archaea, we hypothesize that the ANME-1GBa group is adapted to thrive at high temperatures. We designed specific 16S rRNA gene-targeted primers for the ANME-1 cluster to detect all phylogenetic groups within this cluster, including the deeply branching ANME-1GBa group. The primers were successfully tested both in silico and in experiments with sediment samples where ANME-1 phylotypes had previously been detected. The primers were further used to screen for the ANME-1 microorganisms in diffuse vent fluid samples from deep-sea hydrothermal vents in the Pacific Ocean, and sequences belonging to the ANME-1 cluster were detected in four individual vents. Phylotypes belonging to the ANME-1GBa group dominated in clone libraries from three of these vents. Our findings provide evidence of existence of a putatively extremely thermophilic group of methanotrophic archaea that occur in geographically and geologically distinct marine hydrothermal habitats.     

Isolation and characterization of S genome specific sequences from Aegilops sect. sitopsis species.

Three S genome specific sequences were isolated from Aegilops sect. sitopsis species using different experimental approaches. Two clones, UTV86 and UTV39, were isolated from a partial genomic library obtained from DNA of Aegilops sharonensis, whereas a third clone, UTV5, was isolated from Aegilops speltoides. The three clones were characterized by sequencing, analysis of methylation, and sequence organization and abundance in some Aegilops and Triticum species. The clones UTV39 and UTV5 belong to the same family of tandem repeated sequences and showed high homology with a sequence already present in nucleotide databases. The UTV86 clone from Ae. sharonensis corresponded to an interspersed low frequency repeated sequence and did not show any significant homology with reported sequences. Southern hybridization experiments, using the cloned sequences as probes, detected polymorphism in the restriction patterns of all the five Aegilops species in section sitopsis. Aegilops speltoides showed the most divergent hybridization pattern. A close relationship was detected between the S genome of Ae. speltoides and the G genome of the wild Triticum timopheevii. In situ hybridization revealed a telomeric and (or) subtelomeric location of the sequences UTV39 and UTV5.     

Purification and properties of the sulfur oxygenase/reductase from the acidothermophilic archaeon, <Emphasis Type="Italic">Acidianus </Emphasis>strain S5

The sulfur oxygenase/reductase (SOR) of Acidianus strain S5 was purified and characterized after expressing the SOR gene in a recombinant strain of Escherichia coli. The N-terminal sequence of the purified SOR protein was the same as the deduced amino acid sequence from previously cloned SOR genes. Enzymatic studies indicated that the SOR catalyzed the conversion of elemental sulfur (S(o)) to sulfite, thiosulfate, and sulfide. The optimal pH and temperature were 5.0 and 70 degrees C, respectively. Comparison of this SOR and that of A. ambivalens revealed several differences between these two SORs. The most striking difference is that the SOR of Acidianus S5 had maximal activity at acidic pH. By application of anti-SOR serum and the Western blot technique, it was found that SOR proteins existed in A. brierleyi and in Acidianus S5 cells cultivated with thiosulfate as the sole energy source, indicating that SOR may also play a role in thiosulfate metabolism.     

Archaeal diversity associated with in situ samplers deployed on hydrothermal vents on the East Pacific Rise (13 degrees N)

To evaluate possible compositional changes in archaeal communities at a deep-sea hydrothermal vent field scale, we examined five different samples obtained after deploying in situ collectors for different times on three spatially separated venting sulphide structures on the East Pacific Rise (13 degrees N). Direct cell counts and whole-cell hybridizations with fluorescently labelled 16S rRNA-based oligonucleotide probes revealed that the relative abundance of archaeal populations represented from 14 to 33% of the prokaryotic community. 16S rDNA sequence analysis of the archaeal clone libraries indicated that a large percentage of clones were closely related to known archaeal isolates recovered from similar habitats. Among the 24 different phylotypes identified, Thermococcales-related sequences were dominant in all the libraries that also included representative genera of orders Methanopyrales, Methanococcales, Archaeoglobales and Desulfurococcales. The presence of most of these phylogenetic groups was confirmed in enrichment cultures performed at temperatures from 60 to 90 degrees C. Additional sequences with no known cultivated relatives grouped with the Marine group I Crenarchaeota, Korarchaeota and Deep-sea Hydrothermal Vent Euryarchaeota (DHVE) within which a novel lineage was identified. Furthermore, the archaeal community composition was distinct from vent to vent within the same vent field and varied within short time scales. This study provides new insights into microbial diversity and distribution at deep-sea hydrothermal vents.     

Effect of acute heat shock on gene expression by human peripheral blood mononuclear cells.

We studied the effect of heat shock on gene expression by normal human cells. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy adults. Paired samples from each subject were subjected to either 20 min of heat shock (43 degrees C) or control (37 degrees C) conditions and then returned to 37 degrees C. RNA was isolated 160 min later, and five representative samples were analyzed on Affymetrix gene chip arrays containing approximately 12,600 probes. A biologically meaningful effect was defined as a statistically significant, twofold or greater difference in expression of sequences that were detected in all five experiments under control (downregulated sequences) or heat shock (upregulated sequences) conditions. Changes occurred in 395 sequences (227 increased by heat shock, 168 decreased), representing 353 Unigene numbers, in every functional category previously implicated in the heat shock response. By RT-PCR, we confirmed the findings for one upregulated sequence (Rad, a G protein) and one downregulated sequence (osteopontin, a cytokine). We conclude that heat shock causes extensive gene expression changes in PBMCs, affecting all functional categories of the heat shock response.     

Novel thermo-acidophilic bacteria isolated from geothermal sites in Yellowstone National Park: physiological and phylogenetic characteristics

Moderately thermophilic acidophilic bacteria were isolated from geothermal (30-83 degrees C) acidic (pH 2.7-3.7) sites in Yellowstone National Park. The temperature maxima and pH minima of the isolates ranged from 50 to 65 degrees C, and pH 1.0-1.9. Eight of the bacteria were able to catalyze the dissimilatory oxidation of ferrous iron, and eleven could reduce ferric iron to ferrous iron in anaerobic cultures. Several of the isolates could also oxidize tetrathionate. Six of the iron-oxidizing isolates, and one obligate heterotroph, were low G+C gram-positive bacteria ( Firmicutes). The former included three Sulfobacillus-like isolates (two closely related to a previously isolated Yellowstone strain, and the third to a mesophilic bacterium isolated from Montserrat), while the other three appeared to belong to a different genus. The other two iron-oxidizers were an Actinobacterium (related to Acidimicrobium ferrooxidans) and a Methylobacterium-like isolate (a genus within the alpha -Proteobacteria that has not previously been found to contain either iron-oxidizers or acidophiles). The other three (heterotrophic) isolates were also alpha-Proteobacteria and appeared be a novel thermophilic Acidisphaera sp. An ARDREA protocol was developed to discriminate between the iron-oxidizing isolates. Digestion of amplified rRNA genes with two restriction enzymes ( SnaBI and BsaAI) separated these bacteria into five distinct groups; this result was confirmed by analysis of sequenced rRNA genes.     

Microbiology of acidic, geothermal springs of Montserrat: environmental rDNA analysis

DNA was extracted from water and sediment samples taken from acidic, geothermal pools on the Caribbean island of Montserrat. 16S rRNA genes were amplified by PCR, cloned, sequenced, and examined to indicate some of the organisms that might be significant components of the in situ microbiota. A clone bank representing the lowest temperature pool that was sampled (33°C) was dominated by genes corresponding to two types of acidophiles: Acidiphilium-like mesophilic heterotrophs and thermotolerant Acidithiobacillus caldus. Three clone types with origins in low- and moderate- (48°C) temperature pools corresponded to bacteria that could be involved in metabolism of sulfur compounds: the aerobic A. caldus and putative anaerobic, moderately thermophilic, sulfur-reducing bacteria (from an undescribed genus and from the Desulfurella group). A higher-temperature sample indicated the presence of a Ferroplasma-like organism, dis-tinct from the other strains of these recently recognized acidophilic, iron-oxidizing members of the Euryarchaeota. Acidophilic Archaea from undescribed genera related to Sulfolobus and Acidianus were predicted to dominate the indigenous acidophilic archaeal population at the highest temperatures. Received: March 19, 2000 / Accepted: August 2, 2000     

Colonization of nascent, deep-sea hydrothermal vents by a novel Archaeal and Nanoarchaeal assemblage

Active deep-sea hydrothermal vents are areas of intense mixing and severe thermal and chemical gradients, fostering a biotope rich in novel hyperthermophilic microorganisms and metabolic pathways. The goal of this study was to identify the earliest archaeal colonizers of nascent hydrothermal chimneys, organisms that may be previously uncharacterized as they are quickly replaced by a more stable climax community. During expeditions in 2001 and 2002 to the hydrothermal vents of the East Pacific Rise (EPR) (9 degrees 50'N, 104 degrees 17'W), we removed actively venting chimneys and in their place deployed mineral chambers and sampling units that promoted the growth of new, natural hydrothermal chimneys and allowed their collection within hours of formation. These samples were compared with those collected from established hydrothermal chimneys from EPR and Guaymas Basin vent sites. Using molecular and phylogenetic analysis of the 16S rDNA, we show here that at high temperatures, early colonization of a natural chimney is dominated by members of the archaeal genus Ignicoccus and its symbiont, Nanoarchaeum. We have identified 19 unique sequences closely related to the nanoarchaeal group, and five archaeal sequences that group closely with Ignicoccus. These organisms were found to colonize a natural, high temperature protochimney and vent-like mineral assemblages deployed over high temperature outflows within 92 h. When compared phylogenetically, several of these colonizing organisms form a unique clade independent of those found in mature chimneys and low-temperature mineral chamber samples. As a model ecosystem, the identification of pioneering consortia in deep-sea hydrothermal vents may help advance the understanding of how early microbial life forms gained a foothold in hydrothermal systems on early Earth and potentially on other planetary bodies.     

Selective phylogenetic analysis targeted at 16S rRNA genes of thermophiles and hyperthermophiles in deep-subsurface geothermal environments

Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76 degrees C) and river water (14 degrees C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82 degrees C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84 degrees C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84 degrees C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.     

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.     

Species Composition of Cultivated and Noncultivated Bacteria from Short Filaments in an Icelandic Hot Spring at 88 degrees C

Samples of short pink-grayish filaments were collected from a hot spring in the Hengill area in southwestern Iceland at 85-88 degrees C, pH 6.9 and 1.7 mg/L sulfide. The species composition was studied by cloning and sequencing small subunit rRNA genes obtained by PCR amplifications from mat DNA. Using 98% sequence similarity as a cutoff value, a total of 5 bacterial operational taxonomic units (OTUs) and 6 archaeal OTUs were detected among 68 bacterial clones and 97 archaeal clones. Database matching showed that 80.5% of the archaeal sequences were 99% similar to Pyrobaculum islandicum and 14.5% were closest to the Korarchaeota clone sequence SRI306. About 87% of the bacterial sequences had the closest database match (99%) to the clone sequence SRI48 but were also found to be 99% identical with hydrogen-oxidizing strains previously isolated in this laboratory from hot springs in the same region. Out of 7 Thermus sequences, 4 were 100% identical to T. scotoductus NMX2 A.1 but 3 represented a new uncultivated Thermus species. Four different media, varying in organic nutrients and phosphate composition were used to isolate 81 aerobic thermophilic heterotrophs. Four isolates were Bacillus spp; but out of 77 Thermus isolates, 42 belonged to T. scotoductus and 35 to T. brockianus. T. scotoductus seemed to be preferably isolated on media low in nutrients and phosphate, whereas for T. brockianus it was the opposite. The T. scotoductus clones and isolates had 99-100% sequence similarity to each other. No T. brockianus sequences were found in the bacterial clone library.     

Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA

High-temperature (>/=60 degrees C) synthetic food waste compost was examined by cultivation-dependent and -independent methods to determine predominant microbial populations. Fluorescent direct counts totaled 6.4 (+/-2.5)x10(10) cells gdw(-1) in a freeze-dried 74 degrees C compost sample, while plate counts for thermophilic heterotrophic aerobes averaged 2.6 (+/-1.0)x10(8) CFU gdw(-1). A pre-lysis cell fractionation method was developed to obtain community DNA and a suite of 16S and 18S rDNA-targeted PCR primers was used to examine the presence of Bacteria, Archaea and fungi. Bacterial 16S rDNA, including a domain-specific 1500-bp fragment and a 300-bp fragment specific for Actinobacteria, was amplified by PCR from all compost samples tested. Archaeal rDNA was not amplified in any sample. Fungal 18S rDNA was only amplified from a separate dairy manure compost that reached a peak temperature of 50 degrees C. Amplified rDNA restriction analysis (ARDRA) was used to screen isolated thermophilic bacteria and a clone library of full-length rDNA fragments. ARDRA screening revealed 14 unique patterns among 63 isolates, with one pattern accounting for 31 of the isolates. In the clone library, 52 unique patterns were detected among 70 clones, indicating high diversity of uncultivated bacteria in hot compost. Phylogenetic analysis revealed that the two most abundant isolates belonged in the genera Aneurinibacillus and Brevibacillus, which are not commonly associated with hot compost. With the exception of one Lactobacillus-type sequence, the clone library contained only sequences that clustered within the genus Bacillus. None of the isolates or cloned sequences could be assigned to the group of obligate thermophilic Bacillus spp. represented by B. stearothermophilus, commonly believed to dominate high-temperature compost. Amplified partial fragments from Actinobacteria, spanning the V3 variable region (Neefs et al. (1990) Nucleic Acids Res. 18, 2237-2242), included sequences related to the genera Saccharomonospora, Gordonia, Rhodococcus and Corynebacterium, although none of these organisms were detected among the isolates or full-length cloned rDNA sequences. All of the thermophilic isolates and sequenced rDNA fragments examined in this study were from Gram-positive organisms.     

Microbial diversity in acid mine drainage of Xiang Mountain sulfide mine, Anhui Province, China

To understand the composition and structure of microbial communities in acid (pH 3.0) mine drainage (AMD) associated with pyrite mine tailings in Anhui Province, China, molecular diversities of 16S rRNA and 18S rRNA genes were examined using a PCR-based cloning approach. Bacterial, archaeal and microeukaryotic clone libraries were constructed. In contrast to typical dominance of autotrophic acidophiles, genus Acidiphilium, which consists of mixotrophic acidophiles capable of chemoorganotrophic and photosynthetic metabolisms, was the largest group in the bacterial clone library. These mixotrophic organisms may be advantageous in the oligotrophic AMD environment of the study site (certain amounts of dissolved organic carbon and light) by switching between two modes of metabolisms. Unexpectedly, a large fraction of bacterial clones (12.7%) were related to the neutrophilic genus Legionella, which can cause Legionnaires’ disease, a potentially lethal pneumonia. The eukaryotic 18S rRNA gene sequences were mostly related to Oxytricha, Nuclearia, and Penicillium. In the archaeal clone library, all the sequences were affiliated to the phylum Crenarchaeota, while the Euryarchaeota was not present.     

劳盆地深海热液喷口沉积物中细菌多样性研究

采用PCR-RFLP技术调查了劳盆地深海热液喷口两位点沉积物中的细菌多样性。结果表明, 在劳盆地深海热液喷口沉积物环境中细菌多样性十分丰富, 样品DY1中发现6个细菌类群, DY2中则存在4个细菌类群, 其中Gammaproteobacteria细菌亚群和Epsilonproteobacteria细菌亚群在两文库中均占据最大比例, 为沉积物样品中的优势菌群。另外, 在克隆文库中还发现了一些与数据库中的已知序列同源性较低的类群, 从而说明劳盆地深海热液喷口沉积物中存在特有的微生物种属。     

Pico-, nano- and microplankton communities in hydrothermal marine coastal environments of the Eolian Islands (Panarea and Vulcano) in the Mediterranean Sea

This paper reports the relationship among pico-, nano- and microplanktoniccommunities observed in two different shallow marine hydrothermalenvironments. Seawater samples from five stations in the coastalarea of the ‘Porto di Levante’ (Vulcano Island)and from three stations off the Island of Panarea (Eolian Islands,Italy) were collected in May and July of 1989, respectively.Microbiological investigations were carried out in order todetermine the density of: (i) the total picoplankton (both autotrophicand heterotrophic): (ii) the total picophytoplankton (autofluorescentpicoplanktonic cells); (iii) the larger phytoplankton (>2µm): (iv) the ‘metabolically active’ cellsof total picoplankton and cyanobacteria; (v) the heterotrophicaerobic bacteria. The peak values of picoplankton and picophytoplanktoncomponents, with an order of magnitude of 10⁹ and 10⁷ cells1¹, respectively, were registered in the wannest water samples(30–75°C) collected from the Vulcano area. At Panarea.eukaryotic picophytoplankton and ‘metabolically active’coccoid cyanobacteria showed an opposite trend. A possible competitionbetween the two groups is to be considered. Cyanobacteria, diatomsand the genus Licmophora. in particular, were prevalent in thewarmest hydrothermal vents of Vulcano. Nano- and microphytoplanktoniccommunities in the offshore waters of the Island of Panareashowed more variability than in Vulcano. Moreover, in the Panareawaters the prevalence of phytoflagellates above the thermoclinewas observed, whereas diatoms were predominant below this layer.     

The adenylate kinases from a mesophilic and three thermophilic methanogenic members of the Archaea.

Adenylate kinase has been isolated from four related methanogenic members of the Archaea. For each, the optimum temperature for enzyme activity was similar to the temperature for optimal microbial growth and was approximately 30 degrees C for Methanococcus voltae, 70 degrees C for Methanococcus thermolithotrophicus, 80 degrees C for Methanococcus igneus, and 80 to 90 degrees C for Methanococcus jannaschii. The enzymes were sensitive to the adenylate kinase inhibitor P1, P5-di(adenosine-5')pentaphosphate, a property that was exploited to purify the enzymes by CIBACRON Blue affinity chromatography. The enzymes had an estimated molecular mass (approximately 23 to 25 kDa) in the range common for adenylate kinases. Each of the enzymes had a region of amino acid sequence close to its N terminus that was similar to the canonical P-loop sequence reported for all adenylate kinases. However, the methanogen sequences lacked a lysine residue that has previously been found to be invariant in adenylate kinases, including an enzyme isolated from the archaeon Sulfolobus acidocaldarius. If verified as a nucleotide-binding domain, the methanogen sequence would represent a novel nucleotide-binding motif. There was no correlation between amino acid abundance and the optimal temperature for enzyme activity.