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A psychrotolerant bacterium, strain T-3 (identified as Psychrobacter piscatorii), that exhibited an extraordinarily high catalase activity was isolated from the drain pool of a plant that uses H(2)O(2) as a bleaching agent. Its cell extract exhibited a catalase activity (19,700 U·mg protein(-1)) that was higher than that of Micrococcus luteus used for industrial catalase production. Catalase was approximately 10% of the total proteins in the cell extract of the strain. The catalase (PktA) was purified homogeneously by only two purification steps, anion exchange and hydrophobic chromatographies. The purified catalase exhibited higher catalytic efficiency and higher sensitivity of activity at high temperatures than M. luteus catalase. The deduced amino acid sequence showed the highest homology with catalase of Psycrobacter cryohalolentis, a psychrotolelant bacterium obtained from Siberian permafrost. These findings suggest that the characteristics of the PktA molecule reflected the taxonomic relationship of the isolate as well as the environmental conditions (low temperatures and high concentrations of H(2)O(2)) under which the bacterium survives. Strain T-3 efficiently produces a catalase (PktA) at a higher rate than Exiguobacterium oxidotolerans, which produces a very strong activity of catalase (EktA) at a moderate rate, in order to adapt to high concentration of H(2)O(2).

It has been found that the alkaliphilic Gram-negative bacterium Pseudomonas alcaliphila AL15-21(T) produces a larger amount of soluble c-type cytochromes at pH 10.0 under air-limited condition than at pH 7.0 under high aeration. Cytochrome c-552 was confirmed as the major c-type cytochrome among three soluble c-type cytochromes in the strain. To understand the physiological function of cytochrome c-552, a P. alcaliphila AL15-21(T) cytochrome c-552 gene deletion mutant without a marker gene was constructed by electrotransformation adjusted in this study for the strain. The maximum specific growth rate and maximum cell turbidity of cells grown at pHs 7.0 and 10.0 under the high-aeration condition did not differ significantly between the wild-type and cytochrome c-552 deletion mutant strains. In the mutant grown at pH 10.0 under low-aeration condition, marked decreases in the maximum specific growth rate (40%) and maximum cell turbidity (25%) compared with the wild type were observed. On the other hand, the oxygen consumption rates of cell suspensions of the mutant obtained by the growth at pH 10 under low-aeration condition were slightly higher than that of the wild type. Considering the high electron-retaining ability of cytochrome c-552, the above observations could be accounted for by cytochrome c-552 acting as an electron sink in the periplasmic space. This may facilitate terminal oxidation in the respiratory system at high pH under air-limited conditions.

Six isolates of Trichoderma polysporum-like species isolated from a moss (Sanionia uncinata) in high arctic wetlands at Spitsbergen Island, Svalbard, Norway, were examined for species identification, effects of temperature on growth, pathogenicity to the moss, and polygalacturonase (PGase) activity. All isolates from Spitsbergen were identified as T. polysporum based on morphology, sequences of the rDNA-ITS regions, and growth response to temperature. In addition, two isolates of T. polysporum from Germany and Japan were used. All isolates from Spitsbergen, Germany and Japan infected epidermal tissues of the moss, but did not cause any symptoms in in vitro inoculation tests at 0 °C. One of the isolates from Spitsbergen and the German isolate showed high activity of PGase, an enzyme produced by many saprotrophic and pathogenic fungi, in a pectin liquid medium. The enzyme activity was evident for both isolates even at 0 °C. The present results document the presence of T. polysporum in the high arctic wetlands. The epidermal infection to the moss and PGase production at 0 °C of the T. polysporum isolates suggest that the species likely inhabits a niche as a saprotroph or an endophyte in the arctic environments.

微生物処理により水産系廃棄物 (ホタテ残渣およびイカゴロ) を分解して減容化した。微生物担体にはカラマツ間伐材チップにソバ殻を加えたものを使用した。タンパク質分解能と脂質分解能を有するBacillus属細菌およびYarrowia属酵母を微生物担体に添加して実験を行った。有用菌を微生物担体に添加することにより，ホタテ残渣およびイカゴロの分解率が高くなった。これら水産系廃棄物の分解率を求めるとともに連続操作に必要な温度，pHおよび水分含量等の最適操作条件を検討した。

Indigo fermentation has been traditionally performed for dyeing textiles in Japan. Limited information is available on the microbiota involved and the succession of the bacterial community structure during indigo reduction. We investigated the bacterial community structure associated with indigo fermentation using denaturing gradient gel electrophoresis and clone library analyses of a PCR-amplified 16S rRNA gene in the early phase of fermentation carried out in our laboratory. A marked substitution of Halomonas spp. by Amphibacillus spp. was observed corresponding to the marked change in the state of indigo reduction. Although the reported indigo-reducing bacteria, Alkalibacterium spp., were not predominant in the early phase of fermentation, they were predominant in fermentation liquor aged for 10 months obtained from Date City, Japan, as determined by culture-dependent and culture-independent analyses. Novel indigo-reducing strains, Amphibacillus spp. strain C40 and Oceanobacillus spp. strain A21, were isolated from fermentation liquor aged for 10 months and from liquor aged for 4 days, respectively. It is considered that, in addition to the strains belonging to the genus Alkalibacterium, strains belonging to genera Amphibacillus and Oceanobacillus play important roles in sustaining the reduced state of indigo during fermentation.

Alkaliphiles grow under alkaline conditions that might be disadvantageous for the transmembrane pH gradient (Delta pH, outside acidic). In this study, the behaviors of extruded protons by the respiration of obligate alkaliphilic Bacillus clarkii K24-1U were investigated by comparison with those of neutralophilic Bacillus subtilis IAM 1026. Although whole-cell suspensions of both Bacillus species consumed oxygen immediately after the addition of air, there were lag times before the suspensions were acidified. Under alkaline conditions, the lag time for B. clarkii significantly increased, whereas that for B. subtilis decreased. In the presence of valinomycin or ETH-157, which disrupts the membrane electrical potential (Delta psi), the cell suspensions of both Bacillus species acidified immediately after the addition of air. Artificial electroneutral antiporters (nigericin and monensin) that eliminate the Delta pH exhibited no significant effect on the lag times of the two Bacillus species except that monensin increased the lag times of B. clarkii. The inhibition of ATPase and the Na(+) channel also exhibited little effects on the lag times. The increased lag time for B. clarkii may represent the Delta psi-dependent proton retention on the outer surface of the cytoplasmic membrane to generate a sufficient Delta pH under alkaline conditions.

Glutaminase from Micrococcus luteus K-3 [Micrococcus glutaminase (Mglu); 456 amino acid residues (aa); 48 kDa] is a salt-tolerant enzyme. Our previous study determined the structure of its major 42-kDa fragment. Here, using new crystallization conditions, we determined the structures of the intact enzyme in the presence and absence of its product L-glutamate and its activator Tris, which activates the enzyme by sixfold. With the exception of a 'lid' part (26-29 aa) and a few other short stretches, the structures were all very similar over the entire polypeptide chain. However, the presence of the ligands significantly reduced the length of the disordered regions: 41 aa in the unliganded structure (N), 21 aa for L-glutamate (G), 8 aa for Tris (T) and 6 aa for both L-glutamate and Tris (TG). L-glutamate was identified in both the G and TG structures, whereas Tris was only identified in the TG structure. Comparison of the glutamate-binding site between Mglu and salt-labile glutaminase (YbgJ) from Bacillus subtilis showed significantly smaller structural changes of the protein part in Mglu. A comparison of the substrate-binding pocket of Mglu, which is highly specific for L-glutamine, with that of Erwinia carotovora asparaginase, which has substrates other than L-glutamine, shows that Mglu has a larger substrate-binding pocket that prevents the binding of L-asparagine with proper interactions.

A very long chain polyunsaturated hydrocarbon, hentriacontanonaene (C31:9), was detected in an eicosapentaenoic acid (EPA)-producing marine bacterium, which was isolated from the mid-latitude seashore of Hokkaido, Japan, and was tentatively identified as mesophilic Shewanella sp. strain osh08 from 16S rRNA gene sequencing. The geometry and position of the double bonds in this compound were determined physicochemically to be all cis at positions 3, 6, 9, 12, 15, 19, 22, 25, and 28. Although C31:9 was detected in all of the seven EPA- or/and docosahexaenoic acid-producing bacteria tested, an EPA-deficient mutant (strain IK-1Δ8) of one of these bacteria had no C31:9. Strain IK-1Δ8 had defects in the pfaD gene, one of the five pfa genes responsible for the biosynthesis of EPA. Although Escherichia coli DH5α does not produce EPA or DHA inherently, cells transformed with the pfa genes responsible for the biosynthesis of EPA and DHA produced EPA and DHA, respectively, but not C31:9. These results suggest that the Pfa protein complex is involved in the biosynthesis of C31:9 and that pfa genes must not be the only genes responsible for the formation of C31:9. In this report, we determined for the first time the molecular structure of the C31:9 and discuss the possible biosynthetic pathways of this compound.

Two Gram-variable, aerobic, motile, rod-shaped, endospore-forming bacterial strains, M4-2T and M4-1, were isolated from soil samples collected from Oblast Magadan, Russian Far East, as micro-organisms antagonistic to the psychrophilic phytopathogenic fungus Typhula ishikariensis. Strains M4-2T and M4-1 were identified as members of the genus Paenibacillus by phenotypic and phylogenetic analyses based on 16S rRNA gene sequences. The strains contained anteiso-C15:0 as the major fatty acid (63.0-64.7%) and MK-7 as the major isoprenoid quinone. The DNA G+C contents were 42.8 and 41.7 mol%, respectively. 16S rRNA gene sequence analysis showed that strains M4-2T and M4-1 exhibited high similarities with Paenibacillus macquariensis DSM 2T (99.5 and 99.7%, respectively) and Paenibacillus antarcticus LMG 22078T (99.4 and 99.5%, respectively). There were no clear differences in the phenotypic characteristics and chemotaxonomic and phylogenetic data between the novel isolates and P. macquariensis DSM 2T. DNA-DNA hybridization experiments between strain M4-2T and P. macquariensis DSM 2T and P. antarcticus LMG 22078T revealed reassociation values of 56 and 49%, respectively. Multilocus sequence analysis confirmed the differences between the new isolates and reference strains that were observed with the DNA-DNA hybridization studies. On the basis of the results described above, it is proposed that the isolates represent a novel subspecies of P. macquariensis, Paenibacillus macquariensis subsp. defensor subsp. nov. The type strain is M4-2T (=JCM 14954T=NCIMB 14397T).

A membrane-anchored cytochrome c-550, which is highly expressed in obligately alkaliphilic Bacillus clarkii K24-1U, was purified and characterized. The protein contained a conspicuous sequence of Gly(22)-Asn(34), in comparison with the other Bacillus small cytochromes c. Analytical data indicated that the original and lipase-treated intermediate forms of cytochrome c-550 bind to fatty acids of C(15), C(16) and C(17) chain lengths and C(15) chain length, respectively, and it was considered that these fatty acids are bound to glycerol-Cys(18). Since there was a possibility that the presence of a diacylglycerol anchor contributed to the formation of dimeric states of this protein (20 and 17 kDa in SDS-PAGE), a C18M (Cys(18) --Met)-cytochrome c-550 was constructed. The molecular mass of the C18M-cytochrome c-550 was determined as 15 and 10 kDa in SDS-PAGE and 23 kDa in blue native PAGE. The C18M-cytochrome c-550 bound with or without Triton X-100 formed a tetramer as the original cytochrome c-550 bound with Triton X-100, as determined by gel filtration. The midpoint redox potential of cytochrome c-550 as determined by redox titration was +83 mV, while that determined by cyclic voltammetric measurement was +7 mV. The above results indicate that cytochrome c-550 is a novel cytochrome c.

A novel psychrotolerant, alkalitolerant bacterium, strain Ths, was isolated from a soil sample immersed in hot spring water containing hydrocarbons and grown on a chemically defined medium containing n-tetradecane as the sole carbon source. The isolate grew at 0 degrees C but not at temperatures higher than 45 degrees C; its optimum growth temperature was 27 degrees C. It grew in the pH range of 7-9. The strain utilized C(13)-C(30) n-alkane and fluorene at pH 9 and 4 degrees C. To our knowledge, this is the first report on the bacterium that utilizes a wide range of hydrocarbons at a high pH and a low temperature. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Ths is closely related to genomic species 6 ATCC 17979 (99.1% similarity), genomic species BJ13/TU14 ATCC 17905 (97.8% similarity), genomic species 9 ATCC 9957 (97.6% similarity) belonging to the genus Acinetobacter and to Acinetobacter calcoaceticus JCM 6842(T) (97.5% similarity). DNA-DNA hybridization revealed that the isolate has 62, 25, 18 and 19% relatedness, respectively, to genomic species 6 ATCC 17979, genomic species BJ13/TU14 ATCC 17905, genomic species 9 ATCC 9957 and A. calcoaceticus, respectively.

A novel exopolysaccharide-degrading bacterium, designated strain 10C(T), was isolated from soil from Kitahiroshima city, Hokkaido, Japan. The novel isolate was Gram-negative, strictly aerobic and chemoheterotrophic. The DNA G+C content was 36.9 mol%. Major fatty acids were C(16 : 1)omega7c, iso-C(15 : 0) 2-OH, iso-C(15 : 0) and iso-C(17 : 0 )3-OH. 16S rRNA gene sequence analysis and chemotaxonomic and morphological data indicated that the novel strain clearly belonged to the genus Sphingobacterium. Based on phenotypic properties and DNA-DNA hybridization data, the new isolate was assigned to the genus Sphingobacterium as Sphingobacterium kitahiroshimense sp. nov. The type strain is 10C(T) (=JCM 14970(T)=NCIMB 14398(T)).

Indigo-reducing, obligately alkaliphilic strains A11T, F11 and F12 were isolated from indigo fermentation liquor obtained from Tokushima Prefecture, Shikoku, Japan. The isolates grew at pH 9.0-12.3, but not at pH 7.0-8.0. The optimum pH range for growth was 9.5-11.5. They were Gram-negative, facultatively anaerobic, rod-shaped strains with peritrichous flagella. The isolates grew in 0-14 % (w/v) NaCl, with optimum growth at 1-11 %. They grew at temperatures of 15-35 degrees C with optimum growth at around 20-30 degrees C. dl-Lactate was the major end product from d-glucose. No quinones were detected. The peptidoglycan type was A4 alpha, l-Lys (l-Orn)-d-Asp. The major cellular fatty acids were C16 : 0, C16 : 17c and C18 : 19c. The DNA G+C contents were 47.0-47.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that the isolates belong to the genus Alkalibacterium. DNA-DNA hybridization revealed low relatedness values between the isolates and the three phylogenetically most closely related species, Alkalibacterium olivapovliticus, Alkalibacterium psychrotolerans and Alkalibacterium iburiense (

The effects of H2O2 under low- and high-aeration-level conditions on growth and catalase activity in Exiguobacterium oxidotolerans T-2-2T were investigated. Continuous addition of 5-200 mM H2O2 to the culture medium from the mid-exponential growth phase enhanced the growth of the strain under the low-aeration-level condition, whereas the addition of 5-50 mM H2O2 decreased intracellular specific catalase activity and extracellular total catalases activity. The detection of extracellular catalase by the cells and the decrease in intracellular specific catalase activity and extracellular total catalase activity under the high-aeration-level condition account for the stimulation of growth by the introduced H2O2 and the decrease in catalase activities induced by O(2) from H2O2 in the medium. On the other hand, the addition of H2O2 to the medium prior to the initiation of growth inhibited the growth but increased the specific activity of intracellular catalase in the stationary growth phase. Strain T-2-2T grew when 10 mM H2O2 was added to the medium prior to growth. However, the growth was completely inhibited by the catalase inhibitor 3-amino-1,2,4-triazole (3-AT). The continuous addition of H2O2 at an appropriate concentration from prior to the initiation of growth to the stationary growth phase under the low-aeration-level condition resulted in higher intracellular specific catalase activity and cell growth rate than single H2O2 addition prior to growth.

A soluble class I cytochrome c of an alkaliphile was purified and characterized, and its primary structure was determined. This is the first example of a soluble class I cytochrome c in alkaliphiles. Cells the alkaliphilic gram-negative bacterium Pseudomonas alcaliphila AL15-21(T) grown at pH 10 had a soluble cytochrome c content that was more than twofold that of strain AL15-21(T) cells grown at pH 7 under air-limited conditions. Cytochrome c-552, a soluble cytochrome c with a low molecular weight, was purified from strain AL15-21(T) cells grown at pH 10 under air-limited conditions. Cytochrome c-552 had a molecular mass of 7.5 kDa and exhibited an almost fully reduced state in the resting form, which exhibited absorption maxima at wavelengths of 552, 523 and 417 nm. In the oxidized state, it exhibited an absorption maximum at 412 nm when it was oxidized by ferricyanide, its isoelectric point (pI) was 4.3 and it contained one heme c as a prosthetic group. Cytochrome c-552 was autoreduced at pH 10, and the autoreduction was reproducible. On the other hand, the autoreduction of cytochrome c-552 was not observed at pH 7.0. When pH was increased from 7.0 to 8.3, its midpoint redox potentials (E(m) values) increased from +228 mV to +276 mV as determined by redox titrations, and from +217 mV to +275 mV as determined by cyclic voltammetric measurements. The amino acid sequence deduced by cytochrome c-552 gene analysis revealed that the sequence consists of 96 residues, including 19 residues as an amino-terminal signal peptide. A phylogenetic tree based on amino acid sequence indicated that the protein belongs to group 4, cytochrome c(5) in class I cytochrome c.

Cellular eicosapentaenoic acid (EPA) makes up approximately 3% of total fatty acids in Escherichia coli DH5alpha, a strain that carries EPA biosynthesis genes (pEPADelta1). EPA was increased to 12% of total fatty acids when the host cell co-expressed the vector pGBM3::sa1(vktA), which carried the high-performance catalase gene, vktA. Where this vector was co-expressed, the transformant accumulated a large amount of VktA protein. However, the EPA production of cells carrying the vector, that included the insert lacking almost the entire vktA gene, was approximately 6%. This suggests that the retention of a large DNA insert in the vector and the accumulation of the resulting protein, but not the catalytic activity of VktA catalase, would potentially be able to increase the content of EPA.

Anaerobic degradation of lignin-derived aromatics is an important metabolism for carbon and nutrient cycles in soil environments. Although there are some studies on degradation of lignin-derived aromatics by nitrate- and sulfate-reducing bacteria, knowledge on their degradation under methanogenic conditions are quite limited. In this study, methanogenic microbial communities were enriched from rice paddy field soil with lignin-derived methoxylated monoaromatics (vanillate and syringate) and their degradation intermediates (protocatechuate, catechol and gallate) as the sole carbon and energy sources. Archaeal community analysis disclosed that both aceticlastic (Methanosarcina sp.) and hydrogenotrophic (Methanoculleus sp. and Methanocella sp.) methanogens dominated in all of the enrichments. Bacterial community analysis revealed the dominance of acetogenic bacteria (Sporomusa spp.) only in the enrichments on the methoxylated aromatics, suggesting that Sporomusa spp. initially convert vanillate and syringate into protocatechuate and gallate, respectively, with acetogenesis via O-demethylation. As the putative ring-cleavage microbes, bacteria within the phylum Firmicutes were dominantly detected from all of the enrichments, while the dominant phylotypes were not identical between enrichments on vanillate/protocatechuate/catechol (family Peptococcaceae bacteria) and on syringate/gallate (family Ruminococcaceae bacteria). This study demonstrates the importance of cooperation among acetogens, ring-cleaving fermenters/syntrophs and aceticlastic/hydrogenotrophic methanogens for degradation of lignin-derived aromatics under methanogenic conditions.

A novel exopolysaccharide-producing bacterium, designated strain 9a2T, was isolated from Pacific Ocean sediment. The strain was Gram-stain-negative, motile, strictly aerobic, oxidase- and catalase-positive, and required NaCl for growth. Its major isoprenoid quinone was ubiquinone-8 (Q-8), and its cellular fatty acid profile consisted mainly of C16 : 1ω7c, C18 : 1ω9cand C16 : 0. The DNA G+C content was 46.6 mol%. 16S rRNA gene sequence analysis suggested that strain 9a2Tis a member of the genusAlteromonas. Strain 9a2Texhibited closest phylogenetic affinity toAlteromonas macleodiiNBRC 102226T(99.3 % 16S rRNA gene sequence similarity),A. marinaSW-47T(99.3 %),A. litoreaTF-22T(99.0 %),A. australicaH17T(98.7 %),A. simiduiiBCRC 17572T(98.5 %),A. stellipolarisLMG 21861T(98.3 %) andA. hispanicaF-32T(98.2 %). The DNA–DNA reassociation values between strain 9a2TandA. macleodiiJCM 20772T,A. marinaJCM 11804T,A. litoreaJCM 12188T,A. australicaCIP 109921T,A. simiduiiJCM 13896T,A. stellipolarisLMG 21861TandA. hispanicaLMG 22958Twere below 70 %. Strain 9a2Tcontained phosphatidylethanolamine, phosphatidylglycerol and an unidentified polar lipid. Owing to differences in phenotypic and chemotaxonomic characteristics, phylogenetic analysis based on 16S rRNA gene sequences and DNA–DNA relatedness data, the isolate merits classification as representing a novel species, for which the nameAlteromonasgracilissp. nov. is proposed. The type strain of this species is 9a2T( = JCM 30236T = NCIMB 14947T).

A novel marine bacterium, designated strain 4k5T, was isolated from a sediment sample of the Pacific Ocean. The strain was Gram-stain-negative, strictly aerobic, non-motile, oxidase-positive and catalase-positive and required Na+for growth. Its major isoprenoid quinone was ubiquinone 8 (Q-8), and its cellular fatty acid profile consisted mainly of C18 : 1v9c (71.4 %), C16 : 1v7c (9.1 %) and C18 : 0. The DNA G+C content was 45.3 mol%. 16S rRNA gene sequence analysis suggested that strain 4k5Tis a member of the genusPsychrobacter. Strain 4k5Texhibited the closely phylogenetic affinity toPsychrobacter pacificensisIFO 16270T(99.4 % 16S rRNA gene sequence similarity),P. piscatoriiT-3-2T(97.7 %),P. nivimaris88/2-7T(97.7 %),P. celerSW-238T(97.7 %),P. aestuariiSC35T(97.6 %) andP. vallisCMS39T(97.6 %). DNA–DNA hybridization between strain 4k5TandP. pacificensisNBRC 103191T,P. piscatoriiJCM 15603T.P. nivimarisDSM 16093T,P. celerJCM 12601T,P. aestuariiJCM 16343TandP. vallisDSM 15337Twas 42.5, 47.0, 38.1, 23.7, 9.0 and 27.4 %, respectively. Owing to the significant differences in phenotypic and chemotaxonomic characteristics, phylogenetic analysis based on the 16S rRNA gene sequence and DNA–DNA relatedness data, the isolate merits classification within a novel species, for which the namePsychrobacter oceanisp. nov. is proposed. The type strain is 4k5T( = JCM 30235T = NCIMB 14948T).

A catalase that exhibits a high level of activity and a rapid reaction with organic peroxides has been purified from Exiguobacterium oxidotolerans T-2-2T (EKTA catalase). The amino acid sequence of EKTA catalase revealed that it is a novel clade 1 catalase. Amino acid residues in the active site around the protoheme are conserved in the primary structure of EKTA catalase. Although the general interactions of molecules larger than hydrogen peroxide with catalases are strongly inhibited because of the selection role of long and narrow channels in the substrate reaching the active site, the formation rate of reactive intermediates (compound I) in the reaction of EKTA catalase with peracetic acid is 77 times higher than that of bovine liver catalase (BLC) and 1200 times higher than that of Micrococcus luteus catalase (MLC). The crystal structure of EKTA catalase has been determined and refined to 2.4 A resolution. The main channel structure of EKTA catalase is different from those of BLC and MLC. The rate constant of compound I formation in catalases decreased with an increase in the molecular size of the substrate. For EKTA catalase with a larger bottleneck 15 A from the iron (entrance of narrow channel) in the main channel, a lower rate of reduction in compound I formation rate with an increase in the molecular size of substrates was found. The increase in the rate constant of compound I formation in these catalases was directly proportional to the increase in the size of the bottleneck in the main channel when molecules of substrates larger than H2O2, such as organic peroxides, are used in the reaction. The results indicate that the size of the bottleneck in the main channel in catalase is an important factor in defining the rate of compound I formation corresponding to the molecular size of the substrates, and this was demonstrated. The Leu149-Ile180 and Asp109-Met167 combinations at the entrance of the narrow channel in EKTA catalase determine the size of the bottleneck, and each atom-to-atom distance for the combination of residues was larger than those of corresponding combinations of amino acid residues in BLC and MLC. The combination of these four amino acids is quite specific in EKTA catalase as compared with the combinations in other catalases in the gene database (compared with more than 432 catalase genes in the database).

Two novel exopolysaccharide-producing bacteria, strains S18K6T and S18K5, were isolated from Pacific Ocean sediment. The isolates were Gram-negative, motile, strictly aerobic chemoheterotrophic bacteria. The DNA G+C contents of strains S18K6T and S18K5 were 44.8 and 46.3 mol%, respectively. DNA–DNA relatedness between the two strains was 70 %. Major fatty acids were hexadecanoic acid (C16 : 0), hexadecenoic acid (C16 : 1 ω7c) and octadecenoic acid (C18 : 1 ω7c). 16S rRNA gene sequence, chemotaxonomic and morphological data indicated that these strains clearly belonged to the genus Glaciecola. Based on phenotypic properties and DNA–DNA hybridization data, strains S18K6T and S18K5 are considered to represent a novel species of the genus Glaciecola, for which the name Glaciecola chathamensis sp. nov. is proposed. The type strain is S18K6T (=JCM 13645T=NCIMB 14146T).

Degradation of n-alkanes in diesel oil by Pseudomonas aeruginosa strain WatG (WatG) was verified in soil microcosms. The total petroleum hydrocarbon (TPH) degradation level in two bioaugmentation samples was 51% and 46% for 1 week in unsterilized and sterilized soil microcosms, respectively. The TPH degradation in the biostimulation was of control level (15%). The TPH degradation in aeration-limited samples was clearly reduced when compared with that in aeration-unlimited ones under both sterilized and unsterilized conditions. Addition of WatG into soil microcosms was accompanied by dirhamnolipid production only in the presence of diesel oil. These findings suggest that degradation of n-alkanes in diesel oil in soil microcosms would be facilitated by bioaugmentation of WatG, with production of dirhamnolipid, and also by participation of biostimulated indigenous soil bacteria.

An eicosapentaenoic acid-producing bacterium, previously described asShewanellasp. strain SCRC-2738, was classified by phenotypic characterization, chemotaxonomic analysis, 16S rRNA gene sequence analysis and DNA–DNA hybridization. The isolate was Gram-negative, rod-shaped and motile by using polar flagella. The strain grew at 4–32 °C; the optimum growth temperature was 27 °C. NaCl was required for growth. The major isoprenoid quinones were ubiquinone-7 and ubiquinone-8 and its DNA G+C content was 42·8 mol%. The whole-cell fatty acids mainly (above 5 %) consisted of iso-C13 : 0, iso-C15 : 0, C16 : 0, C16 : 1ω7c, C18 : 1ω7cand C20 : 5ω3 (eicosapentaenoic acid). Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain SCRC-2738Twas related closely (sequence similarities above 99 %) toShewanella marinintestina(99·3 %),Shewanella sairae(99·3 %) andShewanella schlegeliana(99·2 %). DNA–DNA hybridization and phenotypic characteristics confirmed that strain SCRC-2738Tmerited classification as a novel species of the genusShewanella, for which the nameShewanella pneumatophorisp. nov. is proposed. The type strain is SCRC-2738T(=JCM 13187T=NCIMB 14060T).

Although a bioenergetic parameter is unfavorable for production of ATP (DeltapH0), the growth rate and yield of alkaliphilic Bacillus strains are higher than those of neutralophilic Bacillus subtilis. This finding suggests that alkaliphiles possess a unique energy-producing machinery taking advantage of the alkaline environment. Expected bioenergetic parameters for the production of ATP (DeltapH and DeltaPsi) do not reflect the actual parameters for energy production. Certain strains of alkaliphilic Bacillus spp. possess large amounts of cytochrome c when grown at a high pH. The growth rate and yield are higher at pH 10 than at pH 7 in facultative alkaliphiles. These findings suggest that a large amount of cytochrome c at high pHs (e.g., pH 10) may be advantageous for sustaining growth. To date, isolated cytochromes c of alkaliphiles have a very low midpoint redox potential (less than +100 mV) compared with those of neutralophiles (approximately +220 mV). On the other hand, the redox potential of the electron acceptor from cytochrome c, that is, cytochrome c oxidase, seems to be normal (redox potential of cytochrome a=+250 mV). This large difference in midpoint redox potential between cytochrome c and cytochrome a concomitant with the configuration (e.g., a larger negative ion capacity at the inner surface membrane than at the outer surface for the attraction of H+ to the intracellular membrane and a large amount of cyrochrome c) supporting H+-coupled electron transfer of cytochrome c may have an important meaning in the adaptation of alkaliphiles at high pHs. This respiratory system includes a more rapid and efficient H+ and e- flow across the membrane in alkaliphiles than in neutralophiles.

A halotolerant, obligately alkaliphilic bacterium, R-2T, was isolated from the skin of a rainbow trout (Oncorhynchus mykiss), a freshwater fish. The strain is Gram-positive, ferments several carbohydrates, is rod-shaped and motile by peritrichous flagella and produces ellipsoidal spores. The isolate grows at pH 9–10 but not at pH 7–8. This micro-organism grows in 0–22 % (w/v) NaCl at pH 10. Its major cellular fatty acids are iso-C15 : 0, anteiso-C15 : 0 and anteiso-C17 : 0, the major isoprenoid quinone is MK-7 and the DNA G+C content is 38·5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicates that strain R-2T is a member of the genus Oceanobacillus. DNA–DNA hybridization reveals low relatedness between the isolate and Oceanobacillus iheyensis (21·0 %). On the basis of phenotypic characteristics, phylogenetic data and DNA–DNA relatedness data, the isolate should be designated as a novel species, for which the name Oceanobacillus oncorhynchi sp. nov. is proposed. The type strain is R-2T (=JCM 12661T=NCIMB 14022T).

A novel mesophilic, chemolithoautotrophic, sulfur-oxidizing bacterium, designated strain SO07T, was isolated from a microaerobic waste-water biofilm. Chemolithoautotrophic growth was observed with elemental sulfur, sulfide and thiosulfate as sole electron donors and oxygen as electron acceptor. Anaerobic and heterotrophic growth were not observed. Nitrate was not used as a terminal electron acceptor. The optimum pH and temperature for growth were pH 7·5 and 30 °C, respectively. The major isoprenoid quinone was Q-8. The DNA G+C content of strain SO07T was 47·1 mol%. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that strain SO07T formed a monophyletic group in the γ-Proteobacteria with only 89 % similarity to members of the genus Halothiobacillus, its nearest phylogenetic neighbours. In addition, the isolate differed from members of the genus Halothiobacillus in its requirement for and tolerance of NaCl; strain SO07T was unable to grow in NaCl concentrations of more than 180 mM. On the basis of phylogenetic, chemotaxonomic and physiological data, it is proposed that isolate SO07T (=JCM 12417T=ATCC BAA-1033T) represents the type strain of a novel species in a new genus, Thiovirga sulfuroxydans gen. nov., sp. nov.

A halophilic and halotolerant, facultatively alkaliphilic strain, K11(T), was isolated from soil obtained from Oshyamanbe, Oshima, Hokkaido, Japan. The isolate grew at pH 7-10. It was non-motile, Gram-positive and aerobic. Cells comprised straight rods and produced ellipsoidal spores. The isolate grew in 0-20 % NaCl, with optimum growth at 7 % NaCl, and hydrolysed casein, gelatin, starch, DNA and Tweens 20, 40, 60 and 80. The major isoprenoid quinone was menaquinone-7, and the cellular fatty acid profile consisted of significant amounts of C(15) branched-chain acids, iso C(15 : 0) and anteiso C(15 : 0). Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain K11(T) was a member of group 6 [Nielsen et al., FEMS Microbiol Lett 117 (1994), 61-66] (alkaliphiles) of the genus Bacillus. DNA-DNA hybridization revealed a low relatedness (14 %) of the isolate to its closest phylogenetic neighbour, Bacillus clausii. On the basis of phenotypic and chemotaxonomic characteristics, phylogenetic data and DNA-DNA relatedness data, it was concluded that K11(T) (=JCM 12663(T)=NCIMB 14023(T)) merits classification as the type strain of a novel species, for which the name Bacillus oshimensis sp. nov. is proposed.

Bacteria possessing high capacity to degrade gasoline, kerosene, diesel oil, and lubricating oil were screened from several areas of Hokkaido, Japan. Among isolates, two strains, WatG and HokM, which were identified as new strains of Pseudomonas aeruginosa and Serratia marcescens species, respectively, showed relatively high capacity and wide spectrum to degrade the hydrocarbons in gasoline, kerosene, diesel, and lubricating oil. About 90-95% of excess amount of total diesel oil and kerosene added to mineral salts media as a sole carbon source could be degraded by WatG within 2 and 3 weeks, respectively. The same amount of lubricating oil was 60% degraded within 2 weeks. Strain HokM was more capable than WatG in degrading aromatic compounds in gasoline. This strain could also degrade kerosene, diesel, and lubricating oil with a capacity of 50-60%. Thus, these two isolates have potential to be useful for bioremediation of sites highly contaminated with petroleum hydrocarbons.

A novel alkaliphile was isolated from a drain of a fish processing plant. The isolate grew at a pH range of 7–10. Cells were Gram-positive, facultatively aerobic, motile rods with peritrichous flagella. Colonies were orange or yellow in colour. Catalase and oxidase reactions were positive. The isolate grew in 0–12 % NaCl but not above 15 % NaCl. Its cell extract exhibited 567 times higher catalase activity than an Escherichia coli cell extract. The major cellular fatty acids were iso-C13 : 0, anteiso-C13 : 0, iso-C15 : 0, iso-C16 : 0, iso-C17 : 0, anteiso-C17 : 0 and iso-C17 : 1. Its DNA G+C content was 46·7 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing and chemotaxonomic data indicated that strain T-2-2T is a member of the genus Exiguobacterium. DNA–DNA hybridization revealed a low relatedness of the isolate to several phylogenetic neighbours (less than 25 %). On the basis of phenotypic characteristics, phylogenetic data and DNA–DNA relatedness data, the isolate merits classification as a novel species, for which the name Exiguobacterium oxidotolerans sp. nov. is proposed. The type strain is T-2-2T (=JCM 12280T=NCIMB 13980T).

A psychrotolerant, obligately alkaliphilic bacterium, IDR2-2T, which is able to reduce indigo dye was isolated from a fermented polygonum indigo (Polygonum tinctorium Lour.) produced in Date, Hokkaido, using a traditional Japanese method. The isolate grew at pH 9–12 but not at pH 7–8. It was a Gram-positive, facultatively anaerobic, straight rod-shaped bacterium with peritrichous flagella. The isolate grew in 0–17 % (w/v) NaCl but not at NaCl concentrations higher than 18 % (w/v). Its major cellular fatty acids were C14 : 0, C16 : 0, C16 : 19c and C18 : 19c, and its DNA G+C content was 40·6 mol%. dl-lactic acid was the major end-product from d-glucose. No quinones could be detected. The peptidoglycan type was A4β, Orn–d-Glu. A phylogenetic analysis based on 16S rRNA gene sequence data indicated that strain IDR2-2T is a member of the genus Alkalibacterium. DNA–DNA hybridization revealed low relatedness (less than 25 %) between the isolate and two phylogenetically related strains, Alkalibacterium olivapovliticus and Marinilactibacillus psychrotolerans. On the basis of phenotypic characteristics, phylogenetic data and DNA–DNA relatedness data, the isolate merits classification as a novel species, for which the name Alkalibacterium psychrotolerans sp. nov. is proposed. The type strain is IDR2-2T (=JCM 12281T=NCIMB 13981T).

It has been reported that Typhula graminearum and Typhula humulina cause winter damage to cultivated conifer seedlings and hop (Humulus lupulus) roots, respectively, in the European part of Russia. However, the Russian phytopathogist V.G. Potatosova reported that those fungi had the same morphological characteristics as those of Typhula ishikariensis causing snow mold. Since all reports on those fungi are written in Russian, few phytopathologists outside the former Union of Soviet Socialist Republics know about these fungi. We collected sclerotia from winterdamaged pine (Pinus sylvestris) seedlings from the European part of Russia and hop roots from West Siberia. Localities where T. graminearum and T. humulina were reported in the literature overlapped those where sclerotiaforming fungi causing snow mold were recorded in the literature and in the present survey. Our fungal isolates from symptomatic pines and hop roots from both regions showed the same morphological and genetic characteristics as those of ...

A novel moderately thermophilic bacterium, strain TH13T, was isolated from a hot spring in Kamchatka. It was found to be a Gram-positive, facultative aerobe; the straight, non-motile rods grew at 30–64 °C (optimum 54 °C). The isolate was positive for catalase and oxidase tests and reduced nitrate to nitrite, but was negative for H2S production and growth in more than 3 % NaCl (w/v). The isolate grew at pH 7–8, but not at pH values higher than 9. The DNA G+C content was 43·9 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain TH13T was a member of the genus Anoxybacillus. DNA–DNA hybridization revealed a low relatedness (less than 30·2 %) between the isolate and its close phylogenetic neighbours Anoxybacillus pushchinoensis and Anoxybacillus flavithermus. On the basis of phenotypic characteristics, phylogenetic data and DNA–DNA hybridization data, it was concluded that the isolate merited classification as a novel species, for which the name Anoxybacillus voinovskiensis sp. nov. is proposed. The type strain of this species is TH13T (=NCIMB 13956T=JCM 12111T).

A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5 degrees C as minimum growth temperatures in complex and defined media, respectively, was isolated. On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina. The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species. When the bacterium was grown at 5-15 degrees C in a defined medium, it produced a high amount of trans-unsaturated fatty acids. By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids. In the complex medium at 5 degrees C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium. Following a temperature shift from 11 to 5 degrees C, strain L-2 grew better in complex than in defined medium. Furthermore, when the growth temperature was shifted from 0 to 5 degrees C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced. These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism. The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5 degrees C were also studied. The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium. These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.

A facultatively psychrophilic bacterium, strain MD17(T), which hydrolyses lipids at 5 degrees C, was isolated from the Monbetsu coast of the Okhotsk Sea in Hokkaido, Japan, when ice carried by the cold current came to the area. The isolate is an aerobic, non-motile coccobacillus that reduces nitrate to nitrite and hydrolyses Tweens 20, 40, 60 and 80, but not gelatin, DNA or alginic acid. The isolate grows at 0 degrees C, but not at temperatures higher than 36 degrees C; its optimum growth temperature is 25 degrees C. It grows in the presence of 0-10 % NaCl. Its major isoprenoid quinone is ubiquinone-8 (Q-8) and its DNA G+C content is 46.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MD17(T) is closely related to Psychrobacter glacincola DSM 12194(T) (99.0 % similarity) and Psychrobacter immobilis DSM 7229(T) (98.7 % similarity). DNA-DNA hybridization revealed 45.9 % relatedness between strain MD17(T) and P. immobilis ATCC 43116(T) and 33.4 % between strain MD17(T) and P. glacincola ATCC 700754(T). Based on physiological and biochemical characteristics, phylogenetic position (as determined by 16S rRNA gene sequence analysis) and DNA-DNA relatedness, it is concluded that the isolate should be designated as a novel species, for which the name Psychrobacter okhotskensis sp. nov. is proposed. The type strain is MD17(T) (=NCIMB 13931(T)=JCM 11840(T)).

A novel strictly aerobic, heterotrophic, mesophilic bacterium, strain Mb8(T), was isolated from soil in Sapporo City, Hokkaido, Japan. The G+C content of strain Mb8(T) was 66.0 mol%. It had mycolic acids with 44-52 carbon atoms and C16 : 0 and C18 : 1 (9) as the major fatty acids. The major isoprenoid quinone was MK-8(H(2)). The cell wall contained meso-diaminopimelic acid, arabinose and galactose. 16S rDNA, chemotaxonomic and morphological data indicated that this strain clearly belonged to the genus RHODOCOCCUS: Based on phenotypic properties and DNA-DNA hybridization data, strain Mb8(T) (=JCM 11308(T)=NCIMB 13903(T)) has been assigned to the genus Rhodococcus as the type strain of Rhodococcus tukisamuensis sp. nov.

A bacterium, Ikeda-3, that was isolated from the wastewater of a winery was found to produce floating and precipitating exopolysaccharides (EPSs) when grown aerobically in a medium containing sucrose as the sole carbon source. The concentrations of floating and precipitating EPSs were approximately 2.7 g/l and 0.6 g/l, respectively, in a mini-jar fermentor after incubations of the bacterium at 15 degrees C for 5 d in a synthetic medium containing 1% sucrose, 0.01% CaCO3, 0.05% MgSO4, 0.05% K2HPO4, 0.0001% Na2MoO4, 0.05% peptone and 0.82% bicine. A component analysis of two types of EPS suggested that they represent a novel type of sphingan composed of glucose, rhamnose, mannose and mannuroic acid. Glucoronic acid, which is commonly found in sphingans, was absent. From physiological, biochemical and chemotaxonomic characterization, phylogenetic analysis and DNA-DNA relatedness, Ikeda-3 was identified as Novosphingobium rosa. To our knowledge the production of EPS by N. rosa is reported here for the first time.

A novel, facultatively psychrophilic alkaliphile that grows on a chemically defined medium containing n-alkanes as the sole carbon source was isolated from a drain of a fish product-processing plant. The isolate was an aerobic, non-motile, gram-positive bacterium. The bacterium was catalase-positive and oxidase-negative. The cell wall contained meso-diaminopimelic acid, arabinose and galactose; the glycan moiety of the cell wall contained acetyl residues. The G+C content of the DNA was 69.6 mol %. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate was closely related to members of the genus Dietzia (96.1-96.8% similarity). Comparisons of phenotypic and chemotaxonomic characteristics between the isolate and the two known Dietzia species showed that they were very similar. However, the isolate differed from the two known Dietzia species in growth temperature range and certain physiological characteristics. DNA-DNA hybridization revealed that the isolate had 38.4 and 49.7% relatedness, respectively, to Dietzia maris and Dietzia natronolimnaea. On the basis of the physiological and biochemical characteristics, the phylogenetic position as determined by 16S rRNA gene analysis and DNA-DNA relatedness, it is concluded that the isolate should be designated as a novel species, for which the name Dietzia psychralcaliphila sp. nov. is proposed. The type strain is ILA-1T (= JCM 10987T = IAM14896T = NCIMB 13777T).

A novel strain Vibrio aphrogenes sp. nov. strain CA-1004(T) isolated from the surface of seaweed collected on the coast of Mie Prefecture in 1994 [1] was characterized using poly phasic taxonomy including multilocus sequence analysis (MLSA) and a genome based comparison. Both phylogenetic analyses on the basis of 16S rRNA gene sequences and MLSA based on eight protein-coding genes (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA) showed the strain could be placed in the Rumoiensis Glade in the genus Vibrio. Sequence similarities of the 16S rRNA gene and the multilocus genes against the Rumoiensis Glade members, V. rumoiensis, V. algivorus, V. casei, and V. litoralis, were low enough to propose V. aphrogenes sp. nov. strain CA-1004(T) as a separate species. The experimental DNA-DNA hybridization data also revealed that the strain CA-1004(T) was separate from four known Rumoiensis Glade species. The G+C content of the V. aphrogenes strain was determined as 42.1% based on the genome sequence. Major traits of the strain were non motile, halophilic, fermentative, alginolytic, and gas production. A total of 27 traits (motility, growth temperature range, amylase, alginase and lipase productions, and assimilation of 19 carbon compounds) distinguished the strain from the other species in the Rumoiensis Glade. The name V. aphrogenes sp. nov. is proposed for this species in the Rumoiensis Glade, with CA-1004(T) as the type strain (JCM 31643(T) = DSM 103759(T)).

Investigation of microbial interspecies interactions is essential for elucidating the function and stability of microbial ecosystems. However, community-based analyses including molecular-fingerprinting methods have limitations for precise understanding of interspecies interactions. Construction of model microbial consortia consisting of defined-mixed cultures of isolated microorganisms is an excellent method for research on interspecies interactions. In this study, a model microbial consortium consisting of microorganisms that convert acetate into methane directly (Methanosaeta thermophila) and syntrophically (Thermoacetogenium phaeum and Methanothermobacter thermautotrophicus) was constructed and the effects of elevated CO2 concentrations on intermicrobial competition were investigated. Analyses on the community dynamics by quantitative RT-PCR and fluorescent in situ hybridization targeting their 16S rRNAs revealed that high concentrations of CO2 have suppressive effects on the syntrophic microorganisms, but not on the aceticlastic methanogen. The pathways were further characterized by determining the Gibbs free energy changes (ΔG) of the metabolic reactions conducted by each microorganism under different CO2 concentrations. The ΔG value of the acetate oxidation reaction (T. phaeum) under high CO2 conditions became significantly higher than -20 kJ per mol of acetate, which is the borderline level for sustaining microbial growth. These results suggest that high concentrations of CO2 undermine energy acquisition of T. phaeum, resulting in dominance of the aceticlastic methanogen. This study demonstrates that investigation on model microbial consortia is useful for untangling microbial interspecies interactions, including competition among microorganisms occupying the same trophic niche in complex microbial ecosystems.

Metagenomic screening and conventional cultivation have been used to exploit microbial lipolytic enzymes in nature. We used an indigenous forest soil (NS) and oil-fed enriched soil (OS) as microbial and genetic resources. Thirty-four strains (17 each) of lipolytic bacteria were isolated from the NS and OS microcosms. These isolates were classified into the (sub)phyla Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria, all of which are known to be the main microbial resources of commercially available lipolytic enzymes. Seven and 39 lipolytic enzymes were successfully retrieved from the metagenomic libraries of the NS and OS microcosms, respectively. The screening efficiency (a ratio of positive lipolytic clones to the total number of environmental clones) was markedly higher in the OS microcosm than in the NS microcosm. Moreover, metagenomic clones encoding the lipolytic enzymes associated with Alphaproteobacteria, Deltaproteobacteria, Acidobacteria, Armatimonadetes, and Planctomycetes and hitherto-uncultivated microbes were recovered from these libraries. The results of the present study indicate that functional metagenomics can be effectively used to capture as yet undiscovered lipolytic enzymes that have eluded the cultivation-based method, and these combined approaches may be able to provide an overview of lipolytic organisms potentially present in nature.

This chapter focuses on the environmental and taxonomic distributions of gram-positive alkaliphiles. Garbeva et al. developed a polymerase chain reaction (PCR) system for studying the diversity of the species of Bacillus and related taxa using DNA directly obtained from soil. Detection of Bacillus halodurans by this procedure indicated that although the soil samples were slightly acidic, Bacillus halodurans might be one of the major Bacillus species in the soil samples used in that study. Microbial diversities of soda lakes in Africa, Europe, and North America have been detected on the basis of the analysis of DNA clone libraries produced by amplification of obtained DNA as well as from the isolation of microorganisms from the environments. The major gram-negative isolates are members of the gamma subdivision of Proteobacteria. Indigo-reducing bacteria have been isolated by Takahara and Tanabe and identified as Bacillus sp. they have been named Bacillus alcalophilus. This is the only species that can grow at 5°C among the currently known alkaliphilic Bacillus spp. The chapter provides facts that suggest that niches of Bacillus patagoniensis are in soil and in rhizosphere of certain plants. Some of the strains in this group were formally classified as Bacillus. In the next decade, the understanding of the distribution in the environment and of the taxonomic diversities of alkaliphiles will proceed further not only by isolation of novel species of alkaliphiles but also from results of analyses of DNA directly obtained from various environments.

A facultatively psychrophilic bacterium, previously described as Pseudomonas sp. strain E-3, has been reassigned by phenotypic characterization, chemotaxonomic analysis, DNA-DNA hybridization, and 16S rRNA gene phylogenetic analysis. The organism was a gram-negative, aerobic. straight rod with polar flagella. It was catalase positive and oxidase positive, able to grow at -1 degree C but not at 40 degree C, and produced acid from D-glucose under aerobic conditions. The major isoprenoid quinone was ubiquinone-9, and the DNA G + C content was 57.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the bacterium is a member of the genus Pseudomonas and was closest to Pseudomonas fragi. Determination of the DNA-DNA relatedness between strain E-3 and P. fragi revealed too low a level of homology (47.9%-51.3%) to identify them as the same species. On the basis of phenotypic characteristics, phylogenetic analysis, and DNA-DNA relatedness data, it is concluded that strain E-3 represents an individual species. Accordingly, the name Pseudomonas psychrophila is proposed. The type strain is E-3T (= JCM 10889).

Facultatively alkaliphilic Bacillus sp. strain YN-2000 was isolated from an indigo ball. Although the strain has been extensively investigated as a representative strain of alkaliphilic bacillus, its taxonomic position is not yet known. Morphological, biochemical, and physiological characteristics and chemotaxonomic properties indicated that the strain was closely related to Bacillus cohnii; this was confirmed by the high homology of the 16S rRNA sequence and the construction of a phylogenetic tree on the basis of the 16S rRNA sequence and DNA-DNA relatedness data. Strain YN-2000 contained a larger amount of unsaturated fatty acids compared with Bacillus subtilis and the obligate alkaliphile, Bacillus alcalophilus, regardless of its culture pH. When the cells were grown at pH 10, the unsaturated fatty acid content and anteiso-/iso-branched fatty acid ratio became lower than those at pH 7. This result suggests that membrane fluidity decreases when the cells are grown at pH 10 compared to those of pH 7. In the cells of strain YN-2000 grown at pH 10, the cell-surface aspect was rougher, the cell shape was longer, and the cell-surface layer was thicker compared with those of the cells grown at pH 7. The cell-surface structural change might be related to adaptation to an alkaline environment.

Catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1 T , which was isolated from an environment exposed to H 2 O 2 and exhibited high catalase activity, was purified and characterized, and its localization in the cell was determined. Its molecular mass was 230 kDa, and the molecule consisted of four identical subunits. The enzyme, which was not apparently reduced by dithionite, showed a Soret peak at 406 nm in a resting state. The catalytic activity was 527,500 U · mg of protein −1 under standard reaction conditions at 40°C, 1.5 and 4.3 times faster, respectively, than those of the Micrococcus luteus and bovine catalases examined under the same reaction conditions, and showed a broad optimum pH range (pH 6 to 10). The catalase from strain S-1 T is located not only in the cytoplasmic space but also in the periplasmic space. There is little difference in the activation energy for the activity between strain S-1 T catalase and M. luteus and bovine liver catalases. The thermoinstability of the activity of the former catalase were significantly higher than those of the latter catalases. The thermoinstability suggests that the catalase from strain S-1 T should be categorized as a psychrophilic enzyme. Although the catalase from strain S-1 T is classified as a mammal type catalase, it exhibits the unique enzymatic properties of high intensity of enzymatic activity and thermoinstability. The results obtained suggest that these unique properties of the enzyme are in accordance with the environmental conditions under which the microorganism lives.

To examine the distribution of the Na(+)-translocating NADH-quinone reductase (Na(+)-NQR) among marine bacteria, we developed a simple screening method for the detection of this enzyme. By reference to the homologous sequences of the Na(+)-NQR operons from Vibrio alginolyticus and Haemophilus influenzae, a pair of primers was designed for amplification of a part of the sixth ORF (nqr6) of the Na(+)-NQR operon. When PCR was performed using genomic DNA from 13 marine bacteria, a 0.9-kbp fragment corresponding to nqr6 was amplified in 10 strains. Although there were three PCR-negative strains phylogenetically, based on the sequence of the 16S rRNA, these were placed far from the PCR-positive strains. No product was observed in the case of nonmarine bacteria. The nucleotide and predicted amino acid sequences of nqr6 were highly conserved among the PCR-positive marine bacteria. A phylogenetic analysis of marine bacteria, based on nqr6 sequencing, was performed.

The catalase gene, vktA, of the hydrogen peroxide-resistant bacterium Vibrio rumoiensis S-1 has been isolated and sequenced. Plasmid pBSsa1 was obtained by genome library screening and it complemented a catalase-less mutant, Escherichia coli strain UM2, indicating that pBSsa1 contained the catalase gene (vktA). The vktA gene consisted of an open reading frame of 1530 bp encoding a 508 amino-acid protein with a calculated size of 57657.79 Da. The deduced amino acid sequence showed high homology with that of bacterial group III catalases. Investigation of the subcellular catalase localization using an immuno-electron microscopical technique revealed that a larger amount of VktA catalase is present in the periplasmic space than in the cytoplasm. The periplasmic space of this bacterium can therefore be regarded as a reservoir of VktA catalase in V. rumoiensis S-1.