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Isao Yumoto - One of the best experts on this subject based on the ideXlab platform.
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fermentibacillus polygoni gen nov sp nov an Alkaliphile that reduces indigo dye
International Journal of Systematic and Evolutionary Microbiology, 2016Co-Authors: Kikue Hirota, Isao Yumoto, Kenichi AinoAbstract:Facultatively alkaliphilic strains, designated as strains IEB3T and IEB14, were isolated as indigo-reducing strains from a fermented Polygonum indigo (Polygonum tinctorium Lour) liquor sample prepared in our laboratory using a medium containing an indigo fermentation liquor as a sole substrate. The 16S rRNA gene sequence phylogeny and similarity suggested that strains IEB3T and IEB14 exhibit distinctive positions among the members of the genus Bacillus, and their closest neighbour was Bacillus nanhaiisediminis NH3T (similarity: 97.4 %) among the species with validly published names. The 16S rRNA sequence of strain IEB3Twas identical to that of strain IEB14. The cells of the isolates stained Gram-positive and were facultatively anaerobic, straight rods that were motile by a pair of subpolar flagella. Strains IEB3T and IEB14 grew at temperatures between 12 and 40 °C with optimum growth at 30‒33 °C and in the range of pH 7.5-12. Menaquinone-7 (MK-7) was detected as the major isoprenoid quinone. The DNA G+C contents of strains IEB3T and IEB14 were 39.0 and 39.1 mol%, respectively. The whole-cell fatty acid profile mainly (>10 %) consisted of iso-C14:0, iso-C15:0 and anteiso-C15:0. DNA-DNA hybridization revealed a low relatedness value between strain IEB3T and the phylogenetically most closely related species, Bacillus nanhaiisediminis JCM 16507T (<7 % ). On the basis of phenotypic and chemotaxonomic characteristics and phylogenetic data, the isolates represent a novel species within a novel genus, for which the name Fermentibacillus polygoni gen. nov., sp. nov. is proposed. The type strain is IEB3T (=JCM 30817T=NCIMB 14984T).
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amphibacillus iburiensis sp nov an Alkaliphile that reduces an indigo dye
International Journal of Systematic and Evolutionary Microbiology, 2013Co-Authors: Kikue Hirota, Isao Yumoto, Kenichi AinoAbstract:An indigo-reducing alkaliphilic strain, designated strain N314T, was isolated from a fermented polygonum indigo (Polygonum tinctorium Lour.) liquor sample, aged for 10 months, that was obtained from Date City, Iburi Branch, Hokkaido, Japan. The 16S rRNA gene sequence phylogeny suggested that strain N314T is a member of the genus Amphibacillus , with the closest relatives being Amphibacillus indicireducens (98.9 % similarity to the type strain) and Amphibacillus xylanus (98.0 % similarity to the type strain), the only species with 16S rRNA gene sequence similarities higher than 97 % to strain N314T. The cells of the isolate stained Gram-positive and were facultatively anaerobic, straight rods that were motile by means of peritrichous flagella. The strain grew at 26–39 °C with optimum growth at 36 °C. It grew at pH 8.0–9.1, with optimum growth at pH 8.9–9.1. No isoprenoid quinone was detected, and the DNA G+C content was 38.4 mol%. The whole-cell fatty acid profile consisted mainly of iso-C15 : 0 and anteiso-C15 : 0. Analysis of DNA–DNA hybridization with the type strains of A. indicireducens and A. xylanus revealed 29±2 % and 10±2 % relatedness, respectively. Owing to differences in phenotypic characteristics from reported species of the genus A. and results of phylogenetic analyses based on 16S rRNA gene sequences and DNA–DNA relatedness data, the isolate merits classification within a novel species, for which the name Amphibacillus iburiensis sp. nov. is proposed. The type strain is N314T ( = JCM 18529T = NCIMB 14823T).
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oceanobacillus polygoni sp nov a facultatively Alkaliphile isolated from indigo fermentation fluid
International Journal of Systematic and Evolutionary Microbiology, 2013Co-Authors: Kikue Hirota, Isao Yumoto, Yoshinobu Nodasaka, Yoshiko HanaokaAbstract:A facultatively alkaliphilic, lactic-acid-producing and halophilic strain, designated SA9T, was isolated from a fermented Polygonum indigo (Polygonum tinctorium Lour.) liquor sample prepared in a laboratory. The 16S rRNA gene sequence phylogeny suggested that strain SA9T was a member of the genus Oceanobacillus with the closest relative being Oceanobacillus profundus KCCM 42318T (99.3 % 16S rRNA gene sequence similarity). Cells of strain SA9T stained Gram-positive and were facultative anaerobic straight rods that were motile by peritrichous flagella. The strain grew between 5 and 48 °C (optimum, 35 °C) and at pH 7–12 (optimum, pH 9). The isoprenoid quinone detected was menaquinone-7 (MK-7) and the DNA G+C content was 40.6±0.9 mol%. The whole-cell fatty acid profile mainly consisted of iso-C15 : 0, anteiso-C15 : 0, C16 : 0 and anteiso-C17 : 0. DNA–DNA hybridization with Oceanobacillus profundus DSM 18246T revealed a DNA–DNA relatedness value of 23±2 %. On the basis of the differences in phenotypic and chemotaxonomic characteristics, and the results of phylogenetic analyses based on 16S rRNA gene sequences and DNA–DNA relatedness data from recognized species of the genus Oceanobacillus , strain SA9T merits classification as a representative of a novel species of the genus Oceanobacillus , for which the name Oceanobacillus polygoni sp. nov. is proposed. The type strain is SA9T ( = JCM 17252T = NCIMB 14684T). An emended description of the genus Oceanobacillus is also provided.
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oceanobacillus indicireducens sp nov a facultative Alkaliphile that reduces an indigo dye
International Journal of Systematic and Evolutionary Microbiology, 2013Co-Authors: Kikue Hirota, Isao Yumoto, Kenichi Aino, Yoshinobu NodasakaAbstract:An indigo-reducing facultatively alkaliphilic and halophilic strain, designated strain A21T, was isolated from a fermented Polygonum indigo (Polygonum tinctorium Lour.) liquor sample aged for 4 days prepared in a laboratory. 16S rRNA gene sequence phylogeny suggested that strain A21T was a member of the genus Oceanobacillus with the closest relative being the type strain of Oceanobacillus chironomi (similarity: 96.0 %). The cells of the isolate stained Gram-positive and were facultatively anaerobic straight rods that were motile by peritrichous flagella. The strain grew between 18 and 48 °C with optimum growth at 39 °C. It grew in the pH range of 7–12. It hydrolysed casein, gelatin and Tween 20 but not Tweens 40, 60 and 80, starch or DNA. No isoprenoid quinone was detected and the DNA G+C content was 39.7 mol%. The whole-cell fatty acid profile mainly consisted of iso-C15 : 0, anteiso-C15 : 0 and C16 : 0. DNA–DNA hybridization experiments with O. chironomi revealed 13 % relatedness. Owing to the differences in phenotypic and chemotaxonomic characteristics, and phylogenetic analyses based on 16S rRNA gene sequences and DNA–DNA relatedness data from reported Oceanobacillus species, the isolate merits classification as a representative of a novel species, for which the name Oceanobacillus indicireducens sp. nov. is proposed. The type strain is A21T ( = JCM 17251T = NCIMB 14685T). The description of the genus Oceanobacillus is also emended.
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amphibacillus indicireducens sp nov an Alkaliphile that reduces an indigo dye
International Journal of Systematic and Evolutionary Microbiology, 2013Co-Authors: Kikue Hirota, Isao Yumoto, Kenichi Aino, Naoki Morita, Yoshinobu NodasakaAbstract:Two indigo-reducing alkaliphilic strains, designated strain C40T and strain N214, were isolated from a fermented Polygonum Indigo (Polygonum tinctorium Lour.) liquor sample aged for 10 months and obtained from Date City, Hokkaido, Japan. 16S rRNA gene sequence phylogeny suggested that strains C40T and N214 were members of the genus Amphibacillus with the closest relative being Amphibacillus xylanus JCM 7361T (97.5 % 16S rRNA gene sequence similarity with strain C40T), which is the only strain having a 16S rRNA gene sequence similarity higher than 97 % with strain C40T. Cells of strain C40T were Gram-stain-positive, facultatively anaerobic, straight rods that were motile by means of peritrichous flagella. The strains grew between 17 and 39 °C (optimum, 35 °C) and in the pH range of 9.0–12.0. No isoprenoid quinone was detected and the DNA G+C content was 37.5–37.7 mol%. The whole-cell fatty acid profile mainly consisted of iso-C15 : 0 and anteiso-C15 : 0. DNA–DNA hybridization of strain C40T with Amphibacillus xylanus JCM 7361T revealed a DNA–DNA relatedness value of 10±3 %. Owing to the differences in phenotypic characteristics and phylogenetic analyses based on 16S rRNA gene sequences, as well as DNA–DNA relatedness data from reported species of the genus Amphibacillus , the isolates merit classification as a novel species in the genus Amphibacillus , for which the name Amphibacillus indicireducens sp. nov. is proposed. The type strain is C40T ( = JCM 17250T = NCIMB 14686T). An additional strain of the species is N214. An emended description of the genus Amphibacillus is provided.
Terry A. Krulwich - One of the best experts on this subject based on the ideXlab platform.
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The ATP Synthase a-subunit of Extreme Alkaliphiles Is a Distinct Variant MUTATIONS IN THE CRITICAL Alkaliphile-SPECIFIC RESIDUE LYS-180 AND OTHER RESIDUES THAT SUPPORT Alkaliphile OXIDATIVE PHOSPHORYLATION
The Journal of biological chemistry, 2010Co-Authors: Makoto Fujisawa, Terry A. Krulwich, Jun Liu, Oliver J. Fackelmayer, David HicksAbstract:A lysine residue in the putative proton uptake pathway of the ATP synthase a-subunit is found only in alkaliphilic Bacillus species and is proposed to play roles in proton capture, retention and passage to the synthase rotor. Here, Lys-180 was replaced with alanine (Ala), glycine (Gly), cysteine (Cys), arginine (Arg), or histidine (His) in the chromosome of alkaliphilic Bacillus pseudofirmus OF4. All mutants exhibited octylglucoside-stimulated ATPase activity and β-subunit levels at least as high as wild-type. Purified mutant F1F0-ATP synthases all contained substantial a-subunit levels. The mutants exhibited diverse patterns of native (no octylglucoside) ATPase activity and a range of defects in malate growth and in vitro ATP synthesis at pH 10.5. ATP synthesis by the Ala, Gly, and His mutants was also impaired at pH 7.5 in the presence of a protonophoric uncoupler. Thus Lys-180 plays a role when the protonmotive force is reduced at near neutral, not just at high pH. The Arg mutant exhibited no ATP synthesis activity in the Alkaliphile setting although activity was reported for a K180R mutant of a thermoAlkaliphile synthase (McMillan, D. G., Keis, S., Dimroth, P., and Cook, G. M. (2007) J. Biol. Chem. 282, 17395–17404). The hypothesis that a-subunits from extreme Alkaliphiles and the thermoAlkaliphile represent distinct variants was supported by demonstration of the importance of additional Alkaliphile-specific a-subunit residues, not found in the thermoAlkaliphile, for malate growth of B. pseudofirmus OF4. Finally, a mutant B. pseudofirmus OF4 synthase with switched positions of Lys-180 (helix 4) and Gly-212 (helix 5) retained significant coupled synthase activity accompanied by proton leakiness.
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F1F0-ATP synthases of alkaliphilic bacteria: lessons from their adaptations
Biochimica et biophysica acta, 2010Co-Authors: David Hicks, Jun Liu, Makoto Fujisawa, Terry A. KrulwichAbstract:Abstract This review focuses on the ATP synthases of alkaliphilic bacteria and, in particular, those that successfully overcome the bioenergetic challenges of achieving robust H + -coupled ATP synthesis at external pH values > 10. At such pH values the protonmotive force, which is posited to provide the energetic driving force for ATP synthesis, is too low to account for the ATP synthesis observed. The protonmotive force is lowered at a very high pH by the need to maintain a cytoplasmic pH well below the pH outside, which results in an energetically adverse pH gradient. Several anticipated solutions to this bioenergetic conundrum have been ruled out. Although the transmembrane sodium motive force is high under alkaline conditions, respiratory alkaliphilic bacteria do not use Na + - instead of H + -coupled ATP synthases. Nor do they offset the adverse pH gradient with a compensatory increase in the transmembrane electrical potential component of the protonmotive force. Moreover, studies of ATP synthase rotors indicate that Alkaliphiles cannot fully resolve the energetic problem by using an ATP synthase with a large number of c -subunits in the synthase rotor ring. Increased attention now focuses on delocalized gradients near the membrane surface and H + transfers to ATP synthases via membrane-associated microcircuits between the H + pumping complexes and synthases. Microcircuits likely depend upon proximity of pumps and synthases, specific membrane properties and specific adaptations of the participating enzyme complexes. ATP synthesis in Alkaliphiles depends upon Alkaliphile-specific adaptations of the ATP synthase and there is also evidence for Alkaliphile-specific adaptations of respiratory chain components.
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three putative cation proton antiporters from the soda lake Alkaliphile alkalimonas amylolytica n10 complement an alkali sensitive escherichia coli mutant
Microbiology, 2007Co-Authors: Yi Wei, Jun Liu, Terry A. KrulwichAbstract:Attempts to identify members of the antiporter complement of the alkali- and saline-adapted soda lake Alkaliphile Alkalimonas amylolytica N10 have used screens of DNA libraries in antiporter-deficient Escherichia coli KNabc. Earlier screens used Na+ or Li+ for selection but only identified one NhaD-type antiporter whose properties were inconsistent with a robust role in pH homeostasis. Here, new screens using elevated pH for selection identified three other putative antiporter genes that conferred resistance to pH ≥8.5 as well as Na+ resistance. The three predicted gene products were in the calcium/cation antiporter (CaCA), cation/proton antiporter-2 (CPA2) and cation/proton antiporter-1 (CPA1) families of membrane transporters, and were designated Aa-CaxA, Aa-KefB and Aa-NhaP respectively, reflecting homology within those families. Aa-CaxA conferred the poorest Na+ resistance and also conferred modest Ca2+ resistance. Aa-KefB and Aa-NhaP inhibited growth of a K+ uptake-deficient E. coli mutant (TK2420), suggesting that they catalysed K+ efflux. For Aa-NhaP, the reversibility of the growth inhibition by high K+ concentrations depended upon an organic nitrogen source, e.g. glutamine, rather than ammonium. This suggests that as well as K+ efflux is catalysed by Aa-NhaP. Vesicles of E. coli KNabc expressing Aa-NhaP, which conferred the strongest alkali resistance, exhibited K+/H+ antiport activity in a pH range from 7.5 to 9.5, and with an apparent K m for K+ of 0.5 mM at pH 8.0. The properties of this antiporter are consistent with the possibility that this soda lake Alkaliphile uses K+()/H+ antiport as part of its alkaline pH homeostasis mechanism and part of its capacity to reduce potentially toxic accumulation of cytoplasmic K+ or respectively, under conditions of high osmolarity or active amino acid catabolism.
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sequence analysis and functional studies of a chromosomal region of alkaliphilic bacillus firmus of4 encoding an abc type transporter with similarity of sequence and na exclusion capacity to the bacillus subtilis natab transporter
Extremophiles, 1999Co-Authors: Yi Wei, Arthur A Guffanti, Terry A. KrulwichAbstract:A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced Alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revealed that, as in B. subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter. In the Alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function. This is in contrast to the two-gene natAB operon and to another homolog from B. subtilis, the yhaQP genes. Like natAB, however, the Alkaliphile natCAB catalyzes Na+ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na+ extrusion. The full 14.1-kb fragment of Alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na+/H+ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E. coli mutants by subclones of the 14.1-kb piece. There were a total of 12 ORFs whose closest and significant homologs were genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B. subtilis homologs.
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ph tolerance in bacillus Alkaliphiles versus non Alkaliphiles
Novartis Foundation Symposium 221 - Bacterial Responses to Ph, 1999Co-Authors: Terry A. Krulwich, Arthur A Guffanti, Masahiro ItoAbstract:Monovalent cation/proton antiporters that catalyse electrogenic uptake of H+ in exchange for cytoplasmic K+ and/or Na+ are centrally involved in bacterial pH homeostasis under alkaline challenge. Systematic attempts have identified some, but not yet all, of the genes encoding such antiporters that participate in pH homeostasis in the neutrophilic Bacillus subtilis and the extremely alkaliphilic Bacillus firmus OF4. In each organism there are at least three distinct antiporters involved in pH homeostasis. They differ in cation requirement, with pH homeostasis specifically utilizing Na+/H+ antiport in the Alkaliphile and using either Na+ or K+/H+ antiport in B. subtilis. Some of the antiporters involved in pH homeostasis are constitutive and are in place to respond to sudden pH shifts, but there is also an inducible component. At least two sets of homologous antiporters (NhaC and Mrp/Pha) function in both Alkaliphiles and neutrophiles. An additional antiporter of a different transport protein family, the Gram-positive tetracycline-metal/H+ antiporter, is important in pH homeostasis in B. subtilis but has not yet been shown to be present in any Alkaliphile. There are also differences outside of the antiporters themselves that contribute to the greater capacity of the Alkaliphiles for pH homeostasis, including cation re-entry capacity and possible surface properties.
Noriyuki Koyama - One of the best experts on this subject based on the ideXlab platform.
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cloning sequencing and functional expression in escherichia coli of the gene for a p type na atpase of a facultatively anaerobic Alkaliphile exiguobacterium aurantiacum
Biochimica et Biophysica Acta, 2005Co-Authors: Yuusuke Suzuki, Sumie Ueno, Rieko Ohnuma, Noriyuki KoyamaAbstract:Abstract Cloning and sequencing of the gene encoding a P-type Na + -ATPase of a facultatively anaerobic Alkaliphile, Exiguobacterium aurantiacum , were conducted. The structural gene was composed of 2628 nucleotides. The deduced amino acid sequence (876 amino acid residues; Mr, 96,664) suggested that the enzyme possesses 10 membrane-spanning regions. When the amino acid sequences of the four putative membrane regions, M4, M5, M6 and M8, of BL77/1 ATPase were aligned with those of fungal Na + -ATPase, Na + /K + -ATPase, H + -ATPases and sarcoplasmic reticulum Ca 2+ -ATPase, it exhibited the highest homology with Ca 2+ -ATPase except M5 region. By the transformation of Escherichia coli with the expression vector (pQE30) containing the ATPase gene, the enzyme was functionally expressed in E. coli membranes.
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possible involvement of a single histidine residue in the p type na atpase of a facultatively anaerobic Alkaliphile exiguobacterium aurantiacum
Current Microbiology, 2001Co-Authors: Atsuko Hirano, Noriyuki KoyamaAbstract:Effect of various inhibitors on the P-type Na+-ATPase of a facultatively anaerobic Alkaliphile, Exiguobacterium aurantiacum, was examined. The ATPase was extremely sensitive to p-chloromercuriphenylsulfonic acid, a modifier of SH-group. The enzyme was also sensitive to diethylpyrocarbonate, and analysis of the inhibition kinetics by the drug indicated that modification of a single histidine residue per ATPase molecule was sufficient to inactivate the enzyme.
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characterization of a p type na atpase of a facultatively anaerobic Alkaliphile exiguobacterium aurantiacum
Journal of Biological Chemistry, 2000Co-Authors: Sumie Ueno, Naoko Kaieda, Noriyuki KoyamaAbstract:A facultatively anaerobic Alkaliphile, Exiguobacterium aurantiacum, possesses a P-type Na(+)-stimulated ATPase in the membrane (Koyama, N. (1999) Curr. Microbiol. 39, 27-30). In this study, we attempted to purify and characterize the enzyme. The ATPase appears to consist of a single polypeptide with an apparent molecular mass of 100 kDa. The enzyme exhibited an optimum pH for activity at around 9. The enzyme was strongly inhibited by vanadate (50% inhibition observed at 3 microm) and forms an acylphosphate intermediate, suggesting a P-type ATPase. The enzyme, when reconstituted into soybean phospholipid vesicles, exhibited ATP-dependent (22)Na(+) uptake, which was completely inhibited by gramicidin. The reconstituted vesicles exhibited a generation of membrane potential (positive, inside). The enzyme is likely to be involved in an electrogenic transport of Na(+).
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Purification and properties of the membrane-bound NADH oxidase of a facultatively anaerobic Alkaliphile
Archives of Microbiology, 1999Co-Authors: Tomoko Hamada, Takayoshi Wakagi, Hirotaka Shiba, Noriyuki KoyamaAbstract:A membrane-bound NADH oxidase of an anaerobic Alkaliphile, M-12 (a strain of Amphibacillus sp.), was solubilized with decanoyl N-methylglucamide and purified by chromatography on DEAE-Sepharose and hydroxyapatite. The purified enzyme appears to consist of a single polypeptide component with an apparent molecular mass of 56 kDa. The enzyme catalyzed the oxidation of NADH with the formation of H2O2 and exhibited a specific activity of 46 μmol NADH min–1 (mg protein)–1. NADPH did not serve as a substrate for the enzyme. The Km for NADH was estimated to be 0.05 mM. The enzyme exhibited a pH dependence for activity, with a pH optimum at approximately 9.5. The enzyme required a high concentration of salt and exhibited maximum activity in the presence of 600 mM NaCl.
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presence of na stimulated v type atpase in the membrane of a facultatively anaerobic and halophilic Alkaliphile
Fems Microbiology Letters, 1998Co-Authors: Naoko Kaieda, Takayoshi Wakagi, Noriyuki KoyamaAbstract:It was found that a facultatively anaerobic and halophilic Alkaliphile, M-12 (Amphibacillus sp.), possesses a Na+-stimulated ATPase in the membrane. The ATPase activity was inhibited by NO−3 and SCN− which are the inhibitors of V-type ATPase, but not by azide and vanadate, inhibitors of F-type ATPase and P-type ATPase, respectively. Upon the incubation of the membrane in buffer containing ATP and MgCl2, several polypeptides were released from the membrane. Among them, two major polypeptides with apparent molecular masses of 79 and 55 kDa crossreacted with an antiserum against the catalytic units (subunits A and B) of V-type ATPase from Enterococcus hirae. The N-terminal amino acid sequences of the 79 and 55 kDa polypeptides showed high similarity to those of subunits A and B of V-type ATPase from Enterococcus hirae, respectively. M-12 is likely to possess a V-type Na+-ATPase.
Kikue Hirota - One of the best experts on this subject based on the ideXlab platform.
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fermentibacillus polygoni gen nov sp nov an Alkaliphile that reduces indigo dye
International Journal of Systematic and Evolutionary Microbiology, 2016Co-Authors: Kikue Hirota, Isao Yumoto, Kenichi AinoAbstract:Facultatively alkaliphilic strains, designated as strains IEB3T and IEB14, were isolated as indigo-reducing strains from a fermented Polygonum indigo (Polygonum tinctorium Lour) liquor sample prepared in our laboratory using a medium containing an indigo fermentation liquor as a sole substrate. The 16S rRNA gene sequence phylogeny and similarity suggested that strains IEB3T and IEB14 exhibit distinctive positions among the members of the genus Bacillus, and their closest neighbour was Bacillus nanhaiisediminis NH3T (similarity: 97.4 %) among the species with validly published names. The 16S rRNA sequence of strain IEB3Twas identical to that of strain IEB14. The cells of the isolates stained Gram-positive and were facultatively anaerobic, straight rods that were motile by a pair of subpolar flagella. Strains IEB3T and IEB14 grew at temperatures between 12 and 40 °C with optimum growth at 30‒33 °C and in the range of pH 7.5-12. Menaquinone-7 (MK-7) was detected as the major isoprenoid quinone. The DNA G+C contents of strains IEB3T and IEB14 were 39.0 and 39.1 mol%, respectively. The whole-cell fatty acid profile mainly (>10 %) consisted of iso-C14:0, iso-C15:0 and anteiso-C15:0. DNA-DNA hybridization revealed a low relatedness value between strain IEB3T and the phylogenetically most closely related species, Bacillus nanhaiisediminis JCM 16507T (<7 % ). On the basis of phenotypic and chemotaxonomic characteristics and phylogenetic data, the isolates represent a novel species within a novel genus, for which the name Fermentibacillus polygoni gen. nov., sp. nov. is proposed. The type strain is IEB3T (=JCM 30817T=NCIMB 14984T).
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amphibacillus iburiensis sp nov an Alkaliphile that reduces an indigo dye
International Journal of Systematic and Evolutionary Microbiology, 2013Co-Authors: Kikue Hirota, Isao Yumoto, Kenichi AinoAbstract:An indigo-reducing alkaliphilic strain, designated strain N314T, was isolated from a fermented polygonum indigo (Polygonum tinctorium Lour.) liquor sample, aged for 10 months, that was obtained from Date City, Iburi Branch, Hokkaido, Japan. The 16S rRNA gene sequence phylogeny suggested that strain N314T is a member of the genus Amphibacillus , with the closest relatives being Amphibacillus indicireducens (98.9 % similarity to the type strain) and Amphibacillus xylanus (98.0 % similarity to the type strain), the only species with 16S rRNA gene sequence similarities higher than 97 % to strain N314T. The cells of the isolate stained Gram-positive and were facultatively anaerobic, straight rods that were motile by means of peritrichous flagella. The strain grew at 26–39 °C with optimum growth at 36 °C. It grew at pH 8.0–9.1, with optimum growth at pH 8.9–9.1. No isoprenoid quinone was detected, and the DNA G+C content was 38.4 mol%. The whole-cell fatty acid profile consisted mainly of iso-C15 : 0 and anteiso-C15 : 0. Analysis of DNA–DNA hybridization with the type strains of A. indicireducens and A. xylanus revealed 29±2 % and 10±2 % relatedness, respectively. Owing to differences in phenotypic characteristics from reported species of the genus A. and results of phylogenetic analyses based on 16S rRNA gene sequences and DNA–DNA relatedness data, the isolate merits classification within a novel species, for which the name Amphibacillus iburiensis sp. nov. is proposed. The type strain is N314T ( = JCM 18529T = NCIMB 14823T).
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oceanobacillus polygoni sp nov a facultatively Alkaliphile isolated from indigo fermentation fluid
International Journal of Systematic and Evolutionary Microbiology, 2013Co-Authors: Kikue Hirota, Isao Yumoto, Yoshinobu Nodasaka, Yoshiko HanaokaAbstract:A facultatively alkaliphilic, lactic-acid-producing and halophilic strain, designated SA9T, was isolated from a fermented Polygonum indigo (Polygonum tinctorium Lour.) liquor sample prepared in a laboratory. The 16S rRNA gene sequence phylogeny suggested that strain SA9T was a member of the genus Oceanobacillus with the closest relative being Oceanobacillus profundus KCCM 42318T (99.3 % 16S rRNA gene sequence similarity). Cells of strain SA9T stained Gram-positive and were facultative anaerobic straight rods that were motile by peritrichous flagella. The strain grew between 5 and 48 °C (optimum, 35 °C) and at pH 7–12 (optimum, pH 9). The isoprenoid quinone detected was menaquinone-7 (MK-7) and the DNA G+C content was 40.6±0.9 mol%. The whole-cell fatty acid profile mainly consisted of iso-C15 : 0, anteiso-C15 : 0, C16 : 0 and anteiso-C17 : 0. DNA–DNA hybridization with Oceanobacillus profundus DSM 18246T revealed a DNA–DNA relatedness value of 23±2 %. On the basis of the differences in phenotypic and chemotaxonomic characteristics, and the results of phylogenetic analyses based on 16S rRNA gene sequences and DNA–DNA relatedness data from recognized species of the genus Oceanobacillus , strain SA9T merits classification as a representative of a novel species of the genus Oceanobacillus , for which the name Oceanobacillus polygoni sp. nov. is proposed. The type strain is SA9T ( = JCM 17252T = NCIMB 14684T). An emended description of the genus Oceanobacillus is also provided.
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oceanobacillus indicireducens sp nov a facultative Alkaliphile that reduces an indigo dye
International Journal of Systematic and Evolutionary Microbiology, 2013Co-Authors: Kikue Hirota, Isao Yumoto, Kenichi Aino, Yoshinobu NodasakaAbstract:An indigo-reducing facultatively alkaliphilic and halophilic strain, designated strain A21T, was isolated from a fermented Polygonum indigo (Polygonum tinctorium Lour.) liquor sample aged for 4 days prepared in a laboratory. 16S rRNA gene sequence phylogeny suggested that strain A21T was a member of the genus Oceanobacillus with the closest relative being the type strain of Oceanobacillus chironomi (similarity: 96.0 %). The cells of the isolate stained Gram-positive and were facultatively anaerobic straight rods that were motile by peritrichous flagella. The strain grew between 18 and 48 °C with optimum growth at 39 °C. It grew in the pH range of 7–12. It hydrolysed casein, gelatin and Tween 20 but not Tweens 40, 60 and 80, starch or DNA. No isoprenoid quinone was detected and the DNA G+C content was 39.7 mol%. The whole-cell fatty acid profile mainly consisted of iso-C15 : 0, anteiso-C15 : 0 and C16 : 0. DNA–DNA hybridization experiments with O. chironomi revealed 13 % relatedness. Owing to the differences in phenotypic and chemotaxonomic characteristics, and phylogenetic analyses based on 16S rRNA gene sequences and DNA–DNA relatedness data from reported Oceanobacillus species, the isolate merits classification as a representative of a novel species, for which the name Oceanobacillus indicireducens sp. nov. is proposed. The type strain is A21T ( = JCM 17251T = NCIMB 14685T). The description of the genus Oceanobacillus is also emended.
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amphibacillus indicireducens sp nov an Alkaliphile that reduces an indigo dye
International Journal of Systematic and Evolutionary Microbiology, 2013Co-Authors: Kikue Hirota, Isao Yumoto, Kenichi Aino, Naoki Morita, Yoshinobu NodasakaAbstract:Two indigo-reducing alkaliphilic strains, designated strain C40T and strain N214, were isolated from a fermented Polygonum Indigo (Polygonum tinctorium Lour.) liquor sample aged for 10 months and obtained from Date City, Hokkaido, Japan. 16S rRNA gene sequence phylogeny suggested that strains C40T and N214 were members of the genus Amphibacillus with the closest relative being Amphibacillus xylanus JCM 7361T (97.5 % 16S rRNA gene sequence similarity with strain C40T), which is the only strain having a 16S rRNA gene sequence similarity higher than 97 % with strain C40T. Cells of strain C40T were Gram-stain-positive, facultatively anaerobic, straight rods that were motile by means of peritrichous flagella. The strains grew between 17 and 39 °C (optimum, 35 °C) and in the pH range of 9.0–12.0. No isoprenoid quinone was detected and the DNA G+C content was 37.5–37.7 mol%. The whole-cell fatty acid profile mainly consisted of iso-C15 : 0 and anteiso-C15 : 0. DNA–DNA hybridization of strain C40T with Amphibacillus xylanus JCM 7361T revealed a DNA–DNA relatedness value of 10±3 %. Owing to the differences in phenotypic characteristics and phylogenetic analyses based on 16S rRNA gene sequences, as well as DNA–DNA relatedness data from reported species of the genus Amphibacillus , the isolates merit classification as a novel species in the genus Amphibacillus , for which the name Amphibacillus indicireducens sp. nov. is proposed. The type strain is C40T ( = JCM 17250T = NCIMB 14686T). An additional strain of the species is N214. An emended description of the genus Amphibacillus is provided.
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THE ROLE OF MONOVALENT CATION/PROTON ANTIPORTERS IN Na+-RESISTANCE AND pH HOMEOSTASIS IN
2015Co-Authors: Alkaliphile Versus, Jianbo Cheng, A Neutralophile, Terry Ann Krulwich, Arthur A GuffantiAbstract:Both neutralophilic Bacillus subtilis and alkaliphilic Bacillus firmus OF4 depend upon electrogenic Na+/H+ antiporters, which are energized by the gradients established by respiration-coupled proton extrusion, to achieve Na+-resistance and pH homeostasis when the external pH is very alkaline. The interplay of proton and sodium cycles is discussed. In B. subtilis, pH homeostasis, up to pH9, can be achieved using K+ when Na+ is unavailable or when the gene encoding the Na+/H+ antiporter that is involved in Na+-dependent pH homeostasis is disrupted. That gene is a member of the tetracycline efflux family of genes. A second gene, encoding a Na+/H+ antiporter that functions in Na+-resistance, has been identified, and candidates for the K+/H+ antiporter genes are under investigation. Aggregate Na+/H+ antiport activity in B. subtilis is as much as 10 times lower than in the Alkaliphile, and the neutralophile cannot regulate its internal pH upon a shift to pH10.5. Upon such a shift, there is a pronounced reduction in the generation of a primary electrochemical proton gradient. The Alkaliphile, by contrast, maintains substantial driving forces and regulates its internal pH in an exclusively Na+-coupled manner upon shifts to either pH8.7 or 10.5. One gene locus has been identified and a second locus has been inferred as encoding relevant antiporter activities. Prokaryotic patterns of Na+ translocation relevant to Na+-resistance or pH homeostasis Monovalent cation/proton antiporters, and Na+/H+ antiporters in particular, have been known or proposed to play a large variety of important physiological roles, including resistance to elevated levels of Na+ in the medium, pH homeostasis, osmoregulation an
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sequence analysis and functional studies of a chromosomal region of alkaliphilic bacillus firmus of4 encoding an abc type transporter with similarity of sequence and na exclusion capacity to the bacillus subtilis natab transporter
Extremophiles, 1999Co-Authors: Yi Wei, Arthur A Guffanti, Terry A. KrulwichAbstract:A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced Alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revealed that, as in B. subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter. In the Alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function. This is in contrast to the two-gene natAB operon and to another homolog from B. subtilis, the yhaQP genes. Like natAB, however, the Alkaliphile natCAB catalyzes Na+ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na+ extrusion. The full 14.1-kb fragment of Alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na+/H+ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E. coli mutants by subclones of the 14.1-kb piece. There were a total of 12 ORFs whose closest and significant homologs were genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B. subtilis homologs.
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ph tolerance in bacillus Alkaliphiles versus non Alkaliphiles
Novartis Foundation Symposium 221 - Bacterial Responses to Ph, 1999Co-Authors: Terry A. Krulwich, Arthur A Guffanti, Masahiro ItoAbstract:Monovalent cation/proton antiporters that catalyse electrogenic uptake of H+ in exchange for cytoplasmic K+ and/or Na+ are centrally involved in bacterial pH homeostasis under alkaline challenge. Systematic attempts have identified some, but not yet all, of the genes encoding such antiporters that participate in pH homeostasis in the neutrophilic Bacillus subtilis and the extremely alkaliphilic Bacillus firmus OF4. In each organism there are at least three distinct antiporters involved in pH homeostasis. They differ in cation requirement, with pH homeostasis specifically utilizing Na+/H+ antiport in the Alkaliphile and using either Na+ or K+/H+ antiport in B. subtilis. Some of the antiporters involved in pH homeostasis are constitutive and are in place to respond to sudden pH shifts, but there is also an inducible component. At least two sets of homologous antiporters (NhaC and Mrp/Pha) function in both Alkaliphiles and neutrophiles. An additional antiporter of a different transport protein family, the Gram-positive tetracycline-metal/H+ antiporter, is important in pH homeostasis in B. subtilis but has not yet been shown to be present in any Alkaliphile. There are also differences outside of the antiporters themselves that contribute to the greater capacity of the Alkaliphiles for pH homeostasis, including cation re-entry capacity and possible surface properties.
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role of the nhac encoded na h antiporter of alkaliphilic bacillus firmus of4
Journal of Bacteriology, 1997Co-Authors: Masahiro Ito, Arthur A Guffanti, D M Ivey, J Zemsky, Terry A. KrulwichAbstract:Application of protoplast transformation and single- and double-crossover mutagenesis protocols to alkaliphilic Bacillus firmus OF4811M (an auxotrophic strain of B. firmus OF4) facilitated the extension of the sequence of the previously cloned nhaC gene, which encodes an Na+/H+ antiporter, and the surrounding region. The nhaC gene is part of a likely 2-gene operon encompassing nhaC and a small gene that was designated nhaS; the operon is preceded by novel direct repeats. The predicted Alkaliphile NhaC, based on the extended sequence analysis, would be a membrane protein with 462 amino acid residues and 12 transmembrane segments that is highly homologous to the deduced products of homologous genes of unknown function from Bacillus subtilis and Haemophilus influenzae. The full-length version of nhaC complemented the Na+-sensitive phenotype of an antiporter-deficient mutant strain of Escherichia coli but not the alkali-sensitive growth phenotypes of Na+/H+-deficient mutants of either alkaliphilic B. firmus OF4811M or B. subtilis. Indeed, NhaC has no required role in alkaliphily, inasmuch as the nhaC deletion strain of B. firmus OF4811M, N13, grew well at pH 10.5 at Na+ concentrations equal to or greater than 10 mM. Even at lower Na+ concentrations, N13 exhibited only a modest growth defect at pH 10.5. This was accompanied by a reduced capacity to acidify the cytoplasm relative to the medium compared to the wild-type strain or to N13 complemented by cloned nhaC. The most notable deficiency observed in N13 was its poor growth at pH 7.5 and Na+ concentrations up to 25 mM. During growth at pH 7.5, NhaC is apparently a major component of the relatively high affinity Na+/H+ antiport activity available to extrude the Na+ and to confer some initial protection in the face of a sudden upshift in external pH, i.e., before full induction of additional antiporters. Consistent with the inference that NhaC is a relatively high affinity, electrogenic Na+/H+ antiporter, N13 exhibited a defect in diffusion potential-energized efflux of 22Na+ from right-side-out membrane vesicles from cells that were preloaded with 2 mM Na+ and energized at pH 7.5. When the experiment was conducted with vesicles loaded with 25 mM Na+, comparable efflux was observed in preparations from all the strains.
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Energetic problems of extremely alkaliphilic aerobes
Biochimica et biophysica acta, 1996Co-Authors: Terry A. Krulwich, Masahiro Ito, Arthur A Guffanti, Raymond Gilmour, Michael G. Sturr, David HicksAbstract:Over a decade of work on extremely alkaliphilic Bacillus species has clarified the extraordinary capacity that these bacteria have for regulating their cytoplasmic pH during growth at pH values well over 10. However, a variety of interesting energetic problems related to their Na+-dependent pH homeostatic mechanism are yet to be solved. They include: (1) the clarification of how cell surface layers play a role in a category of Alkaliphiles for which this is the case; (2) identification of the putative, electrogenic Na+/H+ antiporter(s) that, in at least some Alkaliphiles, may completely account for a cytoplasmic pH that is over 2 pH units lower than the external pH; (3) the determination of whether specific modules or accessory proteins are essential for the efficacy of such antiporters; (4) the mechanistic basis for the increase in the transmembrane electrical potential at the high external pH values at which the potential-consuming antiporter(s) must be most active; and (5) an explanation for the Na+-specificity of pH homeostasis in the extremely alkaliphilic bacilli as opposed to the almost equivalent efficacy of K+ for pH homeostasis in at least some non-alkaliphilic aerobes. The current status of such studies and future strategies will be outlined for this central area of Alkaliphile energetics. Also considered, will be strategies to elucidate the basis for robust H+-coupled oxidative phosphorylation by Alkaliphiles at pH values over 10. The maintenance of a cytoplasmic pH over 2 units below the high external pH results in a low bulk electrochemical proton gradient (Δp). To bypass this low Δp, Na+-coupling is used for solute uptake even by Alkaliphiles that are mesophiles from environments that are not especially Na+-rich. This indicates that these bacteria indeed experience a low Δp, to which such coupling is an adaptation. Possible reasons and mechanisms for using a H+-coupled rather than a Na+-coupled ATP synthase under such circumstances will be discussed.