Isolation and characterization of mesophilic,oxalate-degrading<Emphasis Type="Italic"> Streptomyces</Emphasis> from plant rhizosphere and forest soils

The present work was aimed at the isolation of additional new pure cultures of oxalate-degrading Streptomyces and its preliminary characterization for further work in the field of oxalate metabolism and taxonomic studies. Mesophilic, oxalate-degrading Streptomyces were enriched and isolated from plant rhizosphere and forest soil samples. Strains were examined for cultural, morphological (spore chain morphology, spore mass colour, diffusible and melanin pigment production), physiological (antibiosis, growth in the presence of inhibitory compounds, assimilation of organic acids and enzyme substrates) and chemotaxonomic characters (cellular lipid components and diagnostic cell-wall diamino acid). The taxonomic data obtained were analysed by using the simple matching (S_SM) and Jaccard (S_J) coefficients, clustering was achieved using the UPGMA algorithm. All strains were able to utilize sodium-, potassium-, calcium- and ammonium-oxalate salts. Based on the results of numerical taxonomy, isolates were grouped into five cluster groups with a 70% S_SM similarity level. Streptomyces rochei was the most common of the cluster groups, with a Willcox probability of P>0.8. Streptomyces antibioticus, S. anulatus, S. fulvissimus, S. halstedii and S. violaceusniger are newly reported as oxalate-utilizing Streptomyces.Electronic Supplementary Material  Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.     

Cluster formation in liverwort-associated methylobacteria and its implications

Pink-pigmented methylotropic bacteria of the genus Methylobacterium inhabit the surfaces of plant organs. In bryophytes, these methylobacteria enhance cell growth, but the nature of this plant–microbe interaction is largely unknown. In this study, methylobacteria were isolated from the upper surface of the free-living thalli of the liverwort Marchantia polymorpha L. Identification of one strain by 16S ribosomal RNA (rRNA) gene-targeted polymerase chain reaction (PCR) and other data show that these microbes represent an undescribed species of the genus Methylobacterium (Methylobacterium sp.). The growth-promoting activity of these wild-type methylobacteria was tested and compared with that of the type strain Methylobacterium mesophilicum. Both types of methylobacteria stimulated surface expansion of isolated gemmae from Marchantia polymorpha by about 350%. When suspended in water, the liverwort-associated bacteria (Methylobacterium sp.) formed dense clusters of up to 600 cells. In liquid cultures of Methylobacterium mesophilicum, single cells were observed, but no clustering occurred. We suggest that the liverwort-associated methylobacteria are co-evolved symbionts of the plants: Cluster formation may be a behavior that enhances the survival of the epiphytic microbes during periods of drought of these desiccation-tolerant lower plants.     

Shinella zoogloeoides BC026对吡啶的降解特性研究

从首钢焦化厂的污水处理系统中分离1株能以吡啶为唯一碳、氮源的细菌BC026,它具有自絮凝特性,对卡那霉素、氨苄青霉素和壮观霉素具有抗性,可在阿须贝无氮培养基中良好生长.通过16S rRNA序列分析和Biolog微生物鉴定系统鉴定,确定该菌为Shinella zoogloeoides.纯菌对单基质的降解实验表明,在30℃、180 r/min和pH为7的条件下,当投菌量为0.1 g/L时,BC026可在17 h内将400 mg/L吡啶完全降解;在吡啶初始浓度为99～1 806 mg/L的无机盐培养基中,BC026均能保持降解活性,较高初始浓度的吡啶对BC026的生长产生一定抑制,但BC026在适应后对吡啶的降解速率较快;降解最适温度为30～35℃,最适pH为8.BC026对吡啶的代谢途径研究表明：降解的第一步是断开吡啶的2条C—N链,生成氨氮和戊二醛,随后戊二醛被氧化为戊二酸,并最终转化为二氧化碳和水;吡啶中的氮有59.5%转化成氨氮.     

1,2,4-三氯苯矿化菌的鉴定与功能分析

采用同位素示踪法从氯苯污染土壤中筛选到能高效降解1,2,4-三氯苯(1,2,4-TCB)为CO₂的细菌E3和F2,根据其表型特征、16S rDNA序列和荧光原位杂交(FISH)分析,鉴定E3和F2为博德特氏菌（Bordetella sp.）.E3和F2的16S rDNA序列相似性为100%;它们与菌株Bordetella sp. QJ2-5同源性最高,与Bordetella petrii(GDH030510)最接近,同源性分别为100%和99.4%.E3和F2能在含1,2,4-TCB的无机盐培养基中彻底降解1,2,4-TCB生成CO₂,30 d降解率达到90%以上,其中58%和46%矿化为CO₂,同时形成一定量的微生物生物量.     

Isolation of five Rubrobacter strains from biodeteriorated monuments

In the last few years, the microbial colonisation of mural paintings in ancient monuments has been attracting the attention of microbiologists and conservators. The genus Rubrobacter is commonly found in biodeteriorated monuments, where it has been reported to cause rosy discolouration. However, to date, only three species of this genus have been isolated, all from thermophilic environments. In this paper, we studied three monuments: the Servilia and Postumio tombs in the Roman Necropolis of Carmona (Spain), and Vilar de Frades church (Portugal), in search of Rubrobacter strains. In all cases, biodeterioration and the formation of efflorescences were observed, and five Rubrobacter strains were isolated. These isolates showed different physiology and migration in denaturing gradient gel electrophoresis, suggesting they might represent new species within this genus. The isolates reproduced some biodeterioration processes in the laboratory and revealed their biomediation in crystal formation.     

Moss-associated methylobacteria as phytosymbionts: an experimental study

Methylotrophic bacteria inhabit the surface of plant organs, but the interaction between these microbes and their host cells is largely unknown. Protonemata (gametophytes) of the moss Funaria hygrometrica were cultivated in vitro under axenic conditions and the growth of the protonemal filaments recorded. In the presence of methylobacteria (different strains of Methylobacterium), average cell length and the number of cells per filament were both enhanced. We tested the hypothesis that auxin (indole-3-acetic acid, IAA), secreted by the epiphytic bacteria and taken up by the plant cells, may in part be responsible for this promotion of protonema development. The antiauxin parachlorophenoxyisobutyric acid (PCIB) was used as a tool to analyze the role of IAA and methylobacteria in the regulation of cell growth. In the presence of PCIB, cell elongation and protonema differentiation were both inhibited. This effect was compensated for by the addition of different Methylobacterium strains to the culture medium. Biosynthesis and secretion of IAA by methylobacteria maintained in liquid culture was documented via a colorimetric assay and thin layer chromatography. Our results support the hypothesis that the development of Funaria protonemata is promoted by beneficial phytohormone-producing methylobacteria, which can be classified as phytosymbionts.     

Non-specific association between filamentous bacteria and fungus-growing ants

Fungus-growing ants and their fungal cultivar form a highly evolved mutualism that is negatively affected by the specialized parasitic fungus Escovopsis. Filamentous Pseudonocardia bacteria occurring on the cuticle of attine ants have been proposed to form a mutualistic interaction with these ants in which they are vertically transmitted (i.e. from parent to offspring colonies). Given a strictly vertical transmission of Pseudonocardia, the evolutionary theory predicts a reduced genetic variability of symbionts among ant lineages. The aim of this study was to verify whether actinomycetes, which occur on Acromyrmex octospinosus leaf-cutting ants, meet this expectation by comparing their genotypic variability with restriction fragment length polymorphisms. Multiple actinomycete strains could be isolated from both individual ant workers and colonies (one to seven strains per colony). The colony specificity of actinomycete communities was high: Only 15% of all strains were isolated from more than one colony, and just 5% were present in both populations investigated. Partial sequencing of 16S ribosomal deoxyribonucleic acid of two of the isolated strains assigned both of them to the genus Streptomyces. Actinomycetes could also be isolated from workers of the two non-attine ant species Myrmica rugulosa and Lasius flavus. Sixty-two percent of the strains derived from attine ants and 80% of the strains isolated from non-attine ants inhibited the growth of Escovopsis. Our data suggest that the association between attine ants and their actinomycete symbionts is less specific then previously thought. Soil-dwelling actinomycetes may have been dynamically recruited from the environment (horizontal transmission), probably reflecting an adaptation to a diverse community of microbial pathogens.     

2株海洋石油降解细菌的降解能力

为实施海洋石油污染的生物治理,从厦门储油码头油污水中分离得到了2株石油降解菌.它们能够在以柴油、萘或芘为唯一碳源的培养基中生长,并适应于海洋环境的温度、pH和盐度.它们对萘都有很强的降解能力,3d内降解率可达87.53%和84.01%;7 d内对芘降解率分别为8.35%、5.37%.经16S rDNA同源性分析表明2菌株之间及其与施氏假单胞菌的同源性皆为99%;Biolog生化鉴定也表明它们为不同的菌株.通过兼并PCR扩增,序列分析发现2种菌编码完全相同的萘双加氧酶基因,该基因与其它假单胞菌的萘双加氧酶大亚基有98%同源性.     

Phylogenetically clustering of rhizobia by genome structure: application to unclassified Rhizobium

Introduction The rhizobia can change nitrogen in the air into ammonia and provide nitrogenous nutrition to the plant. This will make us independent from the use of nitrogenous fertilizers so as to minimize pollution of water tables, lakes and rivers and r…     

城市污水处理厂四环素抗性菌的筛选及其抗性基因分析

城市污水处理厂中抗生素抗性菌的富集和扩散对受纳水体及公共卫生安全造成严重的威胁.本研究从上海某城市污水处理厂活性污泥中筛选得到一株四环素抗性菌株TC-1,基于形态特征、生理生化特性和16S r DNA结果鉴定,菌株TC-1属于节杆菌属(Arthrobacter sp.).对节杆菌属菌株的全基因组分析表明,菌株TC-1含有多种抗生素抗性基因,包括四环素、氯霉素、大环内酯类和青霉素抗性基因.对比节杆菌属抗生素抗性基因的基因簇分析表明,这些抗性基因在节杆菌属中具有普遍存在性,表明其来源于相关菌株的遗传而非单个菌株的偶然获得.鉴于节杆菌属是潜在的病原性微生物,该结论可为废水生物处理系统中含抗生素抗性基因的致病微生物环境行为和风险评估提供参考.     

Intrageneric differences in the four stereoisomers of stenusine in the rove beetle genus, Stenus (Coleoptera, Staphylinidae)

Most species of the rove beetle genus Stenus employ the spreading alkaloid stenusine as an escape mechanism on water surfaces. In the case of danger, they emit stenusine from their pygidial glands, and it propels them over the water very quickly. Stenusine is a chiral molecule with four stereoisomers: (2′R,3R)-, (2′S,3R)-, (2′S,3S)-, and (2′R,3S)-stenusine. The percentile ratio of these four isomers is only known for the most common species of the genus: Stenus comma. With the intention of determining the stereoisomer ratios of five additional species from the two subgenera, Stenus and Hypostenus, we used GC/mass spectrometry measurements with a chiral phase. The results showed that the ratio differs among the genus. These findings can be a basis for chemotaxonomy. It is also possible that the biological function of stenusine, e.g., as antibiotic or fungicide, varies with changing stereoisomer composition.

Indigenous bacteria may interfere with the biocontrol of plant diseases

Prodigiosin is a reddish antibiotic pigment that plays an important role in the biocontrol of plant diseases by the bacterium Serratia marcescens. However, its activity is unstable under agricultural conditions; further, it can be degraded by various environmental factors. To examine the effect of epiphytic microbes on the stability of prodigiosin used for biological control processes, we collected a total of 1,280 bacterial isolates from the phylloplane of cyclamen and tomato plants. Approximately 72% of the bacterial strains isolated from the cyclamen plants and 66% of those isolated from the tomato plants grew on minimal agar medium containing 100 μg ml⁻¹ prodigiosin. Certain isolates obtained from both plant species exhibited prodigiosin-degrading activity. We compared the 16S rRNA gene sequences derived from the isolates with sequences in a database. The comparison revealed that the sequences determined for the prodigiosin-degrading isolates were homologous to those of the genera Pseudomonas, Caulobacter, Rhizobium, Sphingomonas, Janthinobacterium, Novosphingobium, and Rathayibacter. These results indicate that indigenous epiphytic microorganisms may interfere with the interaction between plant pathogens and biocontrol agents by degrading the antibiotics produced by the agents.     

Augmentation of tribenuron methyl removal from polluted soil with Bacillus sp. strain BS2 and indigenous earthworms

Tribenuron methyl (TBM) is a member of the sulfonylurea herbicide family and is widely used worldwide. In this study, TBM-degrading bacteria were enriched with TBM as potential carbon, nitrogen or sulfur source, and 44 bacterial isolates were obtained. These isolates were phylogenetically diverse, and were grouped into 25 operational taxonomic units and 14 currently known genera. Three representatives, Bacillus sp. strain BS2, Microbacterium sp. strain BS3, and Cellulosimicrobium sp. strain BS11, were selected, and their growth and TBM removal from culture broth were investigated. In addition, indigenous earthworms were collected and applied to augment TBM degradation in lab-scale soil column experiments. Results demonstrated that Bacillus sp. strain BS2 and earthworms significantly increased TBM removal during soil column experiments.     

多粘类芽孢杆菌GA1产絮凝剂的培养基和分段培养工艺

对1株从土壤中筛选的产絮凝剂微生物GA1进行了研究.该菌株经形态学特征、生理生化反应及16S rDNA序列(GenBank序列登陆号为DQ166375)相似性分析鉴定为多粘类芽孢杆菌,并命名为Paenibacillus polymyxa GA1.对其进行了产絮凝剂培养条件和培养工艺的研究.结果表明:GA1产絮凝剂的最佳培养基成分(g/L)为蔗糖40.0、酵母浸膏4.0、K₂HPO₄5.0、KH₂PO₄、2.0、NaCl 0.1、MgSO₄ 0.2.研究了该菌株产絮凝剂的最佳培养条件,包括培养基的初始pH、培养温度、摇床速度和接种量.同时针对其产絮凝剂和菌体生长的关系,首次将分段培养工艺应用于GA1产絮凝剂中,即在培养的初期24h内采用菌体生长最佳培养条件,在培养后期采用菌体产絮凝剂的最佳培养条件.结果表明,采用分段培养的工艺,既可保证GA1絮凝剂的产量,又能缩短培养周期.     

石油降解菌的分离鉴定及4株芽胞杆菌种间效应

通过富集培养和分离纯化的方法,从天津大港油田石油污染土壤和渤海海上钻井平台洗油污水中分离出6株石油降解细菌,生理生化试验及16S r DNA序列分析鉴定表明,它们分别属于Bacillus芽胞杆菌属(S1、S2、S3、S4)、Pseudomonas假单胞菌属(W1)和Ochrobactrum苍白杆菌属(W2),其中,S3具有最高的烷烃(41.3%)和芳烃(30.9%)降解率,从石油污染场地中筛选出的内源微生物对本油田石油的降解效果优于外源物种.对4株芽胞杆菌属菌株构建微生物组进行石油降解实验,结果表明,由S1和S4构成的微生物组F3具有最高的烷烃(50.5%)和芳烃(54.0%)降解率,比单菌降解率分别提高了69.9%和156.1%,同时比最优降解单菌S3的降解率分别高出22.1%和74.6%,而由S2和S3构成的微生物组F4对烷烃和芳烃的降解率最低,分别为18.5%和18.9%,比单菌降解率降低了55.3%和39.0%,实验表明同菌属微生物种间对石油的降解同时存在协同促进和拮抗抑制作用,芽胞杆菌属内亲缘性近的菌株之间对石油降解主要表现为促进作用.     

Bioleaching of spent Ni-Cd batteries and phylogenetic analysis of an acidophilic strain in acidified sludge

Biohydrometallurgy is a novel method to recycle discarded batteries, in which sewage sludge is used as microorganisms and culture due to the presence of indigenous Thiobacilli. A two-step continuous flow leaching system consisting of an acidifying reactor and a leaching reactor was introduced to achieve the bioleaching of spent nickel-cadmium (Ni-Cd) batteries. The acid supernatant produced in the acidifying reactor by the microorganisms with ferrous ions as the substrate was conducted into the leaching reactor to dissolve electrode materials. The efficiency of a batch treatment of batteries was examined. The results showed that the complete dissolution of two AA-sized Ni-Cd batteries with 0.6 L/d acid supernatant took about 30, 20, and 35 days for Ni, Cd, and Co, respectively. But the dissolution ability of the three metals was different. Cd and Co can be leached mostly for pH below 4.0 while the complete dissolution of Ni can be achieved for pH below 2.5. Meanwhile, a strain (named Thiooxidans. WL) accounting for the reduction of pH in the acidified sludge was isolated and sequenced. It was identified to be 100% similar to Acidithiobacillus ferrooxidans strain Tf-49 based on 16S rDNA sequence analysis. The relevant phylogenetic tree constructed indicates that the strain should be classified into genus Acidithiobacillus ferrooxidans.     

青藏铁路沿线土壤可培养微生物种群多样性分析

选择青藏铁路沿线不同海拔高度的10个地点采集土壤样品,采用3种不同的培养基分离培养其中的微生物;提取各土壤样品可培养微生物菌体基因组DNA,进行16S rDNA的PCR扩增,对可培养微生物种群多样性进行分析.结果表明,该地区可培养微生物的数量为1×106～8×106 CFU/g.16S rDNA序列分析发现,分离培养的微生物主要包括厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和变形菌门γ亚群(Gammaproteobacteria) 3个类群,分别占菌株总数的53.1%,29.6%和16.0%.其中,Bacillus sp.为优势菌属,占菌株总数的29.6%;Bacillus simplex为优势种,占菌株总数的12.3%.      

Spatial heterogeneity of cyanobacterial communities and genetic variation of Microcystis populations within large, shallow eutrophic lakes (Lake Taihu and Lake Chaohu, China)

Cyanobacteria, specifically Microcystis, usually form massive blooms in eutrophic freshwater lakes. Cyanobacterial samples were collected from eight sites of both Lake Taihu and Lake Chaohu in late summer to determine the diversity and distribution pattern of cyanobacteria and Microcystis in large, shallow, entropic lakes with significant spatial heterogeneity and long-term Microcystis bloom. Molecular methods based on denaturing gradient gel electrophoresis and clone library analysis were used. A similar heterogeneous distribution pattern of cyanobacteria in both lakes was observed. Most parts of these two lakes with high trophic level were dominated by Microcystis. However, in the regions with low trophic levels as well as low concentrations of chlorophyll a, Synechococcus occupied a considerable percentage. Different morphospecies and genotypes dominated the bloom-forming Microcystis populations in these two lakes. Microcystis viridis and Microcystis novacekii were dominant in Lake Chaohu, whereas Microcystis flos-aquae was dominant in Lake Taihu. Only 2 of the13 Microcystis operational taxonomic units were shared between these two lakes. Analysis of molecular variance based on 16S to 23S internal transcribed spacer sequences indicated the significant genetic differentiation of Microcystis between these two lakes (F_st = 0.19, p < 0.001). However, only 19.46% of the genetic variability was explained by the population variation between lakes, whereas most (80.54%) of the genetic variability occurred within the lakes. Phylogenetic analysis revealed no phylogeographic structure of Microcystis population in these two lakes, as illustrated by their cosmopolitan nature. Our results revealed that spatial heterogeneity within lakes has more impact on the cyanobacterial diversity than geographical isolation in a local scale.     

大麦虫幼虫肠道菌群对聚苯乙烯泡沫塑料降解

聚苯乙烯（polystyrene,PS）是广泛使用且难以降解的主要塑料材料之一.在本研究中,选择大麦虫幼虫（Zophobas morio）对PS进行生物降解,发现大麦虫幼虫在为期50 d的培养时间仅摄取PS,能够保持较高存活率（73%）.使用傅里叶变换红外光谱分析大麦虫的虫粪,证实了虫粪中的PS形成了新的含氧官能团.凝胶渗透色谱分析表明,大麦虫摄入PS后其虫粪的重均分子量（M_w）和数均分子量（M_n）均向减小的方向偏移,表明大麦虫摄入的PS发生了降解.使用16S rRNA基因测序技术,分析PS、麦麸和麦麸加PS这3种不同的饲喂条件下大麦虫的肠道细菌,结果表明在不同的摄食条件下,Z.morio的肠道菌群表现出显著差异.在PS饲喂条件下,大麦虫肠道中具有降解PS功能的优势菌群,例如克雷伯氏菌（Klebsiella）和柠檬酸杆菌属（Citrobacter）等显著富集.以PS为唯一碳源,对大麦虫肠道微生物进行为期90 d的体外培养实验,结果发现具有PS降解功能的肠道菌群显著富集.本研究证明了肠道细菌在大麦虫幼虫降解PS的过程中发挥了重要作用,为PS的生物降解提供了新的理论依据与应用思路.     

Isolation of a Pseudomonas Stutzeri strain that degrades 1,2,4-trichlorobenzene and characterization of its degradative plasmid

The genetic information encoding metabolic pathways for xenobiotic compounds in bacteria often resides on catabolic plasmids. The aim of the present work was to know the location of the genes for degrading 1,2,4-trichlorobenzen. In this paper a 1,2,4-trichlorobenzene-degrading strain THSL-1 was isolated from the soil of Tianjin Chemical Plant using 1,2,4-trichlorobenzene as the sole carbon source. The strain was identified as Pseudomonas stutzeri through morphologic survey and 16S rDNA sequence determination. A plasmid was discovered from strain THSL-1 by using the alkali lysis method. When the plasmid was transformed into E. coli. JM109 by the CaCl₂ method, the transformant could grow using 1,2,4-trichlorobenzene as the sole carbon source and had the degradation function of 1,2,4-trichlorobenzene. Therefore, it could be deemed that the plasmid carried the degradative genes of 1,2,4-trichlorobenzene. The average size of the plasmid was finally determined to be 40.2 Kb using selectively three kinds of restricted inscribed enzymes (HindIII, BamHI, and XholI) for single cutting and double cutting the plasmid pTHSL-1, respectively. __________ Translated from China Environmental Science, 2005, 25(4): 385–388 [译自: 中国环境科学]