Catabolism of Substituted Benzoic Acids by Streptomyces Species

Four thermotolerant actinomycetes from soil, identified as Streptomyces albulus 321, Streptomyces sioyaensis P5, Streptomyces viridosporus T7A, and Streptomyces sp. V7, were grown at 45°C in media containing either benzoic acid or hydroxyl- and methoxyl-substituted benzoic acids as the principal carbon sources. Benzoic acid was converted to catechol; p-hydroxybenzoic, vanillic, and veratric acids were converted to protocatechuic acid; and m-hydroxybenzoic acid was converted to gentisic acid. Catechol, protocatechuic acid, and gentisic acid were cleaved by catechol 1,2-dioxygenase, protocatechuate 3,4-dioxygenase, and gentisate 1,2-dioxygenase, respectively. Dioxygenases appeared only in induced cultures. m-Hydroxybenzoic, m-anisic, and p-anisic acids were gratuitous inducers of dioxygenases in some strains. One strain converted vanillic acid to guaiacol.     

海洋动植物共附生微生物的分离和抗菌活性研究

从海参、海胆、海葵、海兔、石莼、羊栖菜、裙带菜分离得到125种共附生海洋微生物,以6种敏感菌为指示菌,从中获得具有抑菌活性的细菌21株,放线菌8株,真菌2株。21株抑菌海洋细菌中芽孢杆菌属为7株,占33．3％,弧菌属为11种,占52．2％,其余3株为假单孢杆菌属,占14．5％。8株抑菌海洋放线菌中链霉菌属为5株,占62．5％,小单孢菌属为3株,占36．5％。2株抑菌海洋真菌均为青霉属。     

Patterns of antimicrobial activities from soil actinomycetes isolated under different conditions of pH and salinity

AIMS: To evaluate the patterns of the production of antimicrobial compounds by diverse collection of actinomycetes isolated from different geographies under alternative conditions of pH and salinity in the media. METHODS AND RESULTS: Actinomycetes were grouped based on their method of isolation and their phenotype diversity was determined by total fatty acid analysis. A total of 335 representative isolates, including 235 Streptomyces species and 100 actinomycetes from other taxa, were screened for the production of antimicrobial activities against a panel of bacteria, filamentous fungi and yeasts, including some of clinical relevance. Production of antimicrobial activities was detected in 230 strains. In the case of the genus Streptomyces, 181 antimicrobial activities (77% of the tested isolates) were recorded. The activities observed among the other actinomycetes taxa were lower (49% of the tested isolates). CONCLUSIONS: The results of this study support the idea that species of actinomycetes isolated in alternative selective conditions of pH and salinity present a significant capacity to produce compounds with antibacterial or antifungal activity. The best group of isolates in terms of production of active secondary metabolites was the one isolated in saline conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The results demonstrate that these actinomycetes strains isolated in alternative selective conditions of pH and salinity and collected from diverse geographical locations present a significant capacity to produce compounds with antibacterial or antifungal activity.     

Screening of nonpolyenic antifungal metabolites produced by clinical isolates of actinomycetes

The purpose of this work was to screen clinical isolates of actinomycetes producing nonpolyenic antifungals. This choice was made to limit the problem of rediscovery of well-known antifungal families, especially polyenic antifungals. One hundred and ten strains were tested, using two diffusion methods and two test media, against three yeast species and three filamentous fungi. Among 54 strains (49%) showing antifungal activity, five strains belonging to the genus Streptomyces were active against all test organisms and appeared promising. These results indicate that clinical and environmental isolates of actinomycetes could be an interesting source of antifungal bioactive substances. The production of nonpolyenic antifungal substances by these five active isolates was investigated using several criteria: antibacterial activity, ergosterol inhibition, and UV-visible spectra of active extracts. One active strain responded to all three selection criteria and produced potentially nonpolyenic antifungal metabolites. This strain was retained for further investigation, in particular, purification, structure elucidation, and mechanism of action of the active product.     

连作花生田根际土壤优势微生物的分离和鉴定

【目的】从不同连作年限的花生田根际土壤中分离优势微生物并进行鉴定,为研究花生连作后优势微生物的变化奠定基础。【方法】采用土壤稀释分离法从不同连作年限花生根际土壤中分离优势细菌、真菌和放线菌,结合菌株形态特征、培养性状、生理生化特征及16S rDNA序列分析对细菌、放线菌进行鉴定,通过形态特征、培养特征和分子鉴定方法对优势真菌进行鉴定。【结果】从连作花生田根际土壤中分离鉴定出7种优势细菌、7种优势真菌和7种优势放线菌。7种优势细菌分别为Leifsonia xyli、氯酚节杆菌(Arthrobacterchlorophenolicus)、黄色微杆菌(Microbacterium flavescens)、鞘氨醇单胞菌属(Sphingomonas sp.)、巴斯德菌属(Pasteurella sp.)、简单芽孢杆菌(Bacillus simplex)和巨大芽孢杆菌(Bacillus megaterium)。7种优势真菌分别为枝状枝孢菌(Cladosporium cladosporioides)、产紫青霉(Penicillium purpurogenum)、哈茨木霉有性型(Hypocrea lixii)、Exophiala pisciphila、微紫青霉(Penicillium janthinellum)、曲霉(Aspergillus sp.)和大丽轮枝菌(Verticillium dahliae)。7种优势放线菌分别为紫红链霉菌(Streptomyces violaceoruber)、华丽黄链霉菌(Streptomyces flaveus)、Streptomyces panaciterrae、不产色链霉菌(Streptomyces achromogenes)、假浅灰链霉菌(Streptomyces pseudogriseolus)、纤维素链霉菌(Streptomyces cellulosae)和金色链霉菌(Streptomyces aureus)。【结论】本研究是第一次系统的从连作花生根际土中分离鉴定优势微生物,种植花生后根际土壤中优势微生物的种类发生了明显变化,但变化没有规律。     

Microbial catabolism of vanillate: decarboxylation to guaiacol.

A novel catabolic transformation of vanillic acid (4-hydroxy-3-methoxybenzoic acid) by microorganisms is reported. Several strains of Bacillus megaterium and a strain of Streptomyces are shown to convert vanillate to guaiacol (o-methoxyphenol) and CO2 by nonoxidative decarboxylation. Use of a modified most-probable-number procedure shows that numerous soils contain countable numbers (10(1) to 10(2) organisms per g of dry soil) of aerobic sporeformers able to convert vanillate to guaiacol. Conversion of vanillate to guaiacol by the microfloras of most-probable-number replicates was used as the criterion for scoring replicates positive or negative. Guaiacol was detected by thin-layer chromatography. These results indicate that the classic separations of catabolic pathways leading to specific ring-fashion substrates such as protocatechuate and catechol are often interconnectable by single enzymatic transformations, usually a decarboxylation.     

粤北一种虫草分离菌的抗酵母现象

CR 95 1 2是一株分离自粤北一种虫草的无孢丝状真菌 ,经培养初步鉴定为无孢菌目(^{Agonomycetales})的丝核菌属 (^Rhizoctonia)。对包括革兰氏阳性及阴性细菌、放线菌、丝状真菌和酵母菌在内的 2 6株致敏菌株的抗性测定表明 ,CR 95 1 2对包括红酵母属 (^Rhodotorula)、酵母属 (^{Saccharomyces})、毕赤氏酵母属 (Pichia)和隐球酵母属 (^Cryptoc     

Functional Gene-Guided Discovery of Type II Polyketides from Culturable Actinomycetes Associated with Soft Coral Scleronephthya sp

Compared with the actinomycetes in stone corals, the phylogenetic diversity of soft coral-associated culturable actinomycetes is essentially unexplored. Meanwhile, the knowledge of the natural products from coral-associated actinomycetes is very limited. In this study, thirty-two strains were isolated from the tissue of the soft coral Scleronephthya sp. in the East China Sea, which were grouped into eight genera by 16S rDNA phylogenetic analysis: Micromonospora, Gordonia, Mycobacterium, Nocardioides, Streptomyces, Cellulomonas, Dietzia and Rhodococcus. 6 Micromonospora strains and 4 Streptomyces strains were found to be with the potential for producing aromatic polyketides based on the analysis of KS(α) (ketoacyl-synthase) gene in the PKS II (type II polyketides synthase) gene cluster. Among the 6 Micromonospora strains, angucycline cyclase gene was amplified in 2 strains (A5-1 and A6-2), suggesting their potential in synthesizing angucyclines e.g. jadomycin. Under the guidance of functional gene prediction, one jadomycin B analogue (7b, 13-dihydro-7-O-methyl jadomycin B) was detected in the fermentation broth of Micromonospora sp. strain A5-1. This study highlights the phylogenetically diverse culturable actinomycetes associated with the tissue of soft coral Scleronephthya sp. and the potential of coral-derived actinomycetes especially Micromonospora in producing aromatic polyketides.     

Metabolism of cinnamic, p-coumaric, and ferulic acids by Streptomyces setonii

Streptomyces setonii strain 75Vi2 was grown at 45 degrees C in liquid media containing yeast extract and trans-cinnamic acid, p-coumaric acid, ferulic acid, or vanillin. Gas chromatography, thin-layer chromatography, and mass spectrometry showed that cinnamic acid was catabolized via benzaldehyde, benzoic acid, and catechol; p-coumaric acid was catabolized via p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and protocatechuic acid; ferulic acid was catabolized via vanillin, vanillic acid, and protocatechuic acid. When vanillin was used as the initial growth substrate, it was catabolized via vanillic acid, guaiacol, and catechol. The inducible ring-cleavage dioxygenases catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase were detected with an oxygen electrode in cell-free extracts of cultures grown in media with aromatic growth substrates and yeast extract.     

Identification of thermo-acidophilic bacteria isolated from the soil of several Japanese fruit orchards

Aims: To investigate the occurrence and distribution of thermo‐acidophilic bacteria (TAB) associated with various commercial fruit crop soils in Japan and to assess their ability to produce the odorous phenolic compound, guaiacol. Methods and Results: Phylogenetic analysis based on the 5′ end of the 16S rRNA gene (approximately 500 bp), was performed on 62 TAB isolated from the soil of several Japanese fruit orchards. The results suggested that 60 of the bacterial strains analysed belonged to the genus Alicyclobacillus, while the remaining two belonged to the genus Bacillus. The majority of strains (58%) were identified as Alicyclobacillus acidoterrestris. This group partitioned into three phylogenetically distinct subgroups (A–C). Isolates identified as A. acidiphilus (two strains), A. acidoterrestris (36 strains), and A. hesperidum subsp. aigle (one strain), produced guaiacol from vanillic acid. Levels of guaiacol production varied significantly among strains. The guaiacol producing phenotype was conserved among certain species, however no correlation was observed between levels of guaiacol production and 16S rRNA gene‐based phylogenetic relatedness. Conclusions: Alicyclobacillus acidoterrestris and Alicyclobacillus contaminans were widely distributed among various fruit orchards in Japan. Guaiacol production was common at the species/subspecies level; however the amount of guaiacol produced by each strain varied significantly. Significance and Impact of the Study: This study provides a comprehensive phylogenetic survey of Alicyclobacillus species in Japanese fruit orchards. Quality control standards for guaiacol producing Alicyclobacillus have also been described.     

黑曲霉CGMCC0774和朱红密孔菌CGMCC1115两步转化阿魏酸制备生物香兰素

在25 L发酵罐中黑曲霉Aspergillus niger CGMCC0774转化阿魏酸可生成香草酸2.24 g/L,摩尔转化率64.6%;朱红密孔菌Pycnoporus cinnabarinus CGMCC1115转化提取的香草酸可生成香草醛1.45 g/L,摩尔转化率为79.9%。将两步微生物转化有机串联,即用黑曲霉转化液加预先培养的朱红密孔菌Pycnoporus cinnabarinus CGMCC1115菌丝体继续转化,可产香草醛1.06 g/L,对原料阿魏酸的摩尔转化率34.0%。用米糠提取的天然阿魏酸做原料,两步串联微生物转化制备的生物香兰素经13C同位素的分析,符合生物香草素的等同要求。     

后基因组时代的丝状真菌基因组学与代谢工程

丝状真菌不仅是传统发酵工业中抗生素、酶制剂和有机酸的主要生产者,而且也是代谢工程育种中异源蛋白表达的重要细胞工厂。丝状真菌的遗传修饰和代谢工程研究是现代工业生物技术领域最具活力的研究方向之一。特别是与细菌和酵母相比,丝状真菌在细胞生长、营养需求、环境适应性、翻译后修饰、蛋白分泌能力和生物安全性等方面具有显著的优势。文章综述了丝状真菌作为异源蛋白表达系统在基因组学技术研究和代谢工程研究方面的最新进展。作者在分析丝状真菌基因组结构、特点的基础上,阐述了比较基因组学、蛋白质组学、转录组学和代谢组学等对丝状真菌的代谢途径重构、新型蛋白挖掘和代谢工程育种中的作用和意义。另一方面,作者分析了丝状真菌在表达外源蛋白时遇到的瓶颈问题,总结了丝状真菌代谢工程育种中的常用策略包括异源基因的融合表达、反义核酸技术、蛋白分泌途径改造、密码子优化和蛋白酶缺陷宿主的选育等技术和手段。最后,对该领域的发展趋势进行了展望。     

Williopsis californica, Williopsis saturnus, and Pachysolen tannophilus: novel microorganisms for stereoselective oxidation of secondary alcohols

A screening of 416 microorganisms from different taxonomical groups (bacteria, actinomycetes, yeasts, and filamentous fungi) has been performed looking for active strains in the stereoselective oxidation of secondary alcohols. The working collection was composed of 71 bacterial strains, 45 actinomycetes, 59 yeasts, 60 basidiomycetes, 33 marine fungi, and 148 filamentous fungi. All microorganisms selected were mesophilic. Yeasts were the most active microbial group in the whole-cell-catalyzed oxidation. Williopsis californica, Williopsis saturnus, and Pachysolen tannophilus were the strains of greatest interest, both as growing cells and as resting cells. The oxidation of the alcohols takes place when cells are in the stationary growth phase (after 48 h of culture). These three strains are S-stereoselective for the oxidation of racemic secondary alkanols and show stereospecificity in the oxidation of menthol or neo-menthol, whereas iso-menthol is not oxidized. In the case of the 1-tetrahydronaphtol enantiomers, only the S-enantiomer is oxidized. The three strains were immobilized by entrapment using agarose and agar from algae of the Gracilaria genus. The agarose derivatives displayed significant improvement in the stereospecificity of the reactions.     

Phylogenetic analysis of mutualistic filamentous bacteria associated with fungus-growing ants

The attine ant-microbe system is a quadripartite symbiosis, involving a complex set of mutualistic and parasitic associations. The symbiosis includes the fungus-growing ants (tribe Attini), the basidiomycetous fungi the ants cultivate for food, specialized microfungal parasites (in the genus Escovopsis) of the cultivar, and ant-associated mu tualistic filamentous bacteria that secrete antibiotics specifically targeted to suppress the growth of Escovopsis. In this study, we conduct the first phylogenetic analysis of the filamentous mutualistic bacteria (actinomycetes) associated with fungus-growing ants. The filamentous bacteria present on 3 genera of fungus-growing ants (Acromyrmex, Trachy myrmex, and Apterostigma) were isolated from 126 colonies. The isolated actinomycetes were grouped into 3 distinct morphological types. Each morphological type was specific to the ant genus from which it was isolated, suggesting some degree of host specificity. The phylogenetic position of the 3 morphotypes was estimated using 16S rDNA for representative strains. The 8 isolates of actinomycetes sequenced are in the family Pseudonocardiaceae (Actino mycetales) and belong to the genus Pseudonocardia. Transmission electron microscopy examination of the actino mycete associated with the cuticle of Acromyrmex sp. revealed bacterial cells with an outer electron-dense membrane, consistent with actinomycetes in the genus Pseudonocardia. Ant-associated Pseudonocardia isolates did not form a monophyletic group, suggesting multiple acquisitions of actinomycetes by fungus-growing ants over their evolutionary history.     

Impact of the first <Emphasis Type="Italic">Streptomyces</Emphasis> genome sequence on the discovery and production of bioactive substances

An important addition to the field of bacterial genomics is the recent publication of the complete genome sequence of Streptomyces coelicolor. This strain has been for some decades the model organism for streptomycetes and other filamentous actinomycetes, Gram-positive bacteria highly valuable for their ability to produce thousands of bioactive metabolites, many of which have found important applications in medicine and agriculture. We discuss here the impacts that the S. coelicolor genome sequence is likely to have on the production of bioactive metabolites by current industrial strains, on the possible development of future superhost(s) for the production of valuable drugs, and on the search for new bioactive substances from microbial sources.     

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。     

Effect of bioconversion conditions on vanillin production by Amycolatopsis sp. ATCC 39116 through an analysis of competing by-product formation

This study investigated the effects of transformation conditions such as initial pH, the initial concentration of glucose and yeast extract in the medium, and the separate addition of ferulic acid and vanillic acid, on the production of vanillin through an analysis of competing by-product formation by Amycolatopsis sp. ATCC 39116. The extent and nature of by-product formation and vanillin yield were affected by initial pH and different initial concentrations of glucose and yeast extract in the medium, with a high yield of vanillin and high cell density obtained at pH 8.0, 10 g/l glucose, and 8 g/l yeast extract. High concentrations of ferulic acid were found to negatively affect cell density. Additional supplementation of 100 mg/l vanillic acid, a metabolically linked by-product, was found to result in a high concentration of vanillin and guaiacol, an intermediate of vanillin. Via an analysis of the effect of these transformation conditions on competing by-product formation, high concentrations of ferulic acid were transformed with a molar yield to vanillin of 96.1 and 95.2 %, by Amycolatopsis sp. ATCC 39116 and Streptomyces V1, respectively, together with a minor accumulation of by-products. These are among the highest performance values reported in the literature to date for Streptomyces in batch cultures.     

Metabolism of isoeugenol via isoeugenol-diol by a newly isolated strain of Bacillus subtilis HS8

A bacterium designated as HS8 was newly isolated from soil based on its ability to degrade isoeugenol. The strain was identified as Bacillus subtilis according to its 16S rDNA sequence analysis and biochemical characteristics. The metabolic pathway for the degradation of isoeugenol was examined. Isoeugenol-diol, for the first time, was detected as an intermediate from isoeugenol to vanillin by a bacterial strain. Isoeugenol was converted to vanillin via isoeugenol-diol, and vanillin was then metabolized via vanillic acid to guaiacol by strain HS8. These metabolites, vanillin, vanillic acid, and guaiacol, are all valuable aromatic compounds in flavor production. At the same time, the bipolymerization of isoeugenol was observed, which produced dehydrodiisoeugenol and decreased the vanillin yield. High level of vanillic acid decarboxylase activity was detected in cell-free extract. These findings provided a detailed profile of isoeugenol metabolism by a B. subtilis strain for the first time, which would improve the production of valuable aromatic compounds by biotechnology.     

武陵山放线菌多样性

[目的]为了探究武陵山放线菌多样性,以便从新放线菌菌株中发现新的潜在药物先导化合物.[方法]从武陵山采集280份土样,采用纯培养的方法,用4种培养基分离到1134株放线菌.选择其中30株代表菌进行了初步分类鉴定;以3株细菌和7株农作物致病真菌作为指示菌,检测其抑菌活性;利用特异性引物扩增的方法,检测是否具有聚酮合酶(PKS Ⅰ、PKSⅡ)基因、非核糖体多肽合成酶(NRPS)基因和多烯类化合物合成酶(CYP)基因.[结果]分离到的武陵山放线菌中,链霉菌占70%以上,还有小单孢菌等8个科13个属,其中有5个菌株是潜在的新种.选取的30株实验菌对细菌、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23%～60%.[结论]武陵山原始森林土壤中,放线菌多样性很丰富,且存在很多未开发的稀有类群.有抑菌活性的菌株,可用于进一步的药物开发利用.     

Differential Growth Inhibition of Diaporthe and Phomopsis Isolates by the Metabolic Activity of Five Actinomycetes

Fifty-five cultures derived from Diaporthe perithecia and Phomopsis pycnidia found on diverse host plant species collected at different times and sites in Vojvodina, Yugoslavia, showed distinguishing quantitative reactions to the fungistatic activity of five actinomycetes obtained as fortuitous laboratory contaminants coming from field material. Streptomyces albidoflavus , S. albus , S. diastaticus , Streptomyces sp., and Streptoverticillium sp. could be ranked by their growth-inhibitory potential, with S. albus showing the strongest, and Streptomyces sp. the lowest. The responses of the fungi varied depending on the tested actinomycetes, but two major groups could be distinguished: A, which encompased the isolates that were less affected by the proximity of the actinomycetes; and B, with those which exhibited high sensitivity in all the experiments. Group A was typically represented by Diaporthe arctii , Phomopsis longicolla, and the Phomopsis type-1 cultures from Xanthium italicum; group B was typically represented by Diaporthe/Phomopsis helianthi, Phomopsis type-2 cultures from X. italicum , and isolates from Lactuca serriola . The results obtained underscore the dissimilarities between D. arctii and D. helianthi , and corroborate the value of the physiological aspects of congeneric isolates in considering taxonomic problems in the coelomicete genus Phomopsis.