Marine actinomycetes: An ongoing source of novel bioactive metabolites

Actinomycetes are virtually unlimited sources of novel compounds with many therapeutic applications and hold a prominent position due to their diversity and proven ability to produce novel bioactive compounds. There are more than 22,000 known microbial secondary metabolites, 70% of which are produced by actinomycetes, 20% from fungi, 7% from Bacillus spp. and 1–2% by other bacteria. Among the actinomycetes, streptomycetes group are considered economically important because out of the approximately more than 10,000 known antibiotics, 50–55% are produced by this genus. The ecological role of actinomycetes in the marine ecosystem is largely neglected and various assumptions meant there was little incentive to isolate marine strains for search and discovery of new drugs. The search for and discovery of rare and new actinomycetes is of significant interest to drug discovery due to a growing need for the development of new and potent therapeutic agents. Modern molecular technologies are adding strength to the target-directed search for detection and isolation of bioactive actinomycetes, and continued development of improved cultivation methods and molecular technologies for accessing the marine environment promises to provide access to this significant new source of chemical diversity with novel/rare actinomycetes including new species of previously reported actinomycetes.     

Gifted microbes for genome mining and natural product discovery

Actinomycetes are historically important sources for secondary metabolites (SMs) with applications in human medicine, animal health, and plant crop protection. It is now clear that actinomycetes and other microorganisms with large genomes have the capacity to produce many more SMs than was anticipated from standard fermentation studies. Indeed ~90 % of SM gene clusters (SMGCs) predicted from genome sequencing are cryptic under conventional fermentation and analytical analyses. Previous studies have suggested that among the actinomycetes with large genomes, some have the coding capacity to produce many more SMs than others, and that strains with the largest genomes tend to be the most gifted. These contentions have been evaluated more quantitatively by antiSMASH 3.0 analyses of microbial genomes, and the results indicate that many actinomycetes with large genomes are gifted for SM production, encoding 20–50 SMGCs, and devoting 0.8–3.0 Mb of coding capacity to SM production. Several Proteobacteria and Firmacutes with large genomes encode 20–30 SMGCs and devote 0.8–1.3 Mb of DNA to SM production, whereas cultured bacteria and archaea with small genomes devote insignificant coding capacity to SM production. Fully sequenced genomes of uncultured bacteria and archaea have small genomes nearly devoid of SMGCs.     

Pathway Engineering in Secondary Metabolite-Producing Actinomycetes

Abstract

Actinomycetes represent the microbial group richest in production of variable secondary metabolites. These mostly bioactive molecules are the end products of complex multistep biosynthetic pathways. Recent progress in the molecular genetics and biochemistry of the biosynthetic capacities of actinomycetes enables first attempts to redesign these pathways in a directed fashion. However, in contrast to several examples of designed biochemical improvement of primary metabolic processes in microorganisms, none of the products or strains derived from pathway engineering in actinomycetes discussed herein have reached pilot or production scale. The main reasons for this slow progress are the complicated pathways themselves, their complex regulation during the actinomycete cell cycle, and their uniqueness, as most pathways and products are specific for a strain rather than for a given species or larger taxonomic group. However, the modular use of a minimum of very similar enzymes and their conversion of similar intermediates to form the building blocks for the production of a maximum of divergent end products gives hope for the future application of these genetic models for the redesign of complex pathways for modified or new natural products. Several strategies that can be followed to reach this aim are discussed, mainly for the variable 6-deoxyhexose metabolism as an ubiquitously applicable example.     

Actinomycete cytochromes P‐450 involved in oxidative metabolism: Biochemistry and molecular biology∗

The diverse metabolic capability of actinomycetes, together with their widespread presence in terrestrial and aquatic environments, has rendered them instrumental in the breakdown of both man‐made and natural chemicals and in the recycling of carbon in nature. Actinomycetes are also well known for their ability to synthesize a wide variety of novel chemicals of pharmaceutical and industrial value. Cytochromes P‐450, a class of oxidative enzymes found in all organisms, play a central role in both biosynthetic and biodegradative reactions performed by actinomycetes. Herein, we describe recent studies of actinomycetes cytochromes P‐450 made possible by advances realized in biochemistry and molecular biology.     

Microbicidal and anti-inflammatory effects of Actinomadura spadix (EHA-2) active metabolites from Himalayan soils, India

Actinomycetes play an essential role in producing several bioactive compounds. In the present study, microbicidal and anti-inflammatory effects of metabolites from actinomycetes were investigated. Actinomycetes were isolated from north eastern Himalayan soil samples, India. The actinomycetes were investigated for their microbicidal property by conventional method and the active actinomycetes were identified by 16s rDNA sequence analyses. Further the metabolites were extracted and fractionated to evaluate the antimicrobial potency; they were subjected to GC–MS analysis. The active fraction was evaluated for selective toxicity and anti-inflammatory potential. Among isolated actinomycetes, EHA-2 showed potent antimicrobial activity and was identified as Actinomadura spadix. Fraction-8 from ethyl acetate extract of EHA-2 showed 100 % inhibition against Candida sp. (MIC—80 μg/mL) and Enterococcus faecalis (MIC—80 μg/mL). The expression of GAPDH in primary cells and 16s rRNA levels on E. faecalis treated with fraction-8 revealed no toxicity to the primary cells. Fraction-8 also suppressed the paw thickness on carrageenan induced animals and also controlled the release of NO, TNFα and IL-1β levels on LPS induced RAW 264.7 cell lines. GC–MS profile of fraction-8 showed the presence of an antimicrobial agent 3,6 di-isobutyl 2,5 piperazinedione, which is the first report in A. spadix. The actinomycetes isolate EHA-2 can be proceed further to produce antibiotics.     

抗逆放线菌的多样性、功能特性及其在环境修复中的应用

放线菌(actinomycetes)是一类可生存于各种极端环境的特殊菌群,具备较强的抗逆特性。其种类丰富,功能多样,适应性强,已被广泛用于抗生素开发、生物防治和环境修复等领域。放线菌可调节土壤微生物群落结构、介导营养元素转化和植物吸收、催化有机污染物降解及重金属的氧化还原过程,该特性赋予其在土壤改良、地力维持和污染物去除等方面广阔的环境应用前景。本文综述了放线菌的多样性、环境分布以及放线菌对环境改良和污染物去除的特性与机制,结合在环境修复领域的应用现状,总结了放线菌修复技术的优势及发展方向。     

Isolation and enumeration of marine actinomycetes from seawater and sediments in Alexandria

Actinomycetes were isolated from near-shore marine sediments and water at four different sites in Alexandria. Statistical analysis revealed that variation in temperature, pH, and dissolved phosphate were of insignificant values, but that variation in total nitrogen and organic matter were significant. The treatment of sediments and water samples by heat resulted in a selective reduction of the nonactinomycetal heterotrophic microflora. Four selective culture media were used for counting actinomycetes in marine water and sediments. The starch nitrate medium favored the growth of these microorganisms. The diversity and counts of actinomycetes varied with the seasonal variation, and the highest counts were detected in dry warm seasons. The numbers of this bacterial group in sediments exceeded by far their numbers in seawater. A positive correlation was found between population size and location. Actinomycetes were found in the highest numbers in the upper layers (0-20 cm depth). In a few cases, the counts of actinomycetes showed bimodal maxima 0-20 and 60-100 cm deep. Sediments were the best source of marine actinomycetes, and their distribution varied depending on the depth from which samples were collected. The ratio of actinomycetes to the total microflora ranged from 0.48 to 2.29, depending on location.     

Actinomycetes isolated from beehives in Thailand

Actinomycetes are one of the main microbial groups that produce bioactive compounds used as antibiotics. Although bacteria, mold and yeast have frequently been found in bees, the presence of actinomycetes in bee hives had not been previously identified or reported. The aim of our research was to focus on the diversity of actinomycetes in bee hives in Thailand. Bees, brood cells and hive materials were collected from apiaries and natural sources. Thirty-two isolates of actinomycetes were isolated and identified using morphological, physiological, chemical and molecular characterization. Most of the isolates belonged to the genus Streptomyces. Some less frequent isolates were classified in the genera Nonomuraea, Nocardiopsis and Actinomadura.     

Phylogenetic characterization of culturable actinomycetes associated with the mucus of the coral Acropora digitifera from Gulf of Mannar

The marine environment is a virtually untapped source of novel actinomycete diversity and its metabolites. Investigating the diversity of actinomycetes in other marine macroorganisms, like seaweeds and sponges, have resulted in isolation of novel bioactive metabolites. Actinomycetes diversity associated with corals and their produced metabolites have not yet been explored. Hence, in this study we attempted to characterize the culturable actinomycetes population associated with the coral Acropora digitifera. Actinomycetes were isolated from the mucus of the coral wherein the actinomycetes count was much higher when compared with the surrounding seawater and sediment. Actinobacteria-specific 16S rRNA gene primers were used for identifying the isolates at the molecular level in addition to biochemical tests. Amplified ribosomal DNA restriction analysis using three restriction enzymes revealed several polymorphic groups within the isolates. Sequencing and blast analysis of the isolates revealed that some isolates had only 96.7% similarity with its nearest match in GenBank indicating that they may be novel isolates at the species level. The isolated actinomycetes exhibited good antibacterial activity against various human pathogens. This study offers for the first time a prelude about the unexplored culturable actinomycetes diversity associated with a scleractinian coral and their bioactive capabilities.     

海洋放线菌的研究现状及未来展望

【背景】放线菌具有丰富的遗传和功能多样性,其次级代谢产物活性广泛,在临床医疗、农业生产和污染防治等领域都发挥着重要的作用。海洋放线菌由于其特殊的代谢途径,能产生独特的活性天然产物而受到广泛关注。【目的】探究国内外海洋放线菌领域研究的热点和趋势,为后续研究提供参考。【方法】以“marine actinomycetes or marine actinobacteria”为关键词,在Web of Science中检索海洋放线菌领域的文章进行计量分析,使用VOSviewer软件对其关键词、国家、机构、作者、发表时间进行可视化分析。【结果】海洋放线菌领域的文章发表数量总体呈逐年上升趋势,主要集中在微生物学及药学领域,中美两国在论文数量和引用频次上远超其他国家,海洋放线菌领域的研究集中在菌株的分离鉴定、活性天然产物挖掘以及生物信息学等方面。【结论】海洋放线菌在全球范围内愈发受到重视,国内外机构应当加强合作,运用生物信息学技术进一步挖掘活性次级代谢产物,推动海洋放线菌领域进一步发展。     

Actinomycetes: an unexplored microorganisms for plant growth promotion and biocontrol in vegetable crops

Actinomycetes, a Gram positive bacteria, well reported as a source of antibiotics, also possess potential to control various plant pathogens, besides acting as plant growth promoting agent. Chemicals in different forms are extensively being used in vegetable farming, adversely affecting the environment and consumer health. Microbial agent like actinomycetes can substantially replace these harmful chemicals, and have now started finding a place as an important input in to farming practices. Only selected vegetable crops belonging to 11 different families have been explored with use of actinomycetes as biocontrol and plant growth promoting agent till now. It provides ample opportunities to vegetable researchers, to further explore with use of this very important group of microorganisms, in order to achieve even higher production level of safe vegetables. Mycostop and Actinovate are two actinomycetes based formulations globally available for use in vegetable farming as a substitute for chemical formulations. Present review article has summarized the literature available on use of actinomycetes in vegetable farming. Existing wide gap in knowledge, and potential thrust areas for future research have also been projected.     

Actinomycetes inducing phytotoxic or fungistatic activity in a Douglas-fir Forest and in an adjacent area of repeated regeneration failure in Southwestern Oregon

Actinomycetes were isolated from the upper 1 - 3 cm of the soil layer in a well-developed forest and in an adjacent clearcut area where Douglas-fir [Pseudotsuga menziesii (MIRB.) Franco] regeneration had been impaired for two decades. The population density in the clearcut area was two times as high as that in the forested area. The percentage of actinomycetes that inhibited seed germination of the test plants was significantly higher in isolates obtained from the clearcut area than in those obtained from the forested area, and isolates from the clearcut showed five times the phytotoxic effect of those from the forest. Some actinomycete isolates, 4 % from the clearcut and 2.6 % from the forest, significantly reduced in vitro growth of two common ectomycorrhizal fungi of Douglas-fir,Laccaria laccata andHebeloma ovstuliniforme. Two actinomycete isolates from the clearcut reduced fungal growth by 40 % and 73 %. Reduction of the nutrient in the growth medium did not affect the antifungal activity of the actinomycetes. The data support the idea that, along with other factors, phytotoxic and antifungal actinomycetes may suppress natural regeneration or establishment of planted seedlings - either directly or. indirectly - through inhibition of seed germination or of mycorrhizal fungi.     

药用昆虫喙尾琵琶甲肠道抗菌活性放线菌的筛选及鉴定

【目的】探究药用昆虫喙尾琵琶甲Blaps rynchopetera肠道具有抗菌活性的放线菌,为抗菌药物开发提供新的放线菌资源。【方法】结合稀释涂布法和选择培养法从喙尾琵琶甲成虫肠道分离放线菌。以耐甲氧西林金黄色葡萄球菌(methieillin-resistant Staphylococus aureus)、金黄色葡萄球菌、大肠杆菌Escherichia coli、粪肠球菌Enterococcus faecalis、鼠伤寒沙门氏菌Salmonella typhimurium 和铜绿假单胞菌Pseudomonas aeruginosa 6株致病细菌以及黑曲霉Aspergillus niger、青霉菌Penicillium expansum和白色念珠菌Canidia albicans 3株致病真菌为指示菌,通过牛津杯琼脂扩散法测试放线菌次生代谢产物的抗菌活性。随后,采用分子生物学方法进行16S rRNA序列分析鉴定活性显著的18株放线菌并构建系统发育树。【结果】从喙尾琵琶甲成虫肠道中分离到176株共生放线菌,初步活性筛选结果显示其中46株放线菌表现出不同程度的抗菌活性。多株放线菌对致病菌具有广谱的抑菌作用且抑菌圈直径超过阳性对照药。选择抑菌圈直径大于15 mm的18株放线菌进行分子鉴定,结果显示均为链霉属Streptomyces。【结论】喙尾琵琶甲肠道含有丰富的抗菌活性放线菌资源。     

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.     

Isolation and Diversity of Actinomycetes in the Chesapeake Bay

Chesapeake Bay was investigated as a source of actinomycetes to screen for production of novel bioactive compounds. The presence of relatively large populations of actinoplanetes (chemotype II/D actinomycetes) in Chesapeake Bay sediment samples indicates that it is an eminently suitable ecosystem from which to isolate actinomycetes for screening programs. Actinomycetes were isolated from sediment samples collected in Chesapeake Bay with an isolation medium containing nalidixic acid, which proved to be more effective than heat pretreatment of samples. Actinomycete counts ranged from a high of 1.4 × 10⁵ to a low of 1.8 × 10² CFU/ml of sediment. Actinomycetes constituted 0.15 to 8.63% of the culturable microbial community. The majority of isolates from the eight stations studied were actinoplanetes (i.e., chemotype II/D), and 249 of these isolates were obtained in a total of 298 actinomycete isolates. Antimicrobial activity profiles indicated that diverse populations of actinoplanetes were present at each station. DNA hybridization studies showed considerable diversity among isolates between stations, but indicated that actinoplanete strains making up populations at nearby stations were more similar to each other than to populations sampled at distant stations. The diversity of actinoplanetes and the ease with which these organisms were isolated from Chesapeake Bay sediments make this a useful source of these actinomycetes.     

Rapid detection of `rare' actinomycetes in environmental samples

Natural products continue to be a useful source of new leads for the pharmaceutical industry. Actinomycetes are prolific producers of natural products and one strategy to increase the possibility of discovering novel chemical entities is to screen actinomycetes considered 'rare' in the environment and previously under-represented in natural product screening collections. We describe a method using bacteriophage as a marker to detect these actinomycetes in environmental samples. This method allows samples to be pre-screened for the presence of target actinomycetes before lengthy isolation programmes are undertaken.     

波罗的海放线菌的多样性

摘要:【目的】为了探索海洋放线菌的多样性,为发现新的药物先导化合物提供新菌源,我们分析了波罗的海的放线菌多样性及生物活性。【方法】采集100 份底泥样品,用7 种培养基分离放线菌809 株;去掉相同菌株后,选择280 株代表菌进行了初步分类鉴定;采用琼脂扩散法,检测了它们对5 种细菌、真菌的抗菌活性;用API ZYM system 测定了21 种酶的活性。【结果】用海藻糖-脯氨酸培养基和HV 培养基分离的放线菌中,稀有放线菌占60% 和63% ;波罗的海的放线菌有15 个属,其中3 个属是首次从海洋中分离     

广西北部湾放线菌的分离筛选及活性产物的鉴定

为从广西北部湾的泥样和植物中分离海洋放线菌,筛选具有抑菌活性的菌株,分离活性化合物。研究采用普通稀释法分离菌株,对发酵产物进行抑菌活性测试,利用活性追踪分离活性化合物,并通过波谱方法确定化合物结构。结果表明从6个海泥样品和3个植物样品中共分离73株放线菌,筛选得到具有抑香蕉枯萎病和金黄色葡萄球菌活性的菌株7株,并从其中的1株链霉菌 Streptomyces sp.MDCW-126的次级代谢产物中分离鉴定了星形孢菌素。从广西北部湾分离的药用活性菌株资源具有开发和深入研究价值。     

Tetrodotoxin production of actinomycetes isolated from marine sediment

Ten actinomycetes isolated from various marine sediments were investigated for the production of tetrodotoxin (TTX). Tissue culture assay, high performance liquid chromatography and gas chromatography-mass spectrometry confirmed that the nine strains produced TTX. Actinomycetes may be responsible for TTX accumulation in marine environments.     

微波处理对钙质土壤放线菌分离效果的影响

采用平板稀释法研究了微波处理对钙质土壤放线菌分离效果的影响。结果表明:①微波处理对提高放线菌出菌率有促进作用。在高氏1号培养基(GA)上,4种供试土壤经微波处理后放线菌总数较对照增加99.5%～234.1%,微波处理效应均达到极显著水平(P0.01);链霉菌数较对照增加58.3%～377.7%,除3号土样外,微波处理效应均达到极显著水平(P0.01);未鉴定放线菌较对照增加96.7%～304.8%,处理与对照的差异均达到显著水平(P0.05);②不同培养基上的微波处理效应不同。GA培养基上的微波处理效应(Effect of Microwave Irradiation,EMI)最强,放线菌总数及未鉴定属放线菌的微波处理效应均表现为GAGA-Ca,GA培养基上放线菌总数的EMI较GA-Ca培养基提高26.4%～120.9%;③风干土样的微波处理效应大于湿土样。