Streptacidiphilus jiangxiensis sp. nov., a novel actinomycete isolated from acidic rhizosphere soil in China

The taxonomic position of three acidophilic actinomycetes isolated from acidic rhizosphere soil was established using a polyphasic approach. The morphological and chemical properties of the isolates were found to be consistent with their assignment to the genus Streptacidiphilus. Almost complete 16S rRNA gene sequences determined for the strains were aligned with corresponding sequences of representatives of the genera Kitasatospora, Streptacidiphilus and Streptomyces and phylogenetic trees inferred using three tree-making algorithms. The organisms formed a distinct subclade within the Streptacidiphilus 16S rRNA gene tree. They also shared nearly identical phenotypic profiles and rep-PCR fingerprint patterns that readily distinguished them from representatives of the established species of Streptacidiphilus. It is evident from the genotypic and phenotypic data that the three isolates form a new species in the genus Streptacidiphilus. The name proposed for this new species is Streptacidiphilus jiangxiensis, the type strain is isolate 33214T (= AS 4.1857T = JCM 12277T).     

嗜酸丝状放线菌的选择性分离与多样性

摘要:【目的】针对酸性土壤中的嗜酸丝状放线菌,建立有效的选择性分离方法,并了解其多样性。【方法】用不同的样品预处理方式和分离培养基,并添加不同的抑制剂进行分离;根据放线菌的菌落数和出菌率确定最佳分离方法组合。采用最佳分离方法对从江西采集的17份酸性土壤样品进行分离;根据培养特征对分离菌株进行分群,进一步通过对各类群的显微形态观察和pH梯度生长实验确定代表菌株;对代表菌株进行16S rRNA基因序列分析研究其多样性。【结果】嗜酸丝状放线菌的最佳分离方法为：土壤样品经分散差速离心预处理后,涂布添加了放线菌酮、制霉菌素和萘啶酮酸（各50 mg/L）的GTV培养基。用此方法共分离到放线菌369株,归为10个不同的颜色类群,其中6.6%为严格嗜酸放线菌,72.4%为中度嗜酸放线菌,21.0%为耐酸放线菌。52株嗜酸放线菌代表菌株分布于放线菌目中的12个属：链霉菌属(Streptomyces)、小单孢菌属(Micromonospora) 、诺卡氏菌属(Nocardia)、野野村菌属(Nonomuraea) 、韩国生工属(Kribbella) 、小双孢菌属(Microbispora)、马杜拉菌属(Actinomadura)、拟无枝菌酸菌属(Amycolatopsis)、指孢囊菌属(Dactylosporangium)、伦茨氏菌属(Lentzea)、游动四孢菌属(Planotetraspora) 和链嗜酸菌属(Streptacidiphilus),其中链霉菌分离菌株在系统发育树上形成12个不同的进化类群。【结论】所建立的选择性分离方法可用于土壤嗜酸丝状放线菌的高效分离;江西酸性土壤含有丰富多样的嗜酸丝状放线菌种属。     

云南酸性土壤中度嗜酸链霉菌的多样性

[目的]了解酸性土壤环境里中度嗜酸链霉菌的多样性,调查其物种资源.[方法]用分散和差速离心法及选择性分离培养基从14份云南酸性土壤样品中分离到367株具有链霉菌培养特征的放线菌,并进行了颜色分群.从各颜色类群中选取代表菌株共97株,通过显微形态观察和pH梯度生长实验确定其中的中度嗜酸链霉菌.进一步从中筛选出16株中度嗜酸链霉菌代表菌株,进行16SrRNA基因序列的相似性和系统发育分析,并结合基因组DNA-DNA相关性数据.[结果]分离菌株归为12同的颜色类群,其中80%属于中度嗜酸链霉菌,其代表菌株在系统发育树上形成了8个距离较远且与已知种不同的进化分枝,可能代表链霉菌属内至少8个不同的新基因种.[结论]用以上方法筛选出的中度嗜酸链霉菌可归为8个不同于已知种的进化群,说明云南酸性土壤含有丰富多样的中度嗜酸链霉菌新物种.     

Diversity of culturable actinobacteria isolated from marine sponge <Emphasis Type="Italic">Haliclona</Emphasis> sp.

This study describes actinobacteria isolated from the marine sponge Haliclona sp. collected in shallow water of the South China Sea. A total of 54 actinobacteria were isolated using media selective for actinobacteria. Species diversity and natural product diversity of isolates from marine sponge Haliclona sp. were analysed. Twenty-four isolates were selected on the basis of their morphology on different media and assigned to the phylum Actinobacteria by a combination of 16S rRNA gene based restriction enzymes digestion and 16S rRNA gene sequence analysis. The 16S rRNA genes of 24 isolates were digested by restriction enzymes TaqI and MspI and assigned to different groups according to their restriction enzyme pattern. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to the genera Streptomyces, Nocardiopsis, Micromonospora and Verrucosispora; one other isolate was recovered that does not belong to known genera based on its unique 16S rRNA gene sequence. To our knowledge, this is the first report of a bacterium classified as Verrucosispora sp. that has been isolated from a marine sponge. The majority of the strains tested belong to the genus Streptomyces and three isolates may be new species. All of the 24 isolates were screened for genes encoding polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). PKS and NRPS sequences were detected in more than half of the isolates and the different "PKS-I-PKS-II-NRPS" combinations in different isolates belonging to the same species are indicators of their potential natural product diversity and divergent genetic evolution.     

Streptacidiphilus durhamensis sp. nov., isolated from a spruce forest soil

The taxonomic position of three acidophilic actinobacteria, strains FGG38, FGG39 and FSCA67^T, isolated from the fermentation litter layer of a spruce forest soil was established using a polyphasic approach. The strains were shown to have chemotaxonomic and morphological properties consistent with their classification in the genus Streptacidiphilus and formed a distinct phyletic line in the Streptacidiphilus 16S rRNA gene tree being most closely related to Streptacidiphilus albus DSM 41753^T (99.4 % similarity). DNA:DNA relatedness data showed that isolate FSCA67^T and the type strain of S. albus belonged to markedly distinct genomic species. The isolates had many phenotypic properties in common and were distinguished readily from their closest phylogenetic neighbours in the Streptacidiphilus gene tree using a broad range of these features. Based on the combined genotypic and phenotypic data the three isolates are considered to represent a new Streptacidiphilus species. The name Streptacidiphilus durhamensis sp. nov. is proposed for this taxon with isolate FSCA67^T (=DSM 45796^T = KACC 17154^T = NCIMB 14829^T) as the type strain.     

川楝内生放线菌多样性及抗菌活性筛选

【目的】探究药用植物川楝内生放线菌多样性,从中挖掘出新的放线菌菌株,发现新的潜在农业生防和医药先导化合物。【方法】从四川境内的资阳、遂宁以及重庆万州采集川楝的根、茎、叶、果、皮,采用纯培养方法,用4种培养基共分离获得148株放线菌。通过形态学观察筛选出60株放线菌进行RFLP分析,选出代表菌株进行16S r RNA基因序列分析。以3株细菌和6株病原真菌作为指示菌株,检测初筛出的60株菌株的抗菌活性以及聚酮合酶(PKSⅠ、PKSⅡ)基因、非核糖体多肽合成酶(NRPS)基因和卤化酶(Halo)基因。【结果】基于16S r RNA-RFLP分析,60株放线菌被分成10簇,筛选出25株代表菌株分别属于7个属,包括Streptomyces、Micromonospora、Planotetraspora、Streptosporangium、Nocardiopsis、Prauseria、Microbispora,其中链霉菌占73.3%。供试的川楝内生放线菌对细菌、真菌有不同程度的抗菌活性;其中含有4类化合物合成基因的菌株占10%-55%。【结论】药用植物川楝内生放线菌具有丰富的多样性,且不同地区不同部位川楝组织中放线菌的种群存在差异;分离菌株广谱的抗菌活性证明,川楝内生放线菌在次生代谢产物合成方面具有巨大潜力,这为进一步的药物开发提供了丰富的菌种资源。     

Phylogenetic study of the species within the family Streptomycetaceae

Species of the genus Streptomyces, which constitute the vast majority of taxa within the family Streptomycetaceae, are a predominant component of the microbial population in soils throughout the world and have been the subject of extensive isolation and screening efforts over the years because they are a major source of commercially and medically important secondary metabolites. Taxonomic characterization of Streptomyces strains has been a challenge due to the large number of described species, greater than any other microbial genus, resulting from academic and industrial activities. The methods used for characterization have evolved through several phases over the years from those based largely on morphological observations, to subsequent classifications based on numerical taxonomic analyses of standardized sets of phenotypic characters and, most recently, to the use of molecular phylogenetic analyses of gene sequences. The present phylogenetic study examines almost all described species (615 taxa) within the family Streptomycetaceae based on 16S rRNA gene sequences and illustrates the species diversity within this family, which is observed to contain 130 statistically supported clades, as well as many unsupported and single member clusters. Many of the observed clades are consistent with earlier morphological and numerical taxonomic studies, but it is apparent that insufficient variation is present in the 16S rRNA gene sequence within the species of this family to permit bootstrap-supported resolution of relationships between many of the individual clusters.     

Isolation,characterisation and antifungal activity of marine actinobacteria from Goa and Kerala,the west coast of India

In total, 53 marine actinobacteria were isolated from the soils of six different locations in Goa and Kerala, on the west coast of India. All the isolates were screened for their antifungal properties against some phytopathogenic fungi by dual culture experiments. Among the 53 actinobacterial isolates, five isolates inhibited the growth of phytopathogens, namely Colletotrichum falcatum, Thielaviopsis paradoxa and Fusarium semitectum. But none of them were effective against Aspergillus niger, Aspergillus candidus and Aspergillus flavus. The antifungal activity of the actinobacteria was tested by food poisoning techniques, using four different concentrations (0.5%, 1.0%, 1.5% and 2.0%) of cell-free culture filtrates, which showed promising activity (almost 100% inhibition) against three pathogenic and one non-pathogenic fungi at 2% extract concentration. A comparison of the antifungal activity of the actinobacteria was also made with three commercial fungicides, namely hexaconazole, thiophanate methyl and propiconazole. The identity of the antagonistic actinobacteria was confirmed based on the morphological, cultural, biochemical, chemo-taxonomical and physiological characteristics. Among 5 antagonistic isolates, three antagonistic isolates were assigned to the genus Streptomyces, Nocardiopsis (1) and Saccharopolyspora (1).     

Streptacidiphilus hamsterleyensis sp. nov., isolated from a spruce forest soil

Three acidophilic actinobacteria, isolates LSCA2, FGG8 and HSCA14^T, recovered from spruce litter were examined using a polyphasic approach. Chemotaxonomic and morphological properties of the isolates were found to be consistent with their classification in the genus Streptacidiphilus. The isolates were shown to have identical 16S rRNA gene sequences and were most closely related to Streptacidiphilus neutrinimicus DSM 41755^T (99.9 % similarity). However, DNA:DNA relatedness between isolate HSCA14^T and the type strain of S. neutrinimicus was found to be low at 44.0 (±14.1) %. A combination of phenotypic features, including degradative and nutritional characteristics were shown to distinguish the isolates from their nearest phylogenetic neighbours. Data from this study show that the isolates form a novel species in the genus for which the name S. hamsterleyensis sp. nov. is proposed. The type strain is HSCA 14^T (=DSM 45900^T = KACC 17456^T = NCIMB 14865^T).     

Ecophysiology of Fe-cycling bacteria in acidic sediments

Using a combination of cultivation-dependent and -independent methods, this study aimed to elucidate the diversity of microorganisms involved in iron cycling and to resolve their in situ functional links in sediments of an acidic lignite mine lake. Using six different media with pH values ranging from 2.5 to 4.3, 117 isolates were obtained that grouped into 38 different strains, including 27 putative new species with respect to the closest characterized strains. Among the isolated strains, 22 strains were able to oxidize Fe(II), 34 were able to reduce Fe(III) in schwertmannite, the dominant iron oxide in this lake, and 21 could do both. All isolates falling into the Gammaproteobacteria (an unknown Dyella-like genus and Acidithiobacillus-related strains) were obtained from the top acidic sediment zones (pH 2.8). Firmicutes strains (related to Bacillus and Alicyclobacillus) were only isolated from deep, moderately acidic sediment zones (pH 4 to 5). Of the Alphaproteobacteria, Acidocella-related strains were only isolated from acidic zones, whereas Acidiphilium-related strains were isolated from all sediment depths. Bacterial clone libraries generally supported and complemented these patterns. Geobacter-related clone sequences were only obtained from deep sediment zones, and Geobacter-specific quantitative PCR yielded 8 × 10(5) gene copy numbers. Isolates related to the Acidobacterium, Acidocella, and Alicyclobacillus genera and to the unknown Dyella-like genus showed a broad pH tolerance, ranging from 2.5 to 5.0, and preferred schwertmannite to goethite for Fe(III) reduction. This study highlighted the variety of acidophilic microorganisms that are responsible for iron cycling in acidic environments, extending the results of recent laboratory-based studies that showed this trait to be widespread among acidophiles.     

Diversity and physiological properties of root endophytic actinobacteria in native herbaceous plants of Korea

Endophytic actinobacterial diversity in the native herbaceous plant species of Korea was analyzed using a culture-based approach. Sixty one actinobacterial strains were isolated, and assigned to 15 genera based on 16S rRNA gene analysis. The members of the genus Streptomyces comprised 45.9% of the total isolates, followed by Micromonospora (18.8%), Rhodococcus (6.6%), Microbispora (4.9%), and Micrococcus (4.9%). Other minor constituents included members of Microbacterium, Streptacidiphilus, Arthrobacter, Dietzia, Kitasatospora, Herbiconiux, Mycobacterium, Nocardia, Rathayibacter, and Tsukamurella. Among the isolates, 65.6% exhibited at least one hydrolytic enzyme activity out of four, and 45.9% exhibited antagonistic activity against at least one fungal pathogen out of five, thus demonstrating that endophytic actinobacteria can be an important source of bioactive compounds. Notably, most strains of Streptomyces proved active for both enzymatic and antagonistic activities.     

Novel thermo-acidophilic bacteria isolated from geothermal sites in Yellowstone National Park: physiological and phylogenetic characteristics

Moderately thermophilic acidophilic bacteria were isolated from geothermal (30-83 degrees C) acidic (pH 2.7-3.7) sites in Yellowstone National Park. The temperature maxima and pH minima of the isolates ranged from 50 to 65 degrees C, and pH 1.0-1.9. Eight of the bacteria were able to catalyze the dissimilatory oxidation of ferrous iron, and eleven could reduce ferric iron to ferrous iron in anaerobic cultures. Several of the isolates could also oxidize tetrathionate. Six of the iron-oxidizing isolates, and one obligate heterotroph, were low G+C gram-positive bacteria ( Firmicutes). The former included three Sulfobacillus-like isolates (two closely related to a previously isolated Yellowstone strain, and the third to a mesophilic bacterium isolated from Montserrat), while the other three appeared to belong to a different genus. The other two iron-oxidizers were an Actinobacterium (related to Acidimicrobium ferrooxidans) and a Methylobacterium-like isolate (a genus within the alpha -Proteobacteria that has not previously been found to contain either iron-oxidizers or acidophiles). The other three (heterotrophic) isolates were also alpha-Proteobacteria and appeared be a novel thermophilic Acidisphaera sp. An ARDREA protocol was developed to discriminate between the iron-oxidizing isolates. Digestion of amplified rRNA genes with two restriction enzymes ( SnaBI and BsaAI) separated these bacteria into five distinct groups; this result was confirmed by analysis of sequenced rRNA genes.     

新疆青海中度嗜盐放线菌生物多样性初步研究

从我国新疆、青海等地采集数份盐碱土样或泥样,采用淀粉-酪素琼脂培养基、甘油天门冬酰胺琼脂培养基、土壤浸汁琼脂培养基分别从中分离到8株、32株中度嗜盐放线菌菌株。经形态、生理学特性与全细胞壁氨基酸组分分析结果比较,选取其中的14株进行16S rDNA序列分析。就物种多样性而言,新疆、青海分离到中度嗜盐放线菌分布至少有3个科,5个属。其中有拟诺卡氏菌科（Nocardiopsaceae）的拟诺卡氏菌属(Nocardiopsis)和链单孢菌属（Streptomonospora）;假诺卡氏菌科(Pseudonocardiaceae)的普氏菌属(Prauserella)和糖单孢菌属(Saccharomonospora);链霉菌科(Streptomycetaceae)的链霉菌属(Streptomyces)。就地区分布来讲,新疆分离到的中度嗜盐放线菌种类要远高于青海。     

Influences of soil acidity on Streptomyces populations inhabiting forest soils.

The Streptomyces populations inhabiting five acidic forest soils were examined. It was found that lowering the pH of a medium selective for streptomycetes (starch-casein agar) to the pH of the particular soil horizon being plated influenced both the total numbers and types of streptomycetes that were isolated from the soils examined in this study. On the acidified medium both the numbers of streptomycetes and the percentage of total bacteria on the plates represented by streptomycetes increased (as compared with the same medium with a pH of 7.2). These differences were greatest on the isolations from the most acid soils. The largest concentrations of streptomycetes were found in the surface horizon (0 to 15 cm) and the litter layer immediately over the surface mineral horizon. Acidity tolerance tests demonstrated that random samplings of isolates contained acidophilic, neutrophilic, and acidoduric strains, with the largest numbers of acidophiles being found on the acidified media from the most acid soils. There were no differences between overall utilization of selected carbohydrates among the isolates taken from either the neutral or acidic media, although a larger proportion of the acid media isolates produced acid from the carbohydrates. Evidence is presented which indicates that different types of streptomycetes were isolated on the acid media, and possible reasons for the presence of these acid-tolerant populations are discussed.     

Influences of soil acidity on Streptomyces populations inhabiting forest soils.

The Streptomyces populations inhabiting five acidic forest soils were examined. It was found that lowering the pH of a medium selective for streptomycetes (starch-casein agar) to the pH of the particular soil horizon being plated influenced both the total numbers and types of streptomycetes that were isolated from the soils examined in this study. On the acidified medium both the numbers of streptomycetes and the percentage of total bacteria on the plates represented by streptomycetes increased (as compared with the same medium with a pH of 7.2). These differences were greatest on the isolations from the most acid soils. The largest concentrations of streptomycetes were found in the surface horizon (0 to 15 cm) and the litter layer immediately over the surface mineral horizon. Acidity tolerance tests demonstrated that random samplings of isolates contained acidophilic, neutrophilic, and acidoduric strains, with the largest numbers of acidophiles being found on the acidified media from the most acid soils. There were no differences between overall utilization of selected carbohydrates among the isolates taken from either the neutral or acidic media, although a larger proportion of the acid media isolates produced acid from the carbohydrates. Evidence is presented which indicates that different types of streptomycetes were isolated on the acid media, and possible reasons for the presence of these acid-tolerant populations are discussed.     

Isolation and identification of actinobacteria from surface-sterilized wheat roots

This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (Triticum aestivum L.). Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria. Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria. Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including Streptomyces, Microbispora, Micromonospora, and Nocardiodes spp. Many of the Streptomyces spp. were found to be similar, on the basis of their 16S rDNA gene sequence, to Streptomyces spp. that had been isolated from potato scabs. In particular, several isolates exhibited high 16S rDNA gene sequence homology to Streptomyces caviscabies and S. setonii. None of these isolates, nor the S. caviscabies and S. setonii type strains, were found to carry the nec1 pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic. These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.     

聚酮类化合物生物合成途径基因阳性菌株生物多样性研究

从中国云南省采集土样,采用GPY培养基、淀粉_酪素琼脂培养基和甘油天门冬酰胺琼脂培养基分离得到了876株细菌放线菌菌株。经聚酮类化合物基因筛选得到75株Ⅰ型和Ⅱ型聚酮类化合物生物合成基因双阳性的菌株,经抗菌活性、形态、生理等结果分析比较,选取其中的10株进行了16S rDNA序列分析。在物种多样性方面,分离到的菌分布至少有7个科,8个属。其中有链霉菌科的链霉菌属,分枝杆菌科的分枝杆菌属,链孢囊菌科的链孢囊菌属和野野村菌属,诺卡氏菌科的诺卡氏菌属,微球菌亚目的一新属、产碱杆菌科的无色杆菌属和β_亚纲中与紫色杆菌属紧邻的一革兰氏阴性菌新属。综合其表型、基因型特征,初步确定其中8株为新物种。研究表明利用新的思路和程序从自然环境分离、鉴定未知菌是微生物资源开发利用的关键。     

Draft genome sequence of the marine Streptomyces sp. strain PP-C42, isolated from the Baltic Sea

Streptomyces, a branch of aerobic Gram-positive bacteria, represents the largest genus of actinobacteria. The streptomycetes are characterized by a complex secondary metabolism and produce over two-thirds of the clinically used natural antibiotics today. Here we report the draft genome sequence of a Streptomyces strain, PP-C42, isolated from the marine environment. A subset of unique genes and gene clusters for diverse secondary metabolites as well as antimicrobial peptides could be identified from the genome, showing great promise as a source for novel bioactive compounds.     

定殖植物根内和根围放线菌的分离鉴定及其体外抑菌促生效应

【目的】旨在分离、筛选并鉴定体外具抑菌促生作用的定殖于植物根内和根围的放线菌,以期丰富放线菌种质资源,为研制植物病害生防菌剂提供技术依据。【方法】采用稀释涂布平板法分离盐碱地、湿地、工业污染土壤中优势植物根内及其根围中的放线菌;通过平板对峙试验筛选具有抑菌效应的菌株,进而采用Salkowski比色法、CAS平板检测法和无氮源培养法进一步检测抑菌菌株的促生作用;通过形态观测、生理生化特性检测及16SrRNA基因序列分析鉴定菌种。【结果】共分离到链霉菌属(Streptomyces)、诺卡氏菌属(Nocardia)和小单孢菌属(Micromonospora) 3属共283株定殖于植物根内和根围的放线菌,3个采样地中湿地数量最多,均为根围土根内;其中链霉菌属占总数的77%,可分为10个类群。经筛选获得7株抑菌活性和促生效应较强的菌株,其中菌株H6-1抑菌效应最大,其无菌发酵液对尖孢镰刀菌(Fusariumoxysporum)、禾谷镰刀菌(Fusariumgraminearum)、灰葡萄孢菌(Botrytis cinerea)、立枯丝核菌(Rhizoctoniasolani)、轮纹大茎点霉(Macrophomakawatsukai)和瓜类炭疽菌(Colletotrichumorbiculare)的抑制率分别为32.3%、42.6%、48%、72.2%、58.1%和60.5%;而D11-4菌株的促生作用最强,能产吲哚乙酸(22.3 mg/L)、产铁载体(晕圈直径25.2 mm)和固氮。经鉴定这7株放线菌是吸水链霉菌变种(Streptomyces angustmyceticus) H4-6、娄彻氏链霉菌(Streptomyces rochei) S2-2、浑圆链霉菌(Streptomycesglobosus)H6-1、(Streptomycesiakyrus)GD8-4、波卓链霉菌(Streptomyces bottropensis) GH8-6、(Streptomyces paradoxus) H8-2和(Streptomyces coralus) D11-4。【结论】三个生境中定殖于植物根内和根围的放线菌类群丰富且所筛选获得的7株放线菌具有生防潜力,值得进一步研发。