<Emphasis Type="Italic">Pseudonocardia bannaensis</Emphasis> sp. nov., a novel actinomycete isolated from the surface-sterilized roots of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

During the course of our research on new actinobacterial sources, a novel actinomycete strain YIM 63101^T was isolated from the surface-sterilized roots of Artemisia annua L. collected from Xishuangbanna, Yunnan province, south-west China and characterized by using a polyphasic approach. The strain formed well-differentiated aerial and substrate mycelia. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 63101^T belongs to the genus Pseudonocardia, with highest similarity to “Pseudonocardia artemisiae YIM 63587^T” (99.4%). Sequence similarities between strain YIM 63101^T and the other Pseudonocardia species ranged from 97.0 (Pseudonocardia saturnea IMSNU 20052^T) to 94.0% (Pseudonocardia compacta IMSNU 20111^T). The chemotaxonomic characteristics, such as cell wall diaminopimelic acid, whole-cell sugars, fatty acid components and the major menaquinones suggested that the organism belonged to the genus Pseudonocardia. The G + C content of the genomic DNA was 69.4 mol%. Based on comparative analysis of physiological, biochemical and chemotaxonomic data, including low DNA–DNA hybridization results, it is proposed that strain YIM 63101^T represents a novel species of the genus Pseudonocardia, named Pseudonocardia bannaensis sp. nov. The type strain is YIM 63101^T (= CCTCC AA 208077 ^T = DSM 45300^T).     

Isolation and identification of 2alpha,25-dihydroxyvitamin D3, a new metabolite from Pseudonocardia autotrophica 100U-19 cells incubated with Vitamin D3

Pseudonocardia autotrophica converted Vitamin D(3) to 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3). The hydroxylation of Vitamin D(3) with P. autotrophica was enhanced by the addition of cyclodextrin. In this microbial hydroxylation, a new Vitamin D(3) metabolite was observed in the reaction mixture of P. autotrophica and Vitamin D(3), and was isolated in a pure form by several steps of chromatography. The structure of the new metabolite was determined to be 2alpha,25-dihydroxyvitamin D(3) by UV, NMR and mass spectroscopic analyses. Biological evaluation of the new metabolite was conducted by means of several experiments.     

Phylogenetic analysis of the genera Pseudonocardia and Actinobispora based on 16S ribosomal DNA sequences

The 16S rDNA sequences of 11 strains, nine type strains of validated Pseudonocardia species and Actinobispora yunnanensis, and two strains of unnamed Pseudonocardia species, were determined and compared with those of representatives of the family Pseudonocardiaceae. The phylogenetic analysis indicated that all of the validated species of the genera Pseudonocardia and Actinobispora consistently formed a monophyletic unit and separated well from the other genera of the family Pseudonocardiaceae. One unnamed Pseudonocardia strain was related to members of the genus Pseudonocardia, whereas the other unnamed Pseudonocardia strain formed a distinct clade within the radiation of the genus Amycolatopsis.     

Detection and identification of Phytophthora alni

In 2004 Brasier et al. described new species--Phytophthora alni, which was especially aeggressive to alder. Now, this Phytophthora disease of alder is widely distributed in Europe as well as in Poland. In this research note we report on identification and detection of P. alni from water and soil samples using PCR method with species-specific primers. Dilution series of P. alni zoospore were used to test the potential sensitivity of the PCR detection methods. Zoospores of P. alni were produced by flooding of 1-week-old Frozen Pea Medium (FPM) cultures in Petri dishes with 30 ml distilled water. The dishes were incubated at 20 degrees C. After 5 days, sporangial production was checked using a binocular microscope and plates were placed at 4 degrees C for 1 h to enhance zoospore release. Zoospores were counted under the microscope using Burker's cabin. A dilution series of zoospores ranging from 5 to 5000 per 200 microl was prepared in autoclaved distilled water and in 1 g samples of autoclaved soil. DNA was extracted from artificially infected water and soil, and purified using the CleanUp Kit (A&A Biotechnology). Zoospores of P. alni in the water were detected by PCR in 5 x 10(3), 5 x 10(2), 5 x 10(1) concentrations. In case of detecting spores in the artificially infected soil it succeeded only for two highest concentrations, i.e. 5 x 10(3), 5 x 10(2) and only when the DNA was additionally purified.     

中国木层孔菌属三个新记录种

报道了木层孔菌属Phellinus 3个中国新记录种。赤杨木层孔菌Phellinus alni典型特征为菌盖具有较宽的同心环带和清晰的密集环纹菌核;黑木层孔菌P. nigricans具有较大的担孢子;东方木层孔菌P. orienticus具有平伏的子实体,担孢子相对较小。对这3个种进行了详细的描述和显微结构绘图。     

Pectinatus portalensis nov. sp., a relatively fast-growing, coccoidal, novel Pectinatus species isolated from a wastewater treatment plant

The genus Pectinatus is currently composed by two species, Pectinatus cerevisiiphilus and Pectinatus frisingensis , both asociated with beer spoilage. This study describes a novel isolate (strain B6) retrieved from a wastewater treatment plant collecting residues from a large number of wineries. Based on similarity analysis of 16S rRNA gene sequences, strain B6 belongs to the genus Pectinatus . Strain B6 is a strict anaerobe like other Pectinatus species and it presents non-motile, coccoid cells showing a slight oval shape. Strain B6 shows marked physiological differences with other Pectinatus species both in fatty acid composition and carbon source utilization. The most abundant fatty acids found in strain B6 were 18:1 (42.8%) and 16:0 (18.3%) representing a total of over 61% of fatty acids in this microorganism while these fatty acids represented 41.3% in P. cerevisiiphilusT and 2.4% in P. frisingensisT of their total. Fatty acid 15:0 was not significant in strain B6 and represented 28.6% and 13.3% for P. cerevisiiphilusT and P. frisingensisT, respectively. Strain B6 showed a faster growth rate and higher optimum temperature than its relatives P. cerevisiiphilus and P. frisingensis . Strain B6, P. cerevisiiphilus and P. frisingensis could be clearly differentiated by acid production tests from substrates such as esculine and gluconate, and the lack of acid production from rhamnose and fucose among others. G+C mol% content in strain B6 is 36.5%. Based on genotypic and phenotypic differences, strain B6 is proposed as a novel Pectinatus species, P. portalensis nov. sp. Both strain B6 and the two described species of Pectinatus grow on beers and wines. These results provide insights about the origin and reservoirs of Pectinatus species and spoiling alcoholic beverages.     

Pseudonocardia nantongensis sp. nov., a novel endophytic actinomycete isolated from the coastal halophyte Tamarix chinensis Lour

A novel isolate, designated strain KLBMP 1282^T was isolated from the surface-sterilized leaves of a coastal halophyte Tamarix chinensis Lour., collected from Nantong, Jiangsu Province, east of China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that this strain belongs to the genus Pseudonocardia, being most closely related to Pseudonocardia kongjuensis LM 157^T (98.33 %), Pseudonocardia autotrophica IMSNU 20050^T (97.77 %), Pseudonocardia endophytica YIM 56035^T (97.63 %), Pseudonocardia ammonioxydans H9 ^T (97.62 %) and Pseudonocardia compacta IMSNU 20111^T (97.56 %); similarity to other type strains of the genus Pseudonocardia was <97.5 %. Chemotaxonomic data confirmed the affiliation of strain KLBMP 1282^T to the genus Pseudonocardia. Strain KLBMP 1282^T contained MK-8(H₄) as the predominant ubiquinone and iso-C_16:0 as the major fatty acid. The polar lipids detected in strain KLBMP 1282^T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides, one unknown phospholipid and four unknown glycolipids. The DNA G + C content of strain KLBMP 1282^T was 73.1 mol %. The results of DNA–DNA hybridizations and the phylogenetic analysis, together with the phenotypic and biochemical tests, allowed the differentiation of strain KLBMP 1282^T from strains of other recognized Pseudonocardia species. Therefore, strain KLBMP 1282^T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia nantongensis sp. nov. is proposed. The type strain is KLBMP 1282^T (=KCTC 29053^T = NBRC 108677^T).     

Psychrophilic Planococcus maitriensis sp.nov. from Antarctica

An orange pigmented bacterium, S1, was isolated from a cyanobacterial mat sample collected in the vicinity of Schirmacher Oasis, Maitri, the Indian station, in Antarctica. The bacterium is Gram-positive and possesses all the characteristics of the genus Planococcus. It is non-sporulating, motile and has A4alpha type peptidoglycan, MK-7 and MK-8 as the major menaquinones and anteiso-C(15:0) as the major fatty acid. Based on the phylogenetic characteristics, the bacterium S1 is identified as a close relative of Planococcus citreus with which it shares 98.12% similarity at the 16S rRNA gene level but exhibits a low similarity of 52% at the whole genome level. Apart from the above major differences, S1 also exhibits phenotypic differences with Planococcus citreus and other members of the genus Planococcus. Based on these differences, the bacterium S1 is identified as a new species of the genus Planococcus for which the name Planococcus maitriensis is proposed. The type strain of Planococcus maitriensis is S1(T) (= MTCC 4827; DSM 15305).     

Streptomyces daliensis sp. nov. from soil

A novel actinomycete strain YIM 31724^T was isolated from a soil sample collected from Dali, Yunnan Province, People’s Republic of China. The strain is characterized by white to yellow white aerial mycelia, spiral spore chains and smooth spore surface. The cell wall of strain YIM 31724^T contained LL-diaminopimelic acid (A₂pm) and traces of meso-A₂pm. Whole-cell hydrolysates contained mainly glucose and small amounts of galactose and xylose. The menaquinones were MK-9(H₆) (31%) and MK-9(H₈) (69%). Phosphatidylethanolamine was the diagnostic phospholipid. The DNA G+C content of strain YIM 31724^T was 67.2 mol%. Phylogenetic analysis indicated that the strain belongs to the genus Streptomyces, with highest similarity to Streptomyces rimosus subsp. rimosus JCM 4667^T (rRNA gene sequence similarity value of 98.9%) and Streptomyces erumpens DSM 40941^T (rRNA gene sequence similarity value of 98.7%). Based on its phenotypic and genotypic characteristics, including low DNA–DNA hybridization results, strain is proposed as the type strain of a novel species, Streptomyces daliensis sp. nov.     

<Emphasis Type="Italic">Streptomyces tritolerans</Emphasis> sp. nov., a novel actinomycete isolated from soil in Karnataka,India

A Gram-positive, nonmotile, moderately halophilic, alkali and thermotolerant strain designated DAS 165(T), was isolated from a dry land soil sample from the Gulbarga region, Karnataka province, India. The isolate produced yellow substrate mycelia and gray aerial mycelia on most tested media. Strain DAS 165(T) showed growth in the presence of 5 to 7% NaCl and at 45 degrees C. The DNA G + C content was 69.7%. 16S rRNA gene sequence analysis together with these characteristics consistently assigned strain DAS 165(T) to the genus Streptomyces. The 16S rRNA gene sequence analysis revealed that strain DAS 165(T) was most closely related to S. tendae ATCC 19812(T) (D 63873) with a sequence similarity of 99.6% (three nucleotide differences out of 1,517). Strain DAS 165(T) formed a distinct clade based on analysis of the almost complete sequence and 120-nucleotide variable gamma region of the 16S rRNA gene. Despite the high sequence similarity, strain DAS 165(T) was phenotypically different from S. tendae ATCC 19812(T). DNA-DNA hybridization between these strains was 47% showing that strain DAS 165(T) is a distinct genomic species. Phenetic and genetic results support the classification of strain DAS 165(T) as a new species, for which the name S. tritolerans is proposed, with strain DAS 165(T) as the type strain (=DSM 41899(T )= CCTCCAA 206013(T)).     

Distribution and expression of elicitin genes in the interspecific hybrid oomycete Phytophthora alni

Phytophthora alni subsp. alni, P. alni subsp. multiformis, and P. alni subsp. uniformis are responsible for alder disease in Europe. Class I and II elicitin gene patterns of P. alni subsp. alni, P. alni subsp. multiformis, P. alni subsp. uniformis, and the phylogenetically close species P. cambivora and P. fragariae were studied through mRNA sequencing and 3' untranslated region (3'UTR)-specific PCRs and sequencing. The occurrence of multiple 3'UTR sequences in association with identical elicitin-encoding sequences in P. alni subsp. alni indicated duplication/recombination events. The mRNA pattern displayed by P. alni subsp. alni demonstrated that elicitin genes from all the parental genomes are actually expressed in this allopolyploid taxon. The complementary elicitin patterns resolved confirmed the possible involvement of P. alni subsp. multiformis and P. alni subsp. uniformis in the genesis of the hybrid species P. alni subsp. alni. The occurrence of multiple and common elicitin gene sequences throughout P. cambivora, P. fragariae, and P. alni sensu lato, not observed in other Phytophthora species, suggests that duplication of these genes occurred before the radiation of these species.     

Structural analysis and biosynthetic engineering of a solubility-improved and less-hemolytic nystatin-like polyene in Pseudonocardia autotrophica

Polyene antibiotics such as nystatin are a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain an approximately 125.7-kb region of contiguous DNA with a total of 23 open reading frames, which are involved in the biosynthesis and regulation of a structurally unique polyene natural product named NPP. Here, we report the complete structure of NPP, which contains an aglycone identical to nystatin and harbors a unique di-sugar moiety, mycosaminyl-(α1-4)-N-acetyl-glucosamine. A mutant generated by inactivation of a sole glycosyltransferase gene (nppDI) within the npp gene cluster can be complemented in trans either by nppDI-encoded protein or by its nystatin counterpart, NysDI, suggesting that the two sugars might be attached by two different glycosyltransferases. Compared with nystatin (which bears a single sugar moiety), the di-sugar containing NPP exhibits approximately 300-fold higher water solubility and 10-fold reduced hemolytic activity, while retaining about 50% antifungal activity against Candida albicans. These characteristics reveal NPP as a promising candidate for further development into a pharmacokinetically improved, less-cytotoxic polyene antifungal antibiotic.     

南海深海新颖低温脂肪酶的克隆、表达及酶学性质鉴定

从南海深海放线菌Pseudonocardia antitumoralis SCSIO 01299中克隆一个脂肪酶基因 lipaseB5。lipaseB5 基因片段大小为972bp,编码的蛋白质具有323个氨基酸残基并与Pseudonocardia sp. HH130629-09中假定脂肪酶有98%的同源性。以pET28a(+)为载体,将重组质粒转化到E. coli BL21(DE3)中,实现lipaseB5高效表达,并利用Ni-NTA 亲和层析纯化lipaseB5。LipaseB5最适反应底物为对硝基苯酚癸酸酯(p-NPD),最适温度为30℃,最适反应pH为7.5。LipaseB5催化p-NPD水解反应的活性达到140.14U/mg,V_max和K_m分别为109.8μmol/min、0.976mmol/L,催化橄榄油的活力为32.019 7U/mg。LipaseB5在pH7.5~8.5保持良好的pH稳定性;在10~20℃低温下保持较高的酶活力,在10~40℃内具有温度稳定性。LipaseB5对大部分金属离子有很好的耐受性,低浓度的Li⁺、Mg²⁺对该酶活性有促进作用。LipaseB5对高浓度的NaCl有很好的耐受性,在100mmol/L的NaCl存在下,酶活性大约保持在103%。LipaseB5对多种短链醇类有机溶剂和表面活性剂有很好的耐受性,TritonX-100、Tween-80和Tween-20对lipaseB5酶有激活作用。     

Enterobacter radicincitans sp. nov., a plant growth promoting species of the family Enterobacteriaceae

A plant growth promoting bacterial isolate (D5/23T) from the phyllosphere of winter wheat, able to fix atmospheric nitrogen and to produce auxines and cytokinins was investigated in a polyphasic taxonomy approach. Phylogenetic analyses using the 16S rRNA gene sequence of the strain clearly indicated that the strain belonged to the family Enterobacteriaceae, most closely related to Enterobacter cloacae with 99.0% and Enterobacter dissolvens with 98.5% sequence similarity. Phylogenetic analysis derived from the sequence of the rpoB gene showed the highest sequence similarities to Enterobacter cowanii (93.0%) but supported the distinct position of strain D5/23T. The isolate produced a fatty acid pattern typical for members of the family Enterobacteriaceae. On the basis of the phylogenetic analyses, DNA-DNA hybridizations, and the unique physiological and biochemical characteristics, we propose that strain D5/23T represents a new species of the genus Enterobacter for which we propose the name Enterobacter radicincitans sp. nov.     

Platycopia compacta n. sp., the Second Species of Platycopioida (Crustacea: Copepoda) in the Indo-Pacific Region, with Remarks on Development, Feeding, Swimming, and Zoogeography

Platycopia compacta new species (Copepoda: Platycopioida) is described from the hyperbenthic zone in the Tokara Islands, southern Japan. This is the second species of the order in the Indo-Pacific region, and the eighth species of the genus. The present new species is distinguishable from the closely related P. inornata from the Bahamas by (1) the formation of allobasis in the antenna; (2) two unequal setae on the basal exite of the maxillule; (3) the intermaxillipedal process with only 3 pairs of prominences at tip; (4) the fusion of the coxa and basis in leg 3; and (5) the fusion of the second and third endopod segments in female legs 3-5. Copepodid stages III, V, and VI are compared with those of P. orientalis collected from Okinawa, and differ in the developmental patterns of the antennule and legs. The presence of an intermaxillipedal process and gut content analysis revealed that Platycopia is carnivorous. The armature of legs 2-5 of the hyperbenthic genus Platycopia is stouter than that of cavernicolous platycopioids, which indicates that Platycopia may penetrate into loose sediment, whereas the latter group might be more adapted to pelagic life. The horizontal distribution of the genus Platycopia apparently exhibits a full Tethyan track.     

Cetobacterium somerae sp. nov. from human feces and emended description of the genus Cetobacterium

Phenorypic and phylogenetic studies were performed on four isolates of an unidentified gram-negative, microaerotolerant, non-spore-forming, rod-shaped bacterium isolated from the feces of children. The unknown organism was bile resistant and produced acetic acid as the major end product of metabolism of peptides and carbohydrates. It possessed a low DNA G + C content of 31 mol %. Comparative 16S rRNA gene sequencing demonstrated that the four isolates were phylogenetically identical (100% 16S rRNA sequence similarity) and represent a hitherto unknown sub-line within the genus Cetobacterium. The novel bacterium displayed approximately 5% sequence divergence with Cetobacterium ceti, and can be readily distinguished from the latter by physiological and biochemical criteria. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown fecal bacterium be classified in the genus Cetobacterium, as Cetobacterium somerae sp. nov. The proposed type strain of Cetobacterium somerae is WAL 14325(T) (ATCC BAA-474(T) = CCUG 46254T).     

Specificity in the symbiotic association between fungus-growing ants and protective Pseudonocardia bacteria

Fungus-growing ants (tribe Attini) engage in a mutualism with a fungus that serves as the ants' primary food source, but successful fungus cultivation is threatened by microfungal parasites (genus Escovopsis). Actinobacteria (genus Pseudonocardia) associate with most of the phylogenetic diversity of fungus-growing ants; are typically maintained on the cuticle of workers; and infection experiments, bioassay challenges and chemical analyses support a role of Pseudonocardia in defence against Escovopsis through antibiotic production. Here we generate a two-gene phylogeny for Pseudonocardia associated with 124 fungus-growing ant colonies, evaluate patterns of ant-Pseudonocardia specificity and test Pseudonocardia antibiotic activity towards Escovopsis. We show that Pseudonocardia associated with fungus-growing ants are not monophyletic: the ants have acquired free-living strains over the evolutionary history of the association. Nevertheless, our analysis reveals a significant pattern of specificity between clades of Pseudonocardia and groups of related fungus-growing ants. Furthermore, antibiotic assays suggest that despite Escovopsis being generally susceptible to inhibition by diverse Actinobacteria, the ant-derived Pseudonocardia inhibit Escovopsis more strongly than they inhibit other fungi, and are better at inhibiting this pathogen than most environmental Pseudonocardia strains tested. Our findings support a model that many fungus-growing ants maintain specialized Pseudonocardia symbionts that help with garden defence.