Newly Isolated Bacillus gibsonii S-2 Capable of using Sugar Beet Pulp for Alkaline Pectinase Production

Summary A Bacillus strain capable of growing under highly alkaline conditions was isolated from a sample of alkaline soil. The isolate was a Gram-positive, spore-forming, aerobic, alkaliphilic bacterium and was designated as strain S-2. Growth of the strain was observed in the pH range of 7–12 and temperature range of 4–40 °C. Sequence analysis of the 16S rDNA of the strain revealed more than 99% homology with the strains of Bacillus gibsonii. The S-2 strain was confirmed as B. gibsonii by comparing its physiological and biochemical characteristics with the B. gibsonii DSM 8722 strain. The S-2 strain could use sugar beet pulp as the carbon source as well as the pectinase inducer to produce extracellular alkaline pectinase by solid-state fermentation. The maximum polygalacturonase yield of 3600 U/g dry sugar beet pulp was obtained at 35 °C after 48 h of incubation.     

Anaerocolumna sedimenticola sp. nov., isolated from fresh water sediment

Strain CBA3638^T was isolated from the Geum River sediment, Republic of Korea. The cells of strain CBA3638^T were Gram-stain-positive, strictly anaerobic, rod-shaped, and 0.5–1.0 μm wide, and 4.0–4.5 μm long. Optimal growth occurred at 37 °C, pH 7.0, and 1.0% (w/v) NaCl. Based on the 16S rRNA gene sequence, the phylogenetic analysis showed that strain CBA3638^T belongs to the genus Anaerocolumna in the family Lachnospiraceae, and is most closely related to Anaerocolumna cellulosilytica (94.6–95.0%). The DDH value with A. cellulosilytica SN021^T showed 15.0% relatedness. The genome of strain CBA3638^T consisted of one circular chromosome that is 5,500,435 bp long with a 36.7 mol% G?+?C content. The genome contained seven 16S-5S-23S rRNA operons and one antibiotic resistance-related transporter gene (mefA). Quinones were not detected. The predominant cellular fatty acids were C_16:0 and C_14:0 and the polar lipids were diphosphatidylglycerol, phosphatidylcholine, and uncharacterised polar lipids. Based on the polyphasic taxonomic analysis, we propose strain CBA3638^T as a novel species in the genus Anaerocolumna, with the name Anaerocolumna sedimenticola sp. nov. The type strain is CBA3638^T (=?KACC 21652^T?=?DSM 110663^T).

     

A freshwater bacterial strain,Shewanella sp. Lzh-2, isolated from Lake Taihu and its two algicidal active substances,hexahydropyrrolo[1,2-a]pyrazine-1,4-dione and 2, 3-indolinedione

Cyanobacterial blooms have become a serious problem in Lake Taihu during the last 20 years, and Microcystis aeruginosa and Synechococcus sp. are the two dominant species in cyanobacterial blooms of Lake Taihu. A freshwater bacterial strain, Shewanella sp. Lzh-2, with strong algicidal properties against harmful cyanobacteria was isolated from Lake Taihu. Two substances with algicidal activity secreted extracellularly by Shewanella sp. Lzh-2, S-2A and S-2B, were purified from the bacterial culture of strain Lzh-2 using ethyl acetate extraction, column chromatography, and high performance liquid chromatography (HPLC) in turn. The substances S-2A and S-2B were identified as hexahydropyrrolo[1,2-a]pyrazine-1,4-dione and 2, 3-indolinedione (isatin), respectively, based on liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and hydrogen-nuclear magnetic resonance (H-NMR) analyses, making this the first report of their algicidal activity toward cyanobacteria. S-2A (hexahydropyrrolo[1,2-a]pyrazine-1,4-dione) had no algicidal effects against Synechococcus sp. BN60, but had a high level of algicidal activity against M. aeruginosa 9110. The LD50 value of S-2A against M. aeruginosa 9110 was 5.7 μg/ml. S-2B (2, 3-indolinedione) showed a potent algicidal effect against both M. aeruginosa 9110 and Synechococcus sp. BN60, and the LD50 value of S-2B against M. aeruginosa 9110 and Synechococcus sp. BN60 was 12.5 and 34.2 μg/ml, respectively. Obvious morphological changes in M. aeruginosa 9110 and Synechococcus sp. BN60 were observed after they were exposed to S-2A (or S-2B) for 24 h. Approximately, the algicidal activity, the concentration of S-2A and S-2B, and the cell density of Lzh-2 were positively related to each other during the cocultivation process. Overall, these findings increase our knowledge about algicidal substances secreted by algicidal bacteria and indicate that strain Lzh-2 and its two algicidal substances have the potential for use as a bio-agent in controlling cyanobacterial blooms in Lake Taihu.     

Ultrastructure ofDigitalis lanata tissue cultures

Summary The anatomy of twoDigitalis lanata tissue culture strains, S-1 and S-2, has been studied. The cardenolide accumulating cell aggregates consisted of highly vacuolated cells with very lobed nuclei in the periphery and a central part of meristematic cells. Sieve tube elements and companion cells and tracheids were observed in some of the cultures. The effect of gibberellic acid (GA₃) and SAN 9789 on the ultrastructure of the cultures was most apparent as regards the plastids and the amounts of mitochondria and ER. The content of starch seemed to be highest in the cardenolide accumulating strain S-2 grown in darkness (S-2 D) with or without GA3, but considerable amounts were also found in S-1 grown in darkness (S-1 D) with or without GA₃, which did not accumulate cardenolides. The amount of plastoglobuli was increased in S-1 by SAN treatment. It was also higher in S-2 D and S-2 D+GA₃ than in S-1 D and S-1 D+GA₃; i.e., it was high in tissues with blocked carotene synthesis. Many large plastoglobuli were also observed in apparently degenerating cells. The amount of ER seemed relatively high in cardenolide producing cultures. The amount of mitochondria was highly variable, but no correlation with cardenolide accumulation could be found.Abbreviations D dictyosome - ER endoplasmic reticulum - M mitochondrion - N nucleus - P plastid - PG plastoglobule - S starch grain - SC sieve cell - V vacuole - W cell wall - GA₃ gibberellic acid - S-1D strain S-1 cultured in darkness - S-1 L strain S-1 cultured in light - S-2 D strain S-2 cultured in darkness - S-2L8 strain S-2 previously cultured in darkness followed by 8 days in light in the present study - SAN SAN 9789 (Norflurazon) 4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-tolyl)-3(2H)-pyridazinone     

Commonality of self-recognition specificity of S haplotypes between Brassica oleracea and Brassica rapa

We have identified several interspecific pairs of S haplotypes having highly similar SRK and SP11/SCR sequences between Brassica oleracea and Brassica rapa. The recognition specificities of S haplotypes in these pairs were examined with three different methods. Stigmas of interspecific hybrids between an S-32 homozygote in B. oleracea and an S-60 homozygote in B. rapa, which were produced to avoid the interspecific incompatibility between the two species, showed incompatibility to the pollen of an S-8 homozygote in B. rapa and to the pollen of an S-15 homozygote in B. oleracea, while it showed compatibility to the pollen of other S haplotypes, suggesting B. oleracea S-32 and B. rapa S-60 have the same recognition specificity as B. rapa S-8 and B. oleracea S-15. Pollen grains of transgenic S-60 homozygous plants in B. rapa carrying a transgene of SP11-24 from B. oleracea were incompatible to B. rapa S-36 stigma, indicating that B. oleracea S-24 and B. rapa S-36 have the same recognition specificity. Application of the SP11 protein of B. rapa S-41 and S-47 onto the surface of B. oleracea S-64 stigmas and S-12 stigmas, respectively, resulted in the incompatibility reaction to pollen grains of another S haplotype, but application onto the stigmas of other S haplotypes did not, suggesting that B. oleracea S-64 stigmas and S-12 stigmas recognized the B. rapa SP11-41 and SP11-47 proteins as self SP11 proteins, respectively. Besides having evolutionary implications, finding of many interspecific pairs of S haplotypes can provide insight into the molecular mechanism of self-recognition. Comparing deduced amino-acid sequences of SP11 proteins and SRK proteins in the pairs, regions of SP11 and SRK important for self-recognition are discussed.     

Endophytic Bacillus sp. Isolated from the Interior of Balloon Flower Root

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C_15:0 (37%), C_17:0 (5.1%), and iso-C_15:0 (27.7%). DNA G + C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.     

Plasmids in the bacterial assemblage of a dystrophic Lake: Evidence for plasmid-encoded nickel resistance

Sixty-two aerobic bacterial strains isolated from the unproductive dystrophic Lake Skärshultsjön (South Sweden) were screened for plasmids. The lake is considered to be an extreme environment because of its high concentration of persistent but nontoxic humic compounds. One-third of the isolates harbored multiple plasmids usually of similar high molecular weights (>25 Mdal). The plasmid-bearing strains were members of the common aquatic taxaPseudomonas spp.,Acinetobacter sp.,Alcaligenes sp.,Aeromonas/Vibrio group, andEnterobacteriaceae (taxonomy is tentative). The majority of isolates displayed multiple resistance to antibiotics and heavy metals. Some of them were capable of degrading aromatic compounds. Three isolates were chosen for curing experiments. Only strain S-68, anAlcaligenes sp., could be cured of one of its two plasmids. It harbored the two cryptic plasmids pQQ32 and pQQ70 of 32 and ca. 70 Mdal, and the latter was segregated during ethidium bromide treatment. Parental strain S-68 was capable of degrading some of nonchlorinated phenolic compounds and displayed resistance to a broad spectrum of antibiotics and the heavy metals Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, and Hg²⁺. Derivative strain S-68-41 lost its resistance to nickel, suggesting segregated plasmid PQQ70 coded for nickel resistance. Transformation experiments to restore nickel resistance in the cured derivative strain were not successful.     

Microaerobic enrichment of benzene-degrading bacteria and description of Ideonella benzenivorans sp. nov., capable of degrading benzene,toluene and ethylbenzene under microaerobic conditions

In the present study, the bacterial community structure of enrichment cultures degrading benzene under microaerobic conditions was investigated through culturing and 16S rRNA gene Illumina amplicon sequencing. Enrichments were dominated by members of the genus Rhodoferax followed by Pseudomonas and Acidovorax. Additionally, a pale amber-coloured, motile, Gram-stain-negative bacterium, designated B7^T was isolated from the microaerobic benzene-degrading enrichment cultures and characterized using a polyphasic approach to determine its taxonomic position. The 16S rRNA gene and whole genome-based phylogenetic analyses revealed that strain B7^T formed a lineage within the family Comamonadaceae, clustered as a member of the genus Ideonella and most closely related to Ideonella dechloratans CCUG 30977^T. The sole respiratory quinone is ubiquinone-8. The major fatty acids are C_16:0 and summed feature 3 (C_16:1 ω7c/iso-C_15:0 2-OH). The DNA G?+?C content of the type strain is 68.8?mol%. The orthologous average nucleotide identity (OrthoANI) and in silico DNA–DNA hybridization (dDDH) relatedness values between strain B7^T and closest relatives were below the threshold values for species demarcation. The genome of strain B7^T, which is approximately 4.5?Mb, contains a phenol degradation gene cluster, encoding a multicomponent phenol hydroxylase (mPH) together with a complete meta-cleavage pathway including a I.2.C-type catechol 2,3-dioxygenase (C23O) gene. As predicted by the genome, the type strain is involved in aromatic hydrocarbon-degradation: benzene, toluene and ethylbenzene are degraded aerobically and also microaerobically as sole source of carbon and energy. Based on phenotypic characteristics and phylogenetic analysis, strain B7^T is a member of the genus Ideonella and represents a novel species for which the name Ideonella benzenivorans sp. nov. is proposed. The type strain of the species is strain B7^T (=?LMG 32,345^T?=?NCAIM B.02664^T).

     

Effect of Rifampicin and Uracil Depletion on Bacteriophage ϕX174 DNA Synthesis in an E. coli dnaHts Mutant

The fluorescent antibody technique was used to trace an inoculated Nocardia erythropolis strain which was capable of rapidly degrading phthalate esters in soil column and activated sludge systems. The reaction of antibody to Nocardia erythropolis S-1 was highly strain specific, i. e., only one of twelve other strain of N. erythropolis was stained with this fluorescent antibody. All other species of Nocardia and other genera of bacteria and a strain of Candida were not stained. Using this technique it was demonstrated that N. erythropolis S-1 inoculated into activated sludge and soil column systems was successfully distinguished from many other microorganisms in mixed culture systems, and the distribution of this strain was appreciated.     

Metabolism of the Fungicide S-n-Butyl S′-p-tert-Butylbenzyl N-3-Pyridyldithiocarbonimidate (S-1358) in Rats

S-1358 was rapidly absorbed, metabolized and readily excreted via urine and feces from orally dosed rats. Excretion of radioactivity was almost complete within 4 days. The radioactivity was distributed mainly in stomach, intestines, liver and kidneys. It seems that S-1358 and its metabolites do not persist in organs and tissues following a single oral dosing.

Major urinary metabolites of the benzyl-labeled S-1358 were p-(1,1-dimethyl-2-hydroxyethyl)benzyl methyl sulfide [B], p-(1,1-dimethyl-2-hydroxyethyl)benzyl methyl sulfone [A], p-(1-methyl-1-carboxylethyl)benzyl methyl sulfide [D], p-(1-methyl-1-carboxylethyl)benzyl methyl sulfone [C] and their glucuronide conjugates. Fecal metabolites were S-n-butyl S′-(1, 1-dimethyl-2-hydroxyethyl)benzyl N-3-pyridyldithiocarbonimidate [MR], A, B, C and D. These metabolites were also found in the bile. The pyridine-labeled S-1358 gave rise to 2-(3′-pyridylimino)-4-carboxylthiazolidine [HM] and 3-aminopyridine [AP] in the urine, and MR and AP in the feces. Intact S-1358 was a major component of the fecal radioactivity.     

<Emphasis Type="Italic">Halorubrum cibi</Emphasis> sp. nov., an extremely halophilic archaeon from salt-fermented seafood

Strain B31^T is a Gram-staining-negative, motile, and extremely halophilic archaeon that was isolated from salt-fermented seafood. Its morphology, physiology, biochemical features, and 16S rRNA gene sequence were determined. Phylogenetic analysis of its 16S rRNA gene sequence and composition of its major polar lipids placed this archaeon in the genus Halorubrum of the family Halobacteriaceae. Strain B31^T showed 97.3, 97.2, and 96.9 % 16S rRNA similarity to the type strains of Halorubrum alkaliphilum, Hrr. tibetense, and Hrr. vacuolatum, respectively. Its major polar lipids were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and sulfated diglycosyl diether (S-DGD). Genomic DNA from strain B31^T has a 61.7 mol% G+C content. Analysis of 16S rRNA gene sequences, as well as physiological and biochemical tests, identified genotypic and phenotypic differences between strain B31^T and other Halorubrum species. The type strain of the novel species is B31^T (=JCM 15757^T =DSM 19504^T).     

Studies on Temperate Phages of Lactobacillus salivarius

Comparative studies were carried out on the mode of action of the defective temperate phage 208 against the homologous lysogenic strain S-208 and a nonlysogenic strain S-161 of Lactobacillus salivarius. Treatment of both strains with phage 208 led to a specific inhibition of protein synthesis, cell killing without any reversion of protein synthesis, and of viable counts by treatment with trypsin. Killing action of phage 208 followed a single hit kinetics for nonlysogenic S-161 and S-208 No. 006, which is a cured strain of S-208, whereas, two to five hit kinetics was obtained for lysogenic S-208. Phage particles exposed to ultraviolet light (5.7 kergs/cm²) also killed S-161 with a single hit kinetics and S-208 with a 3-hit kinetics. However, the kinetics of killing approached a single hit when the protein synthesis of S-208 was inhibited by chloramphenicol prior to the phage addition. Based upon these results, the possible mechanisms of immunity breakdown and of subsequent cell killing were discussed.     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

CAPS and SCAR markers linked to maintenance of self-incompatibility developed from SP11 in Brassica napus L.

Difficulty in propagating self-incompatible lines on a large scale limits the utilization of self-incompatibility in Brassica napus. The self-incompatible line S-1300 and its maintainer Bing409 were used in this study to develop molecular markers linked to the maintenance for the self-incompatibility of S-1300. The maintenance of Bing409 is controlled by one recessive gene. SLG-specific primer pairs PS5/PS15 and PS3/PS21 cannot be used to discriminate the S haplotypes of Bing409 and S-1300. BrSRK-60-based primer pair SRKa-L and SRKa-R produced one band in S-1300, but no band in Bing409. BrSP11-60-based primer pair SP11a-L and SP11a-R gave rise to one band in S-1300 and Bing409, but their length was different. Compared with SP11-S-1300, SP11-Bing409 had two deletions of 2 and 9 bp. One co-dominant cleaved amplified polymorphic sequence marker was developed; two dominant sequence characterized amplified region markers linked to the maintenance were developed on the 9 bp deletion. The markers co-segregated with self-incompatibility phenotypes in S-1300 × Bing409 F₂, two BC₁ and eight BC₁F₂ populations. We have shown a way to develop PCR markers linked to the S haplotype of B. napus, which could be very helpful for marker-assisted selection in B. napus hybrid breeding.     

Production of thaxtomin a by two species ofStreptomyces causing potato scab

A total of nine isolates of streptomycetes were isolated from scab lesions on potato tubers. Five of nine isolates were pathogenic on potato minitubers. Four of the pathogenic isolates produced thaxtomin A (ThxA) in infected tuber tissues. The lesion surface areas inducing ThxA were highest in treatment of the minitubers with an extract of OMB inoculated with S-66 and S-67, intermediate with that inoculated with S-64 and lowest with S-63. The pathogenic isolates were identified by gray aerial mycelia, melanin pigment productivity, the type of spore chain morphology and carbon utilization asS. scabies strains S-63, S-64 and S-68, andS. acidiscabies strains S-66 and S-67. Strains S-63 and S-64 produced 0.65 and 1.60 mg ThxA per L of OMB, respectively, strains S-66 and S-67 producing similar amounts,viz. 2.36 and 2.10 mg/L, respectively. The optimal temperature for production (by both species) was 28 °C. Production of ThxA byS. scabies strain S-64 andS. acidiscabies strain S-66 was suppressed at least 50-fold at 0.5 and 0.3 % of glucose, respectively. Fructose enhanced the production by both species.     

Streptomyces blattellae,a novel actinomycete isolated from the in vivo of a Blattella germanica

During a screening for novel and useful actinobacteria in desert animal, a new actinomycete was isolated and designated strain TRM63209^T. The strain was isolated from in vivo of a Blattella germanica in Tarim University in Alar City, Xinjiang, north-west China. The strain was found to exhibit an inhibitory effect on biofilm formation by Candida albicans ATCC 18,804. The strain was observed to form abundant aerial mycelium, occasionally twisted and which differentiated into spiral spore chains. Spores of TRM63209^T were observed to be oval-shaped, with a smooth surface. Strain TRM63209^T was found to grow optimally at 28 °C, pH 8 and in the presence of 1% (w/v) NaCl. The whole-cell sugars of strain TRM63209^T were rhamnose ribose, xylose, mannose, galactose and glucose, and the principal polarlipids were found to be diphosphatidylglycerol, phos-phatidylethanolamine, phosphatidylcholine, phosphatidylinositol mannoside, phosphatidylinositol and an unknown phospholipid(L). The diagnostic cell wall amino acid was identified as LL-diaminopimelic acid. The predominant menaquinone was found to be MK-9(H6) (14.64%), MK-9(H2) (19.65%), MK-9(H8) (22.34%), MK-10(H2) (25.37%). The major cellular fatty acids were identified as iso-C16:0, 16:0, anteiso-C15:0, anteiso-C17:0, iso-C15:0 and Sum in Feature 3. Analysis of the 16S rRNA sequence showed that strain TRM63209^T exhibits high sequence similarity to Streptomyces bungoensis strain DSM 41781^T 98.20%. A multi-locus sequence analysis of five house-keeping genes (atpD, gyrB, rpoB, recA and trpB) and phylogenomic analysis also illustrated that strain TRM63209^T should be assigned to the genus Streptomyces. The DNA G?+?C content of the strain was determined to be 70.2 mol%. Average nucleotide identity (ANI) between strain TRM63209^T and S. bungoensis DSM 41781^T, Streptomyces phyllanthi PA1-07^T, Streptomyces longwoodensis DSM 41677^T and Streptomyces caeruleatus NRRL B-24802^T were 82.76%, 82.54%, 82.65%, 84.02%, respectively. Digtal DNA-DNA (dDDH) hybridization were 26.30%, 25.10%, 26.20%, 29.50%, respectively. Therefore, it is concluded that strain TRM63209^T represents a novel species of the genus Streptomyces, for which the name Streptomyces blattelae is proposed. The type strain is TRM63209^T (CCTCC AA 2018093^T?=?LMG 31,403?=?TRM63209^T).

     

Genetic variants of the Bombyx mori silkworm encoding sericin proteins of different lengths

A variant sericin polypeptide originally found by acid gel electrophoresis in the Nd-s mutant strain of the silkworm, Bombyx mori, has been analyzed genetically. The vriant polypeptide (called S-2^v) is encoded by a gene which behaves as a codominant allele of the gene encoding the standard S-2 sericin polypeptide. Linkage analysis locates these alleles at 0.0 map unit on chromosome 11. SDS-polyacrylamide gel electrophoresis shows that the molecular weight of the S-2^v variant polypeptide is lower by approximately 62,500 than that of the S-2 polypeptide. Amino acid analysis indicates that the two sericin polypeptides have similar compositions. These results are consistent with the idea that the variant allele arose by deletion within the S-2 coding sequence in the Src-2 gene locus as the result of unequal recombination.     

<Emphasis Type="Italic">Calculibacillus koreensis</Emphasis> gen. nov., sp. nov., an anaerobic Fe(III)-reducing bacterium isolated from sediment of mine tailings

A strictly anaerobic bacterium, strain B5^T, was isolated from sediment of an abandoned coal mine in Taebaek, Republic of Korea. Cells of strain B5^T were non-spore-forming, straight, Gram-positive rods. The optimum pH and temperature for growth were pH 7.0 and 30°C, respectively, while the strain was able to grow within pH and temperature ranges of 5.5–7.5 and 25–45°C, respectively. Growth of strain B5^T was observed at NaCl concentrations of 0 to 6.0% (w/v) with an optimum at 3.0–4.0% (w/v). The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, an unknown phospholipid and three unknown polar lipids. Strain B5^T grew anaerobically by reducing nitrate, nitrite, ferric-citrate, ferric-nitrilotriacetate, elemental sulfur, thiosulfate, and anthraquinone-2-sulfonate in the presence of proteinaceous compounds, organic acids, and carbohydrates as electron donors. The isolate was not able to grow by fermentation. Strain B5^T did not grow under aerobic or microaerobic conditions. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B5^T is most closely related to the genus Tepidibacillus (T. fermentans STGH^T; 96.3%) and Vulcanibacillus (V. modesticaldus BR^T; 94.6%). The genomic DNA G+C content (36.9 mol%) of strain B5^T was higher than those of T. fermentans STGH^T (34.8 mol%) and V. modesticaldus BR^T (34.5 mol%). Based on its phenotypic, chemotaxonomic, and phylogenetic properties, we describe a new species of a novel genus Calculibacillus, represented by strain B5^T (=KCTC 15397^T =JCM 19989^T), for which we propose the name Calculibacillus koreensis gen. nov., sp. nov.     

Production of (S)-(+)-citramalic acid from itaconic acid by resting cells of Alcaligenes denitrificans strain MCI2775

(S)-(+)-Citramalic-acid-producing activity in microorganisms was studied with resting cells in a reaction mixture containing itaconic acid. Itaconic-acid-utilizing bacteria were found to produce (S)-(+)-citramalic acid from itaconic acid. The strain, which showed the best productivity among those studied, was identified taxonomically as Alcaligenes denitrificans strain MCI2775. (S)-(+)-Citramalic acid produced by this strain was present in a 99.9% enantiometric excess. The culture and reaction conditions for the production were optimized for this strain. Addition of Mn²⁺, d-pantothenic acid and l-leucine to the culture medium enhanced the (S)-(+)-citramalic acid-producing activity. Under optimal conditions, 27 g (S)-(+)-citramalic acid/l was produced in 30 h. The yield to itaconic acid added was 69.0 mol%. Correspondence to: Y. Asano     

Biosynthesis of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate) copolyesters with a high molar fraction of 3-hydroxyvalerate by an insect-symbiotic <Emphasis Type="Italic">Burkholderia</Emphasis> sp. IS-01

Burkholderia sp. IS-01 capable of biosynthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)] copolyesters with a high molar fraction of 3HV was isolated from the gut of the adult longicorn beetle, Moechotypa diphysis. The strain IS-01 was relatively tolerant to high concentrations of levulinic acid and accumulated a poly(13.5 mol% 3HB-co-86.5 mol% 3HV) copolyester when cultivated on a mixture of gluconate (20 g/L) and levulinic acid (12.5 g/L). In this case, the content of the copolyester in the cells was approximately 60.0%. The compositions of the copolyesters were easily regulated by altering the molar ratio of gluconate and levulinic acid in the medium. The organism was found to possess a class I PHA synthase (PhaC) gene (1,881 bp) that encodes a protein with a deduced molecular mass of 68,538 Da that consists of 626 amino acids. The PhaC of this organism was most similar to that of B. cenocepacia PC184 (92% similarity).