Structure,function and biosynthesis of carotenoids in the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus halophilus</Emphasis>

Inhibitor studies and mutant analysis revealed a C₃₀ pathway via 4,4′-diapophytoene and 4,4′-diaponeurosporene to 4,4′-diaponeursoporene-4-oic acid esters related to staphyloxanthin in Halobacillus halophilus. Six genes may be involved in this biosynthetic pathway and could be found in two adjacent gene clusters. Two genes of this pathway could be functionally assigned by functional pathway complementation as a 4,4′-diapophytoene synthase and a 4,4′-diapophytoene desaturase gene. These genes were organized in two operons together with two putative oxidase genes, a glycosylase and an acyl transferase ortholog. Pigment mutants were obtained by chemical mutagenesis. Carotenoid analysis showed that a white mutant accumulated 4,4′-diapophytoene due to a block in desaturation. In a yellow mutant carotenogenesis was blocked at the stage of 4,4′-diaponeurosporene and in an orange mutant at the stage of 4,4′-diaponeurosporene-4-oic acid. The protective function of these pigments could be demonstrated for H. halophilus after inhibition of carotenoid synthesis by initiation of oxidative stress. A degree of oxidative stress which still allowed 50% growth of carotenogenic cells resulted in the death of the cells devoid of colored carotenoids.     

The major carotenoid in all known species of heliobacteria is the C30 carotenoid 4,4′-diaponeurosporene, not neurosporene

The carotenoids of five species of heliobacteria (Heliobacillus mobilis, Heliophilum fasciatum, Heliobacterium chlorum, Heliobacterium modesticaldum, and Heliobacterium gestii) were examined by spectroscopic methods, and the C₃₀ carotene 4,4′-diaponeurosporene was found to be the dominant pigment; heliobacteria were previously thought to contain the C₄₀ carotenoid neurosporene. In addition, trace amounts of the C₃₀ diapocarotenes diapolycopene, diapo-ζ-carotene, diapophytofluene, and diapophytoene were also found. Up to now, diapocarotenes have been found in only three species of chemoorganotrophic bacteria, but not in phototropic organisms. Furthermore, the esterifying alcohol of bacteriochlorophyll g from all known species of heliobacteria was determined to be farnesol (C₁₅) instead of the usual phytol (C₂₀). Heliobacteria may be unable to produce geranylgeranyol (C₂₀). Received: 10 March 1997 / Accepted: 3 June 1997     

Unique Carotenoids in the Terrestrial Cyanobacterium <Emphasis Type="Italic">Nostoc commune</Emphasis> NIES-24: 2-Hydroxymyxol 2′-Fucoside,Nostoxanthin and Canthaxanthin

Cyanobacteria produce some carotenoids. We identified the molecular structures, including the stereochemistry, of all the carotenoids in the terrestrial cyanobacterium, Nostoc commune NIES-24 (IAM M-13). The major carotenoid was β-carotene. Its hydroxyl derivatives were (3R)-β-cryptoxanthin, (3R,3′R)-zeaxanthin, (2R,3R,3′R)-caloxanthin, and (2R,3R,2′R,3′R)-nostoxanthin, and its keto derivatives were echinenone and canthaxanthin. The unique myxol glycosides were (3R,2′S)-myxol 2′-fucoside and (2R,3R,2′S)-2-hydroxymyxol 2′-fucoside. This is only the second species found to contain 2-hydroxymyxol. We propose possible carotenogenesis pathways based on our identification of the carotenoids: the hydroxyl pathway produced nostoxanthin via zeaxanthin from β-carotene, the keto pathway produced canthaxanthin from β-carotene, and the myxol pathway produced 2-hydroxymyxol 2′-fucoside via myxol 2′-fucoside. This cyanobacterium was found to contain many kinds of carotenoids and also displayed many carotenogenesis pathways, while other cyanobacteria lack some carotenoids and a part of carotenogenesis pathways compared with this cyanobacterium.     

New carotenoids from the thermophilic green sulfur bacterium Chlorobium tepidum: 1′,2′-dihydro-γ-carotene, 1′,2′-dihydrochlorobactene, and OH-chlorobactene glucoside ester, and the carotenoid composition of different strains

The complete carotenoid composition of the thermophilic green sulfur bacterium Chlorobium tepidum strain TNO was determined by spectroscopic methods. Major carotenoids were four kinds of carotenes: γ-carotene, chlorobactene, and their 1′,2′-dihydro derivatives (1′,2′-dihydro-γ-carotene and 1′,2′-dihydrochlorobactene). In lesser amounts, hydroxyl γ-carotene, hydroxyl chlorobactene, and their glucoside fatty acid esters were found. The only esterified fatty acid present was laurate, and OH-chlorobactene glucoside laurate is a novel carotenoid. In other strains of C. tepidum, the same carotenoids were found, but the composition varied from strain to strain. The overall pigment composition in cells of strain TNO was 4 mol carotenoids and 40 mol bacteriochlorophyll c per mol bacteriochlorophyll a. The effects of nicotine on carotenoid biosynthesis in C. tepidum differed from those in the thermophilic green nonsulfur bacterium Chloroflexus aurantiacus. Received: 3 February 1997 / Accepted: 6 June 1997     

Screening of a high fibrinolytic enzyme producing strain and characterization of the fibrinolytic enzyme produced from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> LD-8547

A fibrinolytic enzyme producing strain Bacillus subtilis LD-8 was isolated from douchi, a traditional Chinese soybean-fermented food. After mutagenesis treatments by UV, NTG (N-methyl-N′-nitroso-N-nitroso-guanidine) and γ-radiation, a high fibrinolytic enzyme producing strain B. subtilis LD-8547 was obtained. Under optimum condition, LD-8547 was able to yield the average fibrinolytic activity of 4220 U/mL in 15 L fermenter. The strong fibrin-specific enzyme was purified from supernatant of B. subtilis LD-8547 culture broth using the combination of various steps. The optimal temperature and pH value of this fibrinolytic enzyme were 50 °C and 8.0, respectively. The molecular weight was about 30 kDa measured by SDS-PAGE. The amidolytic activity of this fibrinolytic enzyme was inhibited completely by 1 mmol/L phenylmethanesulfonyl fluoride (PMSF), but EDTA and EGTA did not affect the enzyme activity. The apparent K _m and V _max values were 0.521 mmol/L and 0.049 mmol/min, respectively. In vitro assays revealed that the enzyme could catalyze blood clot lysis effectively, indicating that this enzyme could be a useful thrombolytic agent.     

Specific biodegradation of polychlorinated biphenyls (PCBs) facilitated by plant terpenoids

The aim of this study was to examine how plant terpenoids, as natural growth substrates or inducers, would affect the biodegradation of PCB congeners. Various PCB degraders that could grow on biphenyl and several terpenoids were tested for their PCB degradation capabilities. Degradation activities of the PCB congeners, 4,4′-dichlorobiphenyl (4,4′-DCBp) and 2,2′-dichlorobiphenyl (2,2′-DCBp), were initially monitored through a resting cell assay technique that could detect their degradation products. The PCB degraders,Pseudomonas sp. P166 andRhodococcus sp. T104, were found to grow on both biphenyl and terpenoids ((S)-(−) limonene,p-cymene and α-terpinene) whereasArthrobacter sp. B1B could not grow on the terpenoids as a sole carbon source. The B1B strain grown on biphenyl exhibited good degradation activity for 4,4′-DCBp and 2,2′-DCBp, while the activity of strains P166 and T104 was about 25% that of the B1B strain, respectively. Concomitant GC analysis, however, demonstrated that strain T104, grown on (S)-(−) limonene,p-cymene and α-terpinene, could degrade 4,4′-DCBp up to 30%, equivalent to 50% of the biphenyl induction level. Moreover, strain T104 grown on (S)-(−) limonene, could also degrade 2,2′-DCBp up to 30%. This indicates that terpenoids, widely distributed in nature, could be utilized as both growth and/or inducer substrate(s) for PCB biodegradation in the environment.     

The major part of polar carotenoids of the aerobic bacteria Roseococcus thiosulfatophilus RB3 and Erythromicrobium ramosum E5 is not bound to the bacteriochlorophyll a-complexes of the photosynthetic apparatus

The obligate aerobic bacteria Roseococcus thiosulfatophilus RB3 and Erythromicrobium ramosum E5 contain numerous polar carotenoids. The major carotenoid of the strain RB3 was the C₃₀ carotene-dioate (4,4-diapocarotene-4,4-dioate) and the respective diglycosyl ester which have never been isolated before from a bacteriochlorophyll containing bacterium. Strain E5 contains the very polar erythroxanthin sulphate. The major carotenoid bound to reaction center and light-harvesting complexes is bacteriorubixanthinal. Most of the carotenoids of both strains are not bound to the pigment-protein complexes of the photosynthetic apparatus but to the envelope fraction (cytoplasmic membrane and cell wall).Abbreviations Bchl bacteriochlorophyll - MeOH methanol     

<Emphasis Type="Italic">Halarchaeum solikamskense</Emphasis> sp. nov., a thermotolerant neutrophilic haloarchaeon from the foamy products of flotation enrichment of potassium minerals

Three pigmented strains of halophilic archaea, RS94-RS96, were isolated from acidic foamy products of flotation enrichment of potassium minerals (Silvinit Co., Solikamsk, Russia). The cells were gram-negative, nonmotile, pleomorphic ovoids, 1.0−1.5 × 1.5−2.5 μm. The isolates were chemoorganotrophic, obligately aerobic, and catalase-positive. A range of carbohydrates and organic acids was used, as well as amino acids and peptides. The strains were halophiles and thermotolerant neutrophiles. They grew in the media with 15 to 30% NaCl (optimum at 20–22%) and 0.005–0.7 M Mg²⁺ (0.1–0.2 M), at pH 5.0–8.2 (optimum 7.0–7.2) and 25–55°C (optimum at 35–50°C). The major fatty acids were C_16:0, C_18:1, C_18:0, and C_16:1. The membranes contained carotenoid pigments of the bacterioruberin series and polar lipids, mostly as C₂₀,C₂₀ isoprenoid derivates: phosphatidylglyceromethylphosphate, phosphatidylglycerol, and three unidentified sulfated glycolipids of the S-DGD type. The DNA G+C content was 65.1–66.4 mol %. Phylogenetic analysis based on the 16S rRNA gene sequencing revealed that the thermotolerant neutrophilic isolate RS94 (DNA G+C content of 66.4 mol %) was most closely related to the nonpigmented moderate acidophile Halarchaeum acidiphilum MH1-52-1^T (97.3%). Based on its phenotypic and genotypic characteristics, the organism was classified as a new species of the genus Halarchaeum with the proposed name Halarchaeum solikamskense sp. nov. The type strain is RS94^T (= VKPM B-11282^T).     

Fungal bioconversion of toxic polychlorinated biphenyls by white-rot fungus, Phlebia brevispora

Toxic coplanar polychlorinated biphenyls (Co-PCBs) were used as substrates for a degradation experiment with white-rot fungus, Phlebia brevispora TMIC33929, which is capable of degrading polychlorinated dibenzo-p-dioxins. Eleven PCB congener mixtures (7 mono-ortho- and 4 non-ortho-PCBs) were added to the cultures of P. brevispora and monitored by high resolution gas chromatography and mass spectrometry (HRGC/HRMS). Five PCB congeners, 3,3′,4,4′-tetrachlorobiphenyl, 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl were degraded by P. brevispora. To investigate the fungal metabolism of PCB, each Co-PCB was treated separately by P. brevispora and the metabolites were analyzed by gas chromatography and mass spectrometry (GC/MS) and identified on the basis of the GC/MS comparison with the authentic compound. Meta-methoxylated metabolite was detected from the culture containing each compound. Additionally, para-dechlorinated and -methoxylated metabolite was also detected from the culture with 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl, which are mono-ortho-PCBs. In this paper, we identified the congener specific degradation of coplanar PCBs by P. brevispora, and clearly proved for the first time by identifying the metabolites that the white-rot fungus, P. brevispora, transformed recalcitrant coplanar PCBs.     

Photosynthesis,chlorophyll fluorescence,and antioxidant enzyme responses of invasive weed <Emphasis Type="Italic">Mikania micrantha</Emphasis> to <Emphasis Type="Italic">Bemisia tabaci</Emphasis> infestation

In a glasshouse, Bemisia tabaci infestation largely reduced response of photosynthesis to irradiance and CO₂ concentration of Mikania micrantha compared with the non-infested control (C) ones. The maximum irradiance-saturated photosynthetic rate (P _max) and saturation irradiance (SI) of the infested M. micrantha were only 21.3 % and 6.5 % of the C-plants, respectively. B. tabaci infestation led to the reduction of contents of chlorophyll and carotenoids in M. micrantha, which was accompanied with the decrease of actual photosystem 2 (PS2) efficiency (Φ_PS2), efficiency of excitation energy capture by open PS2 reaction centres (F_v′/F_m′), electron transport rate (ETR), and photochemical quenching (q_P). Moreover, superoxide dismutase and catalase activities significantly decreased while proline and glutathione contents significantly increased in infested M. micrantha. Hence B. tabaci infestation not only induced direct damage of photosynthetic apparatus but also altered the antioxidant enzymes activities in M. micrantha, which might as consequences accelerate senescence of this weed.     

A new monocupin quercetinase of Streptomyces sp. FLA: identification and heterologous expression of the queD gene and activity of the recombinant enzyme towards different flavonols

The gene queD encoding quercetinase of Streptomyces sp. FLA, a soil isolate related to S. eurythermus ^T, was identified. Quercetinases catalyze the 2,4-dioxygenolytic cleavage of 3,5,7,3′,4′-pentahydroxyflavone to 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide. The queD gene was expressed in S. lividans and E. coli, and the recombinant hexahistidine-tagged protein (QueDHis₆) was purified. Several flavonols were converted by QueDHis₆, whereas CO formation from the 2,3-dihydroflavonol taxifolin and the flavone luteolin were not observed. In contrast to bicupin quercetinases from Aspergillus japonicus and Bacillus subtilis, and bicupin pirins showing quercetinase activity, QueD of strain FLA is a monocupin exhibiting 35.9% sequence identity to the C-terminal domain of B. subtilis quercetinase. Its native molecular mass of 63 kDa suggests a multimeric protein. A queD-specific probe hybridized with fragments of genomic DNA of four other quercetin degrading Streptomyces strains, but not with DNA of B. subtilis. Potential ORFs upstream of queD probably code for a serine protease and an endoribonuclease; two ORFs downstream of queD may encode an amidohydrolase and a carboxylesterase. This arrangement suggests that queD is not part of a catabolic gene cluster. Quercetinases might play a major role as detoxifying rather than catabolic enzymes.     

Metabolism of 4,4′-dichlorobiphenyl by white-rot fungi Phanerochaete chrysosporium and Phanerochaete sp. MZ142

Degradation experiment of model polychlorinated biphenyl (PCB) compound 4,4′-dichlorobiphenyl (4,4′-DCB) and its metabolites by the white-rot fungus Phanerochaete chrysosporium and newly isolated 4,4′-DCB-degrading white-rot fungus strain MZ142 was carried out. Although P. chrysosporium showed higher degradation of 4,4′-DCB in low-nitrogen (LN) medium than that in potato dextrose broth (PDB) medium, Phanerochaete sp. MZ142 showed higher degradation of 4,4′-DCB under PDB medium condition than that in LN medium. The metabolic pathway of 4,4′-DCB was elucidated by the identification of metabolites upon addition of 4,4′-DCB and its metabolic intermediates. 4,4′-DCB was initially metabolized to 2-hydroxy-4,4′-DCB and 3-hydroxy-4,4′-DCB by Phanerochaete sp. MZ142. On the other hand, P. chrysosporium transformed 4,4′-DCB to 3-hydroxy-4,4′-DCB and 4-hydroxy-3,4′-DCB produced via a National Institutes of Health shift of 4-chlorine. 3-Hydroxy-4,4′-DCB was transformed to 3-methoxy-4,4′-DCB; 4-chlorobenzoic acid; 4-chlorobenzaldehyde; and 4-chlorobenzyl alcohol in the culture with Phanerochaete sp. MZ142 or P. chrysosporium. LN medium condition was needed to form 4-chlorobenzoic acid, 4-chlorobenzaldehyde, and 4-chlorobenzyl alcohol from 3-hydroxy-4,4′-DCB, indicating the involvement of secondary metabolism. 2-Hydroxy-4,4′-DCB was not methylated. In this paper, we proved for the first time by characterization of intermediate that hydroxylation of PCB was a key step in the PCB degradation process by white-rot fungi.     

<Emphasis Type="Italic">Pontibacter salisaro</Emphasis> sp. nov., Isolated from a clay tablet solar saltern in Korea

A Gram-negative, aerobic, rod-shaped, and red-pigmented bacterial strain, HMC5104^T, was isolated from a solar saltern, found in Jeungdo, Republic of Korea (34°59′47″N 126°10′02″E). The major fatty acids were summed feature 4 (comprising iso-C_17:1 I and/or anteiso-C_17:1 B; 37.2%), iso-C_15:0 (20.4%), and iso-C_17.0 30H (15.3%). The DNA G+C content was 46.0 mol%. The major isoprenoid quinone was menaquinone-7 (MK-7). A phylogenetic tree based on 16S rRNA gene sequences showed that strain HMC5104^T formed a lineage within the genus Pontibacter, and was closely related to Pontibacter korlensis (95.9%), P. roseus (94.9%), and P. actiniarum (94.3%). Similarities to all other Pontibacter species were between 95.9–93.9%. On the basis of the evidence presented in this study, strain HMC5104^T represents a novel species of the genus Pontibacter, for which the name Pontibacter salisaro sp. nov. is proposed. The type strain is HMC5104^T (=KCTC 22712^T = NBRC 105731^T).     

Up-regulation of cyclic electron flow and down-regulation of linear electron flow in antisense-<Emphasis Type="Italic">rca</Emphasis> mutant rice

To investigate how excess excitation energy is dissipated in a ribulose-1,5-bisphospate carboxylase/oxygenase activase antisense transgenic rice with net photosynthetic rate (P _N) half of that of wild type parent, we measured the response curve of P _N to intercellular CO₂ concentration (C _i), electron transport rate (ETR), quantum yield of open photosystem 2 (PS2) reaction centres under irradiation (F_v′/F_m′), efficiency of total PS2 centres (Φ_PS2), photochemical (q_P) and non-photochemical quenching (NPQ), post-irradiation transient increase in chlorophyll (Chl) fluorescence (PITICF), and P700⁺ re-reduction. Carboxylation efficiency dependence on C _i, ETR at saturation irradiance, and F_v′/F_m′, Φ_PS2, and q_P under the irradiation were significantly lower in the mutant. However, NPQ, energy-dependent quenching (q_E), PITICF, and P700⁺ re-reduction were significantly higher in the mutant. Hence the mutant down-regulates linear ETR and stimulates cyclic electron flow around PS1, which may generate the ΔpH to support NPQ and q_E for dissipation of excess excitation energy.     

Characterization of a multifunctional feather-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> isolated from forest soil

In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO₃, 0.1% (w/v) K₂HPO₄, 0.06% (w/v) KH₂PO₄ and 0.04% (w/v) MgCl₂·6H₂O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml). After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted in free –SH group, soluble protein and amino acids production. The concentration of free –SH group in the culture medium was 15.5 ± 0.2 μM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the concentration of total amino acid produced was 3360.4 μM. Proline (2809.9 μM), histidine (371.3 μM) and phenylalanine (172.0 μM) were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA), phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil.     

The leaves of the common box,Buxus sempervirens (Buxaceae), become red as the level of a red carotenoid,anhydroeschscholtzxanthin, increases

Carotenoids from the leaves of the common box,Buxus sempervirens (Buxaceae), which turn red in late autumn to winter, were analyzed by reversed-phase HPLC. A novel carotenoid, monoanhydroeschscholtzxanthin (3), was isolated from the red-colored leaves. UV-VIS, MS,¹H-NMR and CD spectral data showed that the structure of 3 was (3S)-2′, 3′, 4′, 5′-tetradehydro-4, 5′-retro-β, β-caroten-3-ol. As well as anhydroeschscholtzxanthin (2), the major red carotenoid in the leaves, eschscholtzxanthin (4) was identified. Very small amounts of yellow carotenoids (neoxanthin, violaxanthin, lutein and β-carotene), which are major components of green leaves, were present in the red-colored leaves. The amounts of chlorophylla andb in the leaves decreased markedly during coloration, even at the early stages, whereas those of the yellow carotenoids decreased gradually. In contrast, the content of 2, a red carotenoid, increased steadily during coloration. The biosynthetic pathway of 2 inB. sempervirens was deduced tentatively on the basis of the individual carotenoid contents during autumnal coloration.     

<Emphasis Type="Italic">Halomonas sediminis</Emphasis> sp. nov., a new halophilic bacterium isolated from salt-lake sediment in China

A novel Gram-negative, slightly halophilic, catalase- and oxidase-positive, obligately aerobic bacterium, strain YIM-C248^T, was isolated from a sediment sample collected from a salt-lake in the Qaidam Basin in Qinghai, north-west China. Cells were non-sporulating short rods, occurring singly or as doublets, motile with peritrichous flagella. Growth occurred with 1–15% (w/v) NaCl [optimum 2–4% (w/v) NaCl], at pH 6.0–10.0 (optimum pH 7.5) and at 4–35°C (optimum 25–30°C). The major cellular fatty acids were C_18:1 ω7c, C_12:0 3-OH, cyclo C_19:0 ω8c, C_16:0 and C_16:1. The predominant respiratory quinone was Q-9 and the genomic DNA G + C content was 58.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YIM-C248^T should be assigned to the genus Halomonas. The sequence similarities between the isolate and the type strains of members of the genus Halomonas were in the range of 92.5–97.5%. The combination of phylogenetic analysis, DNA–DNA hybridization data, phenotypic characteristics and chemotaxonomic differences supported the view that strain YIM-C248^T represents a new species of the genus Halomonas, for which the name Halomonas sediminis sp. nov. is proposed, with YIM-C248^T (=CCTCC AA 207031 = KCTC 22167) as the type strain. The GenBank/EMBL/DBBJ accession number for the 16S rRNA gene sequence of strain YIM-C248^T is EU135707.     

<Emphasis Type="Italic">Bacillus kyonggiensis</Emphasis> sp. nov., isolated from soil of a lettuce field

A Gram-positive, rod-shaped, motile, endospore-forming bacterial strain, designated NB22^T, was isolated from soil of a lettuce field in Kyonggi province, South Korea, and was characterized by using a polyphasic taxonomic approach. This novel isolate grew optimally at 30–37°C and pH 8–9. It grew in the presence of 0–4% NaCl (optimum, 1–2%). Comparative 16S rRNA gene sequence analysis showed that strain NB22^T was closely related to members of the genus Bacillus and fell within a coherent cluster comprising B. siralis 171544^T (98.1%) and B. korlensis ZLC-26^T (97.3%). The levels of 16S rRNA gene sequence similarity with respect to other Bacillus species with validly published names were less than 96.4%. Strain NB22^T had a genomic DNA G+C content of 36.3 mol% and the predominant respiratory quinone was MK-7. The peptidoglycan contained meso-diaminopimelic acid. The major cellular fatty acids were iso-C_15:0, anteiso-C_15:0, C_14:0, and C_16:0. These chemotaxonomic results supported the affiliation of strain NB22^T to the genus Bacillus, and the low DNA-DNA relatedness values and distinguishing phenotypic characteristics allowed genotypic and phenotypic differentiation of strain NB22^T from recognized Bacillus species. On the basis of the evidence presented, strain NB22^T is considered to represent a novel species of the genus Bacillus, for which the name Bacillus kyonggiensis sp. nov. is proposed. The type strain is NB22^T (=KEMB 5401-267^T =JCM 17569^T).     

Activation of the Cardiac Ryanodine Receptor by Sulfhydryl Oxidation is Modified by Mg2+ and ATP

The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca²⁺] of 10⁻⁷ m (in the absence of Mg²⁺ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm cis Mg²⁺ (10⁻⁷ m cis Ca²⁺) or by 10 mm cis Mg²⁺ (10⁻³ m cis Ca²⁺), or activated by 4 mm ATP (10⁻⁷ m cis Ca²⁺), also responded to 1 mm cis 4,4′-DTDP with activation and then loss of activity. P _o and mean open time (T _o ) of the maximally activated channels were lower in the presence of Mg²⁺ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg²⁺, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca²⁺ or caffeine (Eager et al., 1997), the activation produced by 1 mm cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation. Received: 8 September 1997/Revised: 20 January 1998