Showing papers in "Extremophiles in 2004"

Geochemical and microbiological evidence for a hydrogen-based, hyperthermophilic subsurface lithoautotrophic microbial ecosystem (HyperSLiME) beneath an active deep-sea hydrothermal field

Abstract: Subsurface microbial communities supported by geologically and abiologically derived hydrogen and carbon dioxide from the Earth's interior are of great interest, not only with regard to the nature of primitive life on Earth, but as potential analogs for extraterrestrial life. Here, for the first time, we present geochemical and microbiological evidence pointing to the existence of hyperthermophilic subsurface lithoautotrophic micro- bial ecosystem (HyperSLiME) dominated by hyper- thermophilic methanogens beneath an active deep-sea hydrothermal field in the Central Indian Ridge. Geo- chemical and isotopic analyses of gaseous components in the hydrothermal fluids revealed heterogeneity of both concentration and carbon isotopic compositions of methane between the main hydrothermal vent (0.08 mM and )13.8& PDB, respectively) and the adjacent diver- gent vent site (0.2 mM and )18.5& PDB, respectively), representing potential subsurface microbial methano- genesis, at least in the divergent vent emitting more 13 C-depleted methane. Extremely high abundance of magmatic energy sources such as hydrogen (2.5 mM) in the fluids also encourages a hydrogen-based, lithoauto- trophic microbial activity. Both cultivation and culti- vation-independent molecular analyses suggested the predominance of Methanococcales members in the superheated hydrothermal emissions and chimney inte- riors along with the other major microbial components of Thermococcales members. These results imply that a HyperSLiME, consisting of methanogens and ferment- ers, occurs in this tectonically active subsurface zone, strongly supporting the existence of hydrogen-driven subsurface microbial communities.

Diversity of Kenyan soda lake alkaliphiles assessed by molecular methods

Abstract: DNA was extracted from water and sediment samples taken from soda lakes of the Kenyan-Tanzanian Rift Valley. DNA was also extracted from microbial enrichment cultures of sediment samples. 16S rRNA genes were amplified by the polymerase chain reaction and microbial diversity was studied using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA amplicons. Cloning and sequencing of single DGGE bands showed that they usually contained mixed amplicons. Several of the amplicon sequences had high identities, up to 99%, with 16S rRNA genes of organisms previously isolated from soda lakes, while others were only distantly related, with identities as low as 82%. Phylogenetic analysis of the sequenced amplicons indicated that sequences were related to the haloarchaeal, Bacillus/Clostridium, Rhodobacterium/Thioalcalovibrio/ Methylobacter, and Cytophaga/Flavobacterium/Bacteroides (CFB) groups and the enterobacteria/Aeromonas/Vibrio part of the γ3 subdivision of the Proteobacteria.

Tetraether-linked membrane monolayers in Ferroplasma spp: a key to survival in acid.

Abstract: Ferroplasma acidarmanus thrives in hot, extremely low pH, metal-rich solutions associated with dissolving metal sulfide ore deposits. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thin layer chromatography analyses of F. acidarmanus membranes indicate that tetraether lipids predominate, with at least three core lipid structures. NMR measurements indicate that the cytoplasmic pH of F. acidarmanus is approximately 5.6. The optimal growth pH is approximately 1.2, and the lowest growth pH is approximately 0.0. Thus, these organisms maintain pH gradients across their membranes that approach 5 pH units. Tetraether lipids were originally thought to be specifically associated with thermophiles but are now known to be widely distributed within the archaeal domain. Our data, in combination with recently published results for thermophilic and mesothermophilic acidophilic archaea, indicate that there may be a stronger association between tetraether lipids and tolerance to acid and/or large metal ion gradients.

Salt-induced changes in lipid composition and membrane fluidity of halophilic yeast-like melanized fungi.

Abstract: The halophilic melanized yeast-like fungi Hortaea werneckii, Phaeotheca triangularis, and the halotolerant Aureobasidium pullulans, isolated from salterns as their natural environment, were grown at different NaCl concentrations and their membrane lipid composition and fluidity were examined. Among sterols, besides ergosterol, which was the predominant one, 23 additional sterols were identified. Their total content did not change consistently or significantly in response to raised NaCl concentrations in studied melanized fungi. The major phospholipid classes were phosphatidylcholine and phosphatidylethanolamine, followed by anionic phospholipids. The most abundant fatty acids in phospholipids contained C16 and C18 chain lengths with a high percentage of C18:2Δ9,12. Salt stress caused an increase in the fatty acid unsaturation in the halophilic H. werneckii and halotolerant A. pullulans but a slight decrease in halophilic P. triangularis. All the halophilic fungi maintained their sterol-to-phospholipid ratio at a significantly lower level than did the salt-sensitive Saccharomyces cerevisiae and halotolerant A. pullulans. Electron paramagnetic resonance (EPR) spectroscopy measurements showed that the membranes of all halophilic fungi were more fluid than those of the halotolerant A. pullulans and salt-sensitive S. cerevisiae, which is in good agreement with the lipid composition observed in this study.

The first evidence of anaerobic CO oxidation coupled with H2 production by a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent.

Abstract: From 24 samples of hydrothermal venting structures collected at the East Pacific Rise (13°N), 13 enrichments of coccoid cells were obtained which grew on CO, producing H2 and CO2 at 80°C. A hyperthermophilic archaeon capable of lithotrophic growth on CO coupled with equimolar production of H2 was isolated. Based on its 16S rRNA sequence analysis, this organism was affiliated with the genus Thermococcus. Other strains of Thermococcales species (Pyrococcus furiosus, Thermococcus peptonophilus, T. profundus, T. chitonophagus, T. stetteri, T. gorgonarius, T. litoralis, and T. pacificus) were shown to be unable to grow on CO. Searches in sequence databases failed to reveal deposited sequences of genes related to CO metabolism in Thermococcales. Our work provides the first evidence of anaerobic CO oxidation coupled with H2 production performed by an archaeon as well as the first documented case of lithotrophic growth of a Thermococcales representative.

Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice, Spitzbergen.

Abstract: The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4 degrees C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses alpha and gamma of Proteobacteria, the Bacillus-Clostridium group, the order Actinomycetales, and the Cytophaga-Flexibacter-Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria gamma subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25 degrees C. Only a few strains were psychrophilic, with an optimal growth at 10-15 degrees C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4 degrees C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, alpha-amylase and beta-galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10 degrees C, and almost no production was detected at higher temperatures (20-30 degrees C). Catalytic activity was detected even below the freezing point of water (at -5 degrees C), demonstrating the unique properties of these enzymes.

Halobacterium noricense sp. nov., an archaeal isolate from a bore core of an alpine Permian salt deposit, classification of Halobacterium sp. NRC-1 as a strain of H. salinarum and emended description of H. salinarum

Abstract: Two rod-shaped haloarchaeal strains, A1 and A2, were isolated from a bore core from a salt mine in Austria. The deposition of the salt is thought to have occurred during the Permian period (225–280 million years ago). The 16S rDNA sequences of the strains were 97.1% similar to that of the type species of the genus Halobacterium, which was also determined in this work. Polar lipids consisted of C20–C20 derivatives of phosphatidylglycerol, methylated phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether and sulfated tetraglycosyl diether. Optimal salinity for growth was 15–17.5% NaCl; Mg++ was tolerated up to a concentration of 1 M. The DNA–DNA reassociation value of strain A1T was 25% with H. salinarum DSM 3754T and 41% with Halobacterium sp. NRC-1, respectively. Based on these results and other properties, e.g. whole cell protein patterns, menaquinone content and restriction patterns of DNA, strains A1 and A2 are members of a single species, for which we propose the name H. noricense. The type strain is A1 (DSM 15987T, ATCC BAA-852T, NCIMB 13967T). Since we present evidence that Halobacterium sp. NRC-1 is a member of H. salinarum, an emended description of H. salinarum is provided.

Phytoplankton diversity and cyanobacterial dominance in a hypereutrophic shallow lake with biologically produced alkaline pH.

Abstract: In this work, we have characterized the diversity of phytoplanktonic species in a highly alkaline and hypereutrophic shallow lake, Santa Olalla (southwestern Spain), the evolution of their relative abundances, and that of several physicochemical parameters over 2 years. In the absence of an external input of alkaline water, Santa Olalla’s stable high pH (average pH 9.52, with several maxima >10.5) is explained by an extremely high photosynthetic primary productivity. A variety of phytoplankton species was observed even during pH maxima. These included several species of green algae, diatoms, and euglenoids and several cyanobacteria from the orders Nostocales and Chroococcales. Quantitatively, cyanobacteria dominated. A blooming event due to Aphanothece clathrata was observed at one pH maximum, during which the diversity as measured by the Shannon-Weaver index was extremely low. Santa Olalla’s cyanobacteria are alkaliphilic and/or extremely alkalitolerant and appear to be responsible for the generation and maintenance of stable high-pH conditions in their environment.

Small heat shock proteins from extremophiles: a review

Abstract: Many microorganisms from extreme environments have been well characterized, and increasing access to genomic sequence data has recently allowed the analysis of the protein families related to stress responses. Heat shock proteins appear to be ubiquitous in extremophiles. In this review, we focus on the family of small heat shock proteins (sHSPs) from extremophiles, which are α-crystallin homologues. Like the α-crystallin eye lens proteins, sHSPs act as molecular chaperones and prevent aggregation of denatured proteins under heat and desiccation stress. Many putative sHSP homologues have been identified in the genomic sequences of all classes of extremophiles. Current studies of shsp gene expression have revealed mechanisms of regulation and activity distinct from other known hsp gene regulation systems. Biochemical studies on sHSPs are limited to thermophilic and hyperthermophilic organisms, and the only two available crystal structures of sHSPs from Methanocaldococcus jannaschii, a hyperthermophilic archaeon and a mesophilic eukaryote, have contributed significantly to an understanding of the mechanisms of action of sHSPs, although many aspects remain unclear.

Characterization and gene cloning of a novel β-mannanase from alkaliphilic Bacillus sp. N16-5

Abstract: An alkaline β-mannanase was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. N16-5. The enzyme had optimum activity at pH 9.5 and 70°C. It was composed of a single polypeptide chain with a molecular weight of 55 kDa deduced from SDS-PAGE, and its isoelectric point was around pH 4.3. The enzyme efficiently hydrolyzed galactomannan and glucomannan, producing a series of oligosaccharides and monosaccharides. The β-mannanase gene (manA) contained an open reading frame (ORF) of 1,479 bp, encoding a 32-amino acids signal peptide, and a mature protein of 461 amino acids, with a calculated molecular mass of 50,743 Da. Strain N16-5 ManA, deduced from the manA ORF, exhibited relatively high amino acid similarity to the members of the glycosyl hydrolase family 5. The eight conserved active-site amino acids in family 5 glycosyl hydrolase were found in the deduced amino acid sequence of strain N16-5 ManA.

A xylan-degrading strain of Sulfolobus solfataricus: isolation and characterization of the xylanase activity.

Abstract: Two strains (O(alpha) and X(2)) of the hyperthermophilic crenarchaeon Sulfolobus solfataricus strain MT4 were selected and isolated for their ability to grow on xylan. O(alpha) and X(2), grown on media containing oat spelt xylan and birchwood xylan as the sole nutrient source, respectively, produced the same thermostable xylanase that was demonstrated to be inducible in xylan cultures. In an oat spelt medium, S. solfataricus O(alpha) underwent interesting morphological changes in the cell envelope, exhibiting mobile appendages not present in the typical coccal shape. The enzyme was prevalently membrane associated and showed a molecular mass of approximately 57.0 kDa. It was also highly thermostable, with a half-life of 47 min at 100 degrees C, and exhibited an optimal temperature and pH of 90 degrees C and 7.0, respectively. Xylo-oligosaccharides were the enzymatic products of xylan hydrolysis, and the smallest degradation product was xylobiose, thus indicating that the enzyme was an endoxylanase. The enzyme was able to bind weakly to crystalline cellulose (Avicel) and more strongly to insoluble xylan in a substrate amount-and temperature-dependent manner.

Bacterial diversity of the Inner Mongolian Baer Soda Lake as revealed by 16S rRNA gene sequence analyses.

Abstract: Bacterial diversity associated with Baer Soda Lake in Inner Mongolia of China was investigated using a culture-independent method. Bacterial 16S rRNA gene libraries were generated using bacterial oligonucleotide primers, and 16S rRNA gene sequences of 58 clones were analyzed phylogenetically. The library was dominated by 16S rDNAs of Gram-negative bacteria (24% α-Proteobacteria, 31% β-Proteobacteria, 33% γ-Proteobacteria, and 2% δ-Proteobacteria), with a lower percentage of clones corresponding to Gram-positive bacteria. Forty cloned sequences were similar to that of known bacterial isolates (>97% sequence similarity), represented by the species of the genera Brevundimonas, Comamonas, Alcaligenes, Stenotrophomonas, and Klebsiella. Eighteen cloned sequences showed less affiliation with known taxa (<97% sequence similarity) and may represent novel taxa.

A phylogenetic analysis of Wadi el Natrun soda lake cellulase enrichment cultures and identification of cellulase genes from these cultures

Abstract: Samples of sediments and surrounding soda soils (SS) from the extremely saline and alkaline lakes of the Wadi el Natrun in the Libyan Desert, Egypt, were obtained in October 2000. Anaerobic enrichment cultures were grown from these samples, DNA isolated, and the bacterial diversity assessed by 16S rRNA gene clone analysis. Clones derived from lake sediments (LS) most closely matched Clostridium spp., Natronoincola histidinovorans, Halocella cellulolytica, Bacillus spp., and the Cytophaga–Flexibacter–Bacteroides group. Similar clones were identified in the SS, but Bacillus spp. predominated. Many of the clones were most closely related to organisms already identified in alkaline or saline environments. Two genomic DNA libraries were made from the pooled LS enrichments and a single SS-enrichment sample. A novel cellulase activity was identified and characterized in each. The lake cellulase ORF encoded a protein of 1,118 amino acids; BLASTP analysis showed it was most closely related to an endoglucanase from Xanthomonas campestris. The soil-cellulase ORF encoded a protein of 634 amino acids that was most closely related to an endoglucanase from Fibrobacter succinogenes.

Thermoactive extracellular proteases of Geobacillus caldoproteolyticus, sp. nov., from sewage sludge.

Abstract: A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65 degrees C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH4Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0-11.0 and a temperature range of 40-90 degrees C, with an optimal pH of 8.0-9.0 and an optimal temperature of 70-80 degrees C, respectively. The protease was stable over the temperature range of 40-70 degrees C and retained 57 and 38% of its activity at 80 and 90 degrees C, respectively, after 1 h.

Genomic characterization of thermophilic Geobacillus species isolated from the deepest sea mud of the Mariana Trench

Abstract: The thermophilic strains HTA426 and HTA462 isolated from the Mariana Trench were identified as Geobacillus kaustophilus and G. stearothermophilus, respectively, based on physiologic and phylogenetic analyses using 16S rDNA sequences and DNA–DNA relatedness. The genome size of HTA426 and HTA462 was estimated at 3.23–3.49 Mb and 3.7–4.49 Mb, respectively. The nucleotide sequences of three independent λ-phage inserts of G. stearothermophilus HTA462 have been determined. The organization of protein coding sequences (CDSs) in the two λ-phage inserts was found to differ from that in the contigs corresponding to each λ insert assembled by the shotgun clones of the G. kaustophilus HTA426 genome, although the CDS organization in another λ insert is identical to that in the HTA426 genome.

Thialkalivibrio halophilus sp. nov., a novel obligately chemolithoautotrophic, facultatively alkaliphilic, and extremely salt-tolerant, sulfur-oxidizing bacterium from a hypersaline alkaline lake.

Abstract: A new chemolithoautotrophic, facultatively alkaliphilic, extremely salt-tolerant, sulfur-oxidizing bacterium was isolated from an alkaline hypersaline lake in the Altai Steppe (Siberia, Russia). According to 16S rDNA analysis and DNA–DNA hybridization, strain HL 17T was identified as a new species of the genus Thialkalivibrio belonging to the γ subdivision of the Proteobacteria for which the name Thialkalivibrio halophilus is proposed. Strain HL 17T is an extremely salt-tolerant bacterium growing at sodium concentrations between 0.2 and 5 M, with an optimum of 2 M Na+. It grew at high concentrations of NaCl and of Na2CO3/NaHCO3 (soda). Strain HL 17T is a facultative alkaliphile growing at pH range 7.5–9.8, with a broad optimum between pH 8.0 and 9.0. It used reduced inorganic sulfur compounds (thiosulfate, sulfide, polysulfide, elemental sulfur, and tetrathionate) as energy sources and electron donors. In continuous culture under energy limitation, thiosulfate was stoichiometrically oxidized to sulfate. In sodium carbonate medium under alkaline conditions, the maximum growth rate was similar, while the biomass yield was lower as compared with the NaCl-grown culture. The maximum sulfur-oxidizing capacity measured in washed cells was higher in the soda buffer independent of the growth conditions. The compatible solute content of the biomass was higher in the sodium chloride-grown culture than in the sodium carbonate/bicarbonate-grown culture. The data suggest that the osmotic pressure differences between soda and NaCl solutions might be responsible for the difference observed in compatible solutes production. This may have important implications in overall energetic metabolism of high salt adaptation.

Isolation of extremophiles with the detection and retrieval of Shewanella strains in deep-sea sediments from the west Pacific.

Abstract: Tests to detect the presence of piezophilic Shewanella strains in the deep-sea sediments of the west, mid- and east Pacific at different depths were done by amplification of previously identified pressure-regulated operons (ORF1,2 and ORF3). The operon fragments were detected in all the deep-sea sediment samples, indicating the broad presence of piezophilic deep-sea Shewanella species or related species in the deep-sea sediments across the Pacific. Extremophiles were isolated from the deep-sea sediment of the west Pacific under atmospheric pressure. Two psychrophilic/psychrotrophic strains, WP2 and WP3, were assigned to the Shewanella genus as determined by their 16S rDNA sequences. WP2 and WP3 were both capable of amplifying pressure-regulated operons; the sequences of the pressure-regulated operons of WP2 and WP3 share high identity between each other, but have more differences from those of S. benthica and S. violacea. The major fatty acids of WP2 and WP3 are 3OH-i-13:0, 14:0, i-15:0, 16:0, 16:1, 18:1, and 20:5. Combined phenotypic analysis, 16S rDNA sequences, and DNA–DNA hybridization results suggest that WP2 and WP3 are two new deep-sea Shewanella species.

Thermococcus marinus sp nov and Thermococcus radiotolerans sp nov., two hyperthermophilic archaea from deep-sea hydrothermal vents that resist ionizing radiation

Abstract: Enrichments for anaerobic, organotrophic hyperthermophiles were performed with hydrothermal chimney samples collected from the Mid-Atlantic Ridge at a depth of 3,550 m (23°22′N, 44°57′W) and the Guaymas Basin (27°01′N, 111°24′W) at a depth of 2,616 m. Positive enrichments were submitted to γ-irradiation at doses of 20 and 30 kGy. Two hyperthermophilic, anaerobic, sulfur-metabolizing archaea were isolated. Strain EJ1T was isolated from chimney samples collected from the Mid-Atlantic Ridge after γ-irradiation at 20 kGy, and strain EJ2T was isolated from the Guaymas Basin after γ-irradiation at 30 kGy. Only strain EJ2T was motile, and both formed regular cocci. These new strains grew between 55 and 95 °C with the optimal temperature being 88 °C. The optimal pH for growth was 6.0, and the optimal NaCl concentration for growth was around 20 g l−1. These strains were obligate anaerobic heterotrophs that utilized yeast extract, tryptone, and peptone as a carbon source for growth. Ten amino acids were essential for the growth of strain EJ1T, such as arginine, aspartic acid, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tyrosine, and valine, while strain EJ2T was unable to grow on a mixture of amino acids. Elemental sulfur or cystine was required for EJ2T growth and was reduced to hydrogen sulfide. Rifampicin inhibited growth for both strains EJ1T and EJ2T. The G+C contents of the genomic DNA were 52.3 and 54.5 mol% for EJ1T and EJ2T, respectively. As determined by 16S rRNA gene sequence analysis, these strains were more closely related to Thermococcus gorgonarius, T. celer, T. guaymasensis, T. profundus, and T. hydrothermalis. However, no significant homology was observed between them with DNA–DNA hybridization. These novel organisms also possess phenotypic traits that differ from those of its closest phylogenetic relatives. Therefore, it is proposed that these isolates, which are amongst the most radioresistant hyperthermophilic archaea known to date with T. gammatolerans (Jolivet et al. 2003a), should be described as novel species T. marinus sp. nov. and T. radiotolerans sp. nov. The type strain of T. marinus is strain EJ1T (=DSM 15227T=JCM 11825T) and the type strain of T. radiotolerans is strain EJ2T (=DSM 15228T=JCM 11826T).

Engineering the thermostability of Trichoderma reesei endo-1,4-β-xylanase II by combination of disulphide bridges

Abstract: Disulphide bridges were introduced in different combinations into the N-terminal region and the single α-helix of mesophilic Trichoderma reesei xylanase II (TRX II). We used earlier disulphide-bridge data and designed new disulphide bridges for the combination mutants. The most stable mutant contained two disulphide bridges (between positions 2 and 28 and between positions 110 and 154, respectively) and the mutations N11D, N38E, and Q162H. With a half-life of ~56 h at 65°C, the thermostability of this sevenfold mutant was ~5,000 times higher than that of TRX II, and the half-life was 25 min even at 75°C. The thermostability of this mutant was ~30 times higher than that of the corresponding mutant missing the bridge between positions 2 and 28. The extensive stabilization at two protein regions did not alter the kinetic properties of the sevenfold mutant from that of the wild-type TRX II. The combination of disulphide bridges enhanced significantly the pH-dependent stability in a wide pH range.

Alkalimonas amylolytica gen. nov., sp. nov., and Alkalimonas delamerensis gen. nov., sp. nov., novel alkaliphilic bacteria from soda lakes in China and East Africa

Abstract: Two related novel alkaliphilic and slightly halophilic bacteria are described. They are strain N10 from Lake Chahannor in China and strain 1E1 from Lake Elmenteita in East Africa. Both strains are strictly aerobic, heterotrophic, alkaliphilic, mesophilic, and require NaCl for growth. The optimal conditions for growth were at pH 10–10.5 and 2–3% (w/v) NaCl. Cells of both strains were Gram-negative, rod-shaped, non-spore-forming, and motile with a single polar flagellum. Cellular fatty acids in both strains were predominantly saturated and mono-unsaturated straight-chain fatty acids (16:0, 16:1ω7c and 18:1ω7c). The major isoprenoid quinone of both strains was Q8. The major polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylglycerol phosphate and phosphatidylethanolamine. The guanine plus cytosine (G+C) content of the DNA was 52.5 mol% and 55.4 mol%, respectively. Phylogenetic analysis revealed that the two strains formed a distinct lineage within the gamma-3 subclass of the Proteobacteria. The strains shared a 16S rDNA sequence similarity of 96.1% and showed less than 93.7% of sequence similarity to any other known species. Based on polyphasic data, the two strains were differentiated from currently recognized genera and represent a new genus, Alkalimonas gen. nov., with two species, Alkalimonas amylolytica sp. nov. (type strain is N10T = AS 1.3430) and Alkalimonas delamerensis sp. nov. ( type strain is 1E1P, T = CBS 391.94). The GenBank accession numbers for the 16S rRNA gene sequence of strains N10 and 1E1 are AF250323 and X92130, respectively.

Identification of archaea and some extremophilic bacteria using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

Abstract: Archaea and a number of groups of environmentally important bacteria, e.g., sulfate-reducing bacteria, anoxygenic phototrophs, and some thermophiles, are difficult to characterize using current methods developed for phenotypically differentiating heterotrophic bacteria. We have evaluated matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS) as a rapid method for identifying different groups of extremophilic prokaryotes using a linear mass spectrometer (Micromass, UK). The instrument is designed to acquire mass-spectral patterns from prokaryotic cell-wall components between masses of 500 and 10,000 Da in a statistically robust manner and create a database that can be used for identification. We have tested 28 archaea (10 genera, 20 spp.) and 42 bacteria (25 genera, 37 spp.) and found that all species yield reproducible, unique mass-spectral profiles. As a whole, the profiles for the archaea had fewer peaks and showed less differentiation compared to the bacteria, perhaps reflecting fundamental differences in cell-wall structure. The halophilic archaea all had consistent patterns that showed little differentiation; however, the software was able to consistently distinguish Halobacterium salinarium, Halococcus dombrowski, and Haloarcula marismortui from one another, although it could not always correctly distinguish four strains of Hb. salinarium from one another. The method was able to reliably identify 105 cells of either Albidovulum inexpectatum or Thermococcus litoralis and could detect as low as 103 cells. We found that the matrix, alpha-cyano-4-hydroxy-cinnamic acid yielded better spectra for archaea than 5-chloro-2-mercapto-benzothiazole. Overall, the method was rapid, required a minimum of sample processing, and was capable of distinguishing and identifying a very diverse group of prokaryotes.

Distribution, diversity and activity of microorganisms in the hyper-alkaline spring waters of Maqarin in Jordan.

Abstract: The hyper-alkaline, high-Ca2+ springs of Maqarin, Jordan, were investigated as an analogue for various microbial processes at the extremely high pH generated by cement and concrete in some underground radioactive waste repositories. Leaching of metamorphic, cementitious phases in Maqarin has produced current, hyper-alkaline groundwater with a maximum pH of 12.9. Six consecutive expeditions were undertaken to the area during 1994–2000. The total number of microorganisms in the alkaline waters was 103–105 cells/ml. Analysis of the 16S-ribosomal ribonucleic acid (rRNA) diversity revealed microorganisms mainly belonging to the Proteobacteria. Obvious similarities between the obtained sequences and sequences from other alkaline sites could not be found. Numerous combinations of culture media compositions were inoculated with spring, seepage and groundwaters and incubated under aerobic and anaerobic conditions with various carbon sources. Assimilation studies were performed using identical radio-labeled carbon sources. Glucose seemed to be the preferred carbon source for assimilation, followed by acetate, lactate, and leucine. The results demonstrate that microorganisms from the hyper-alkaline springs of Maqarin could grow and be metabolically active under aerobic and anaerobic hyper-alkaline conditions. However, the growth and activity found were not vigorous; instead, slow growth, low numbers, and a generally low metabolic activity were found. This suggests that microbial activity will be low during the hyper-alkaline phase of cementitious repositories.

Isolation and characterization of a cellulolytic Geobacillus thermoleovorans T4 strain from sugar refinery wastewater.

Abstract: A novel, cellulolytic, bacterial thermophilic strain, T4, was isolated from sugar refinery wastewater in southern Taiwan. This isolate, a Gram-negative, motile, aerobically growing sporulating rod, can secrete thermostable endocellulase (endo-1,4-β-D-glucanase, EC 3.2.1.4) and hydrolyze carboxymethylcellulose (CMC), phosphoric acid-swollen cellulose, Avicel, filter paper, and salicin. When strain T4 was grown in CMC medium, the cellulolytic enzyme activity in culture supernatants was stable up to 70°C. More than 10% of the original activity was still detectable after heating to 100°C with a pH 7.0 for 1 h. Based on 16S rDNA sequence analysis, DNA base composition, phenotypic and physiological characteristics, as well as DNA–DNA hybridization, strain T4 was classified as Geobacillus thermoleovorans T4 (DSM 14791 = CCRC 17200). We also demonstrated that the type species G. stearothermophilus (DSM 22 = ATCC 12980) could hydrolyze amorphous and crystalline (filter paper) celluloses at a rate of 13 and 14%, respectively, in comparison with strain T4.

Degradation of phenol and toxicity of phenolic compounds: a comparison of cold-tolerant Arthrobacter sp. and mesophilic Pseudomonas putida.

Abstract: Phenol degradation efficiency of cold-tolerant Arthrobacter sp. AG31 and mesophilic Pseudomonas putida DSM6414 was compared. The cold-tolerant strain was cultivated at 10°C, while the mesophile was grown at 25°C. Both strains degraded 200 mg and 400 mg phenol/l within 48–72 h of cultivation, but the cold-tolerant strain produced more biomass than the mesophile. Both strains oxidized catechol by the ortho type of ring fission. Catechol 1,2 dioxygenase (C1,2D) activity was found intra- and extracellularly in the absence and in the presence of phenol. In the presence of 200 mg phenol/l, C1,2D activity of the mesophile was about 1.5- to 2-fold higher than that of the cold-tolerant strain. However, an initial phenol concentration of 400 mg/l resulted in a comparable enzyme activity of the cold-tolerant and the mesophilic strain. The two strains differed significantly in their toxicity pattern towards 12 aromatic (mostly phenolic) compounds at different growth temperatures, which was determined via growth inhibition in the presence of nutrients and toxicants. For the cold-tolerant strain, toxicity was significantly lower at 10°C than at 25°C. The mesophile showed a significantly lower susceptibility to high hydrocarbon concentrations when grown at 25°C compared to 10°C.

Differential pressure resistance in the activity of RNA polymerase isolated from Shewanella violacea and Escherichia coli

Abstract: RNA polymerase was purified from the piezophile Shewanella violacea DSS12, and the transcriptional activity after pressure treatment was compared with that of the mesophile Escherichia coli. Application of pressure at 100 MPa for 30 min reduced the E. coli RNA polymerase activity to 60% of the activity at atmospheric pressure, whereas the S. violacea RNA polymerase maintained full activity, indicating that the S. violacea RNA polymerase is more stable than its E. coli counterpart. This result was supported by the analysis of the strength of subunit interactions of the enzyme from both species, using a high-pressure electrophoresis apparatus, which showed that a pressure of 140 MPa caused dissociation of E. coli RNA polymerase but not that of S. violacea RNA polymerase. On the other hand, the core enzyme of S. violacea RNA polymerase, which lacked the sigma70 factor, was dissociated at 140 MPa. These results suggest that the sigma70 factor is required for stabilization of S. violacea RNA polymerase under high-pressure conditions. In this paper, we provide in vitro evidence for piezoadaptation at the transcriptional level, using purified RNA polymerase from cells of S. violacea and E. coli.

Growth kinetics of haloalkaliphilic, sulfur-oxidizing bacterium Thioalkalivibrio versutus strain ALJ 15 in continuous culture

Abstract: The chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio versutus strain ALJ 15, isolated from a soda lake in Kenya, was grown in a continuous culture, with thiosulfate or polysulfide as growth-limiting energy source and oxygen as electron acceptor, at pH 10 and at pH 0.6, 2 M and 4 M total sodium. The end product of the sulfur-compound oxidation was sulfate. Elemental sulfur and a cell-bound, polysulfide-like compound appeared as intermediates during substrate oxidation. In the thiosulfate-limited culture, the biomass yields and maximum specific growth rates decreased two and three times, respectively, with increasing sodium concentration. The apparent affinity constant measured for thiosulfate and polysulfide was in the micromolar range (Ks=6±3 μM). The maintenance requirement (ms=8±5 mmol S2O32/g dry weight h−1) was in the range of values found for other autotrophic sulfur-oxidizing bacteria. The organism had a comparable maximum specific rate of oxygen uptake with thiosulfate, polysulfide, and sulfide, while elemental sulfur was oxidized at a lower rate. Glycine betaine was the main organic compatible solute. The respiration rates with different species of polysulfides (Sn2−) were tested. All polysulfide species were completely oxidized at high rates to sulfate. Overall data demonstrated efficient growth and sulfur compounds oxidation of haloalkaliphilic chemolithoautotrophic bacteria from soda lakes.

Psychrophilic Pseudomonas syringae requires trans -monounsaturated fatty acid for growth at higher temperature

Abstract: A psychrophilic bacterium, Pseudomonas syringae (Lz4W) from Antarctica, was used as a model system to establish a correlation, if any, between thermal adaptation, trans-fatty acid content and membrane fluidity. In addition, attempts were made to clone and sequence the cti gene of P. syringae (Lz4W) so as to establish its characteristics with respect to the cti of other Pseudomonas spp. and also to in vitro mutagenize the cti gene so as to generate a cti null mutant. The bacterium showed increased proportion of saturated and trans-monounsaturated fatty acids when grown at 28°C compared to cells grown at 5°C, and the membrane fluidity decreased with growth temperature. In the mutant, the trans-fatty acid was not synthesized, and the membrane fluidity also decreased with growth temperature, but the decrease was not to the extent that was observed in the wild-type cells. Thus, it would appear that synthesis of trans-fatty acid and modulation of membrane fluidity to levels comparable to the wild-type cells is essential for growth at higher temperatures since the mutant exhibits growth arrest at 28°C. In fact, the cti null mutant-complemented strain of P. syringae (Lz4W-C30b) that was capable of synthesizing the trans-fatty acid was indeed capable of growth at 28°C, thus confirming the above contention. The cti gene of P. syringae (Lz4W) that was cloned and sequenced exhibited high sequence identity with the cti of other Pseudomonas spp. and exhibited all the conserved features.

Transcriptional analysis of dynamic heat-shock response by the hyperthermophilic bacterium Thermotoga maritima.

Abstract: The thermal stress response of the hyperthermophilic bacterium Thermotoga maritima was characterized using a 407-open reading frame-targeted cDNA microarray. Transient gene expression was followed for 90 min, following a shift from 80°C to 90°C. While some aspects of mesophilic heat-shock response were conserved in T. maritima, genome content suggested differentiating features that were borne out by transcriptional analysis. Early induction of predicted heat-shock operons hrcA-grpE-dnaJ (TM0851-TM0850-TM0849), groES-groEL (TM0505-TM0506), and dnaK-sHSP (TM0373-TM0374) was consistent with conserved CIRCE elements upstream of hrcA and groES. Induction of the T. maritima rpoE/sigW and rpoD/sigA homologs suggests a mechanism for global heat-shock response in the absence of an identifiable ortholog to a major heat-shock sigma factor. In contrast to heat-shock response in Escherichia coli, the majority of genes encoding ATP-dependent proteases were downregulated, including clpP (TM0695), clpQ (TM0521), clpY (TM0522), lonA (TM1633), and lonB (TM1869). Notably, T. maritima showed indications of a late heat-shock response with the induction of a marR homolog (TM0816), several other putative transcriptional regulators (TM1023, TM1069), and two α-glucosidases (TM0434 and TM1068). Taken together, the results reported here indicate that, while T. maritima shares core elements of the bacterial heat-shock response with mesophiles, the thermal stress regulatory strategies of this organism differ significantly. However, it remains to be elucidated whether these differences are related to thermophilicity or phylogenetic placement.

Identification of iron-reducing Thermus strains as Thermus scotoductus.

Abstract: Thermus strain SA-01, previously isolated from a deep (3.2 km) South African gold mine, is closely related to Thermus strains NMX2 A.1 and VI-7 (previously isolated from thermal springs in New Mexico, USA, and Portugal, respectively). Thermus strains SA-01 and NMX2 A.1 have also been shown previously to grow using nitrate, Fe(III), Mn(IV) or S(O) as terminal electron acceptors and to be capable of reducing Cr(VI), U(VI), Co(III), and the quinone-containing compound anthraquinone-2,6-disulfonate. The objectives of this study were to determine the phylogenetic positions of the three known metal-reducing Thermus strains and to determine the phylogenetic significance of metal reduction within the genus Thermus. Phylogenetic analyses of 16S rDNA sequences, BOX PCR genomic fingerprinting, and DNA-DNA reassociation analyses indicated that these strains belong to the previously described genospecies T. scotoductus. The morphologies and lipid fatty acid profiles of these metal-reducing strains are consistent with their identification as T. scotoductus; however, the T. scotoductus strains tested in this study evinced a wide intraspecies variability in some other phenotypic traits, e.g., carbon substrate utilization and pigmentation. Iron reduction occurred in all strains of T. scotoductus tested except the mixotrophic, sulfur-oxidizing strain IT-7254. Thermus strains belonging to other species did not reduce Fe(III) to Fe(II) or reduced it only poorly.

Promoters from a cold-adapted bacterium: definition of a consensus motif and molecular characterization of UP regulative elements.

Abstract: Although low-temperature tolerant micro-organisms were discovered long ago, limited information is still available on the transcription machinery in cold-adapted bacteria. This knowledge represents a necessary background for the investigation of the adaptation of gene-expression mechanisms at low temperatures. The recent development of a shuttle genetic system for the transformation of the cold-adapted Gram-negative bacterium Pseudoalteromonas haloplanktis strain TAC125 has made possible the isolation of the psychrophilic promoters described in this paper. TAC125 genomic DNA fragments were cloned in the shuttle vector and the promoter-containing recombinant clones were selected for their ability to express a promoter-less lacZ gene. The nucleotide sequence of several selected inserts and the transcription start points of the transcribed m-RNAs were determined. A promoter consensus sequence for Pseudoalteromonas haloplanktis TAC125 was proposed on the basis of a sequence comparison between the various active promoters. Furthermore, the identification and the functional characterization of two UP elements from this cold-adapted bacterium are also reported.