Culturable Actinobacteria from the Marine Sponge Hymeniacidon perleve: Isolation and Phylogenetic Diversity by 16S rRNA gene-RFLP Analysis

A total of 106 actinobacteria associated with the marine sponge Hymeniacidon perleve collected from the Yellow Sea, China were isolated using eight different media. The number of species and genera of actinobacteria recovered from the different media varied significantly, underlining the importance of optimizing the isolation conditions. The phylogenetic diversity of the actinobacteria isolates was assessed using 16S rRNA gene amplification–restriction fragment length polymorphism (RFLP) analysis of the 106 strains with different morphologies. The RFLP fingerprinting of selected strains by HhaI-digestion of the 16S rRNA genes resulted in 11 different patterns. The HhaI-RFLP analysis gave good resolution for the identification of the actinobacteria isolates at the genus level. A phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to seven genera of culturable actinobacteria including Actinoalloteichus, Micromonospora, Nocardia, Nocardiopsis, Pseudonocardia, Rhodococcus, and Streptomyces. The dominant genus was Streptomyces, which represented 74% of the isolates. Three of the strains identified are candidates for new species.     

Biologically active secondary metabolites from Actinomycetes

Secondary metabolites obtained from Actinomycetales provide a potential source of many novel compounds with antibacterial, antitumour, antifungal, antiviral, antiparasitic and other properties. The majority of these compounds are widely used as medicines for combating multidrug-resistant Gram-positive and Gram-negative bacterial strains. Members of the genus Streptomyces are profile producers of previously-known secondary metabolites. Actinomycetes have been isolated from terrestrial soils, from the rhizospheres of plant roots, and recently from marine sediments. This review demonstrates the diversity of secondary metabolites produced by actinomycete strains with respect to their chemical structure, biological activity and origin. On the basis of this diversity, this review concludes that the discovery of new bioactive compounds will continue to pose a great challenge for scientists.     

Marine <Emphasis Type="Italic">Streptomyces</Emphasis> as a novel source of bioactive substances

Marine actinobacteria are the most economically as well as biotechnologically valuable prokaryotes. Representative genera of marine actinobacteria include Actinomadura, Aeromicrobium, Dietzia, Gordonia, Marinophilus, Micromonospora, Nonomuraea, Rhodococcus, Saccharomonospora, Saccharopolyspora, Salinispora, Streptomyces, Solwaraspora, Williamsia, Verrucosispora and several others. Among the genera of marine actinobacteria, the genus Streptomyces is represented in nature by the largest number of species and varieties, which differ greatly in their morphology, physiology, and biochemical activities. Marine Streptomyces occur in different biological sources such as fishes, molluscs, sponges, seaweeds and mangroves, besides seawater and sediments. In this review an evaluation is made on the present state of research on marine Streptomyces and its perspectives. The highlights include the production of metabolites such as antibiotics, anticancer compounds, enzymes, enzyme inhibitors and pigments by marine Streptomyces and their application as single cell protein and as probiotics in aquaculture. The marine environment contains a wide range of distinct Streptomyces that are not present in the terrestrial environment. With increasing advancement in science and technology, there would be greater demands in future for new bioactive compounds synthesised by Streptomyces from various marine sources.     

Endophytic Actinobacteria from the Brazilian Medicinal Plant Lychnophora ericoides Mart. and the Biological Potential of Their Secondary Metabolites

Endophytic actinobacteria from the Brazilian medicinal plant Lychnophora ericoides were isolated for the first time, and the biological potential of their secondary metabolites was evaluated. A phylogenic analysis of isolated actinobacteria was accomplished with 16S rRNA gene sequencing, and the predominance of the genus Streptomyces was observed. All strains were cultured on solid rice medium, and ethanol extracts were evaluated with antimicrobial and cytotoxic assays against cancer cell lines. As a result, 92% of the extracts showed a high or moderate activity against at least one pathogenic microbial strain or cancer cell line. Based on the biological and chemical analyses of crude extracts, three endophytic strains were selected for further investigation of their chemical profiles. Sixteen compounds were isolated, and 3‐hydroxy‐4‐methoxybenzamide ( 9 ) and 2,3‐dihydro‐2,2‐dimethyl‐4(1H)‐quinazolinone ( 15 ) are reported as natural products for the first time in this study. The biological activity of the pure compounds was also assessed. Compound 15 displayed potent cytotoxic activity against all four tested cancer cell lines. Nocardamine ( 2 ) was only moderately active against two cancer cell lines but showed strong activity against Trypanosoma cruzi. Our results show that endophytic actinobacteria from L. ericoides are a promising source of bioactive compounds.     

Marine Sediment-Derived Streptomyces Bacteria from British Columbia,Canada Are a Promising Microbiota Resource for the Discovery of Antimicrobial Natural Products

Representatives of the genus Streptomyces from terrestrial sources have been the focus of intensive research for the last four decades because of their prolific production of chemically diverse and biologically important compounds. However, metabolite research from this ecological niche had declined significantly in the past years because of the rediscovery of the same bioactive compounds and redundancy of the sample strains. More recently, a new picture has begun to emerge in which marine-derived Streptomyces bacteria have become the latest hot spot as new source for unique and biologically active compounds. Here, we investigated the marine sediments collected in the temperate cold waters from British Columbia, Canada as a valuable source for new groups of marine-derived Streptomyces with antimicrobial activities. We performed culture dependent isolation from 49 marine sediments samples and obtained 186 Streptomyces isolates, 47 of which exhibited antimicrobial activities. Phylogenetic analyses of the active isolates resulted in the identification of four different clusters of bioactive Streptomyces including a cluster with isolates that appear to represent novel species. Moreover, we explored whether these marine-derived Streptomyces produce new secondary metabolites with antimicrobial properties. Chemical analyses revealed structurally diverse secondary metabolites, including four new antibacterial novobiocin analogues. We conducted structure-activity relationships (SAR) studies of these novobiocin analogues against methicillin-resistant Staphylococcus aureus (MRSA). In this study, we revealed the importance of carbamoyl and OMe moieties at positions 3” and 4” of novobiose as well as the hydrogen substituent at position 5 of hydroxybenzoate ring for the anti-MRSA activity. Changes in the substituents at these positions dramatically impede or completely eliminate the inhibitory activity of novobiocins against MRSA.     

Phytoplankton trigger the production of cryptic metabolites in the marine actinobacterium Salinispora tropica

Filamentous members of the phylum Actinobacteria are a remarkable source of natural products with pharmaceutical potential. The discovery of novel molecules from these organisms is, however, hindered because most of the biosynthetic gene clusters (BGCs) encoding these secondary metabolites are cryptic or silent and are referred to as orphan BGCs. While co-culture has proven to be a promising approach to unlock the biosynthetic potential of many microorganisms by activating the expression of these orphan BGCs, it still remains an underexplored technique. The marine actinobacterium Salinispora tropica, for instance, produces valuable compounds such as the anti-cancer molecule salinosporamide but half of its putative BGCs are still orphan. Although previous studies have used marine heterotrophs to induce orphan BGCs in Salinispora, its co-culture with marine phototrophs has yet to be investigated. Following the observation of an antimicrobial activity against a range of phytoplankton by S. tropica, we here report that the photosynthate released by photosynthetic primary producers influences its biosynthetic capacities with production of cryptic molecules and the activation of orphan BGCs. Our work, using an approach combining metabolomics and proteomics, pioneers the use of phototrophs as a promising strategy to accelerate the discovery of novel natural products from marine actinobacteria.     

Pharmaceutically active secondary metabolites of marine actinobacteria

Marine actinobacteria are one of the most efficient groups of secondary metabolite producers and are very important from an industrial point of view. Many representatives of the order Actinomycetales are prolific producers of thousands of biologically active secondary metabolites. Actinobacteria from terrestrial sources have been studied and screened since the 1950s, for many important antibiotics, anticancer, antitumor and immunosuppressive agents. However, frequent rediscovery of the same compounds from the terrestrial actinobacteria has made them less attractive for screening programs in the recent years. At the same time, actinobacteria isolated from the marine environment have currently received considerable attention due to the structural diversity and unique biological activities of their secondary metabolites. They are efficient producers of new secondary metabolites that show a range of biological activities including antibacterial, antifungal, anticancer, antitumor, cytotoxic, cytostatic, anti-inflammatory, anti-parasitic, anti-malaria, antiviral, antioxidant, anti-angiogenesis, etc. In this review, an evaluation is made on the current status of research on marine actinobacteria yielding pharmaceutically active secondary metabolites. Bioactive compounds from marine actinobacteria possess distinct chemical structures that may form the basis for synthesis of new drugs that could be used to combat resistant pathogens. With the increasing advancement in science and technology, there would be a greater demand for new bioactive compounds synthesized by actinobacteria from various marine sources in future.     

Marine actinomycetes as a source of novel secondary metabolites

A set of 600 actinomycetes strains which were isolated from marine sediments from various sites in the Pacific and Atlantic Oceans were screened for the production of bioactive secondary metabolites. Marine streptomycete strains were found to be producers of well known chemically diverse antibiotics isolated from terrestrial streptomycetes, as in the case of marine Micromonospora strains. New marine members of the rare genus Verrucosispora seem to be a promising source for novel bioactive secondary metabolites as shown in the case of the abyssomicin producing strain AB-18-032.     

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1–M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.     

Unique Actinomycetes from Marine Caves and Coral Reef Sediments Provide Novel PKS and NRPS Biosynthetic Gene Clusters

In the ever-expanding search for novel bioactive molecules and enzymes, marine actinomycetes have proven to be a productive source. While open reef sediment and sponge-associated actinomycetes have been extensively examined, their marine cave counterparts remain unevaluated. Anchialine cave systems in the Bahamas offered an ideal setting to evaluate the occurrence and variation within sediment-associated actinomycete communities. While in close geographical proximity to open reef environments, these systems provide a specialized environmental niche devoid of light and direct exposure to nutrient input. In the present study, selective isolation techniques and molecular methods were used to test the hypothesis that variable distribution of actinomycetes and secondary metabolite gene clusters occur between open reef and marine cave systems. The results indicated that differences exist within the culturable sediment-associated actinomycete communities between marine caves and open reef systems, with members of the genus Streptomyces dominating cultures from open reef sediments and a more diverse suite of actinomycetes isolated from marine cave sediment samples. Within the cave isolates, members of the proposed genus Solwaraspora were the most represented. Based on PKS- and NRPS-gene-targeted PCR amplification and sequencing, geographic variation in the occurrence of these biosynthetic pathways was also observed. These findings indicate that marine cave systems are a lucrative source in the search for novel secondary metabolite producers with biotechnological applications and that environmental and geographic factors likely affect the occurrence of these biosynthetic pathways.     

链霉菌形态分化与次级代谢产物合成的研究进展

链霉菌是一类具有复杂的形态分化周期和强大的次级代谢能力的高GC含量的放线菌,能够利用其初级代谢产生的前体化合物和能量,合成多种结构复杂、功能多样的具有生物活性的次级代谢产物,在农业、食品、畜牧业、工业以及医药研究等领域都具有重要的价值。在链霉菌的形态分化后期常常伴随着次级代谢产物的生物合成,并且两者都受到复杂的网络调控;同时链霉菌的形态对次级代谢产物的产量和种类造成很大影响。对链霉菌生长周期的全面理解将加深对链霉菌形态分化与次级代谢产物合成关系的认识。本文将对链霉菌的形态分化过程、形态分化和抗生素合成两者共同的调控因子以及链霉菌形态与抗生素产量之间的关系进行综述,这将有助于理解抗生素的合成过程,也将会在缩短发酵周期、构建高产工程菌株、新型杀菌剂的研发以及新型抗生素的合成等方面给予我们启发。     

链霉菌次级代谢产物高产菌株的构建策略

随着基因测序技术的发展,人类获得了大量有关链霉菌次级代谢产物合成基因簇的信息,通过合理的构建策略可以激活其中的隐性基因簇或提高基因簇的表达水平,从而获得新的链霉菌次级代谢产物,或显著提高已知次级代谢产物的发酵水平。从基因表达调控、转座子突变、合成生物学方法、组学方法等四个方面,综述了提高链霉菌次级代谢产物产量的构建策略及其研究进展。     

Genome mining of Streptomyces ambofaciens

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.     

海南西海岸红树林伴生植物内生放线菌多样性及其延缓衰老活性初筛

为寻找新型抗衰老药物,该文以海南西海岸红树林伴生植物为研究对象,采用9种不同培养基从7种伴生植物21份样品中分离纯化放线菌,通过PCR扩增,16S rRNA基因序列分析已纯化放线菌的多样性,利用秀丽隐杆线虫(Caenorhabditis elegans)模型筛选菌株来进行延缓衰老活性研究。结果表明:(1)从7种伴生植物21份样品中共分离到26株海洋放线菌,隶属于9科15属,分别为拟诺卡菌属、短状杆菌属、短小杆菌属、Demequina、戈登氏菌属、类诺卡氏菌属、Lysinimicrobium、细杆菌属、假诺卡氏菌属、微球菌属、原小单孢菌属、拟无枝酸菌属、Yimella、北里孢菌属和链霉菌属,其中链霉菌属为优势菌属。(2)经秀丽隐杆线虫模型筛选,发现有2株海洋放线菌的发酵粗提物具有延缓秀丽隐杆线虫衰老的作用。综上结果说明海南西海岸红树林伴生植物中含有丰富多样的药用放线菌资源,为海洋放线菌抗衰老研究奠定了基础。     

Sponge-associated sp. RM66 metabolome induction with N-acetylglucosamine: Antibacterial,antifungal and anti-trypanosomal activities

The marine sponge Amphimedon sp., collected from Hurghada (Egypt) was investigated for its sponge-derived actinomycetes diversity. Nineteen actinomycetes were cultivated and phylogenetically identified using 16S rDNA gene sequencing were carried out. The strains belong to genera Kocuria, Dietzia, Micrococcus, Microbacterium and Streptomyces. Many silent biosynthetic genes clusters were investigated using genome sequencing of actinomycete strains and has revealed in particular the genus Streptomyces that has indicated their exceptional capacity for the secondary metabolites production that not observed under classical cultivation conditions. In this study, the effect of N-acetylglucosamine on the metabolome of Streptomyces sp. RM66 was investigated using three actinomycetes media (ISP2, M1 and MA). In total, twelve extracts were produced using solid and liquid fermentation approaches. Liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data were analysed using metabolomics tools to compare natural product production across all crude extracts. Our study highlighted the elicitation effect of N-acetylglucosamine on the secondary metabolite profiles of Streptomyces sp. RM66. These results highlight the of N-acetylglucosamine application as an elicitor to induce the cryptic metabolites and for increasing the chemical diversity. All the twelve extracts were tested for their antibacterial activity was tested against Staphylococcus aureus NCTC 8325, antifungal activity against Candida albicans 5314 (ATCC 90028) and anti-trypanosomal activity against Trypanosoma brucei brucei. Extract St1 showed the most potent one with activities 2.3, 3.2 and 4.7 ug/ml as antibacterial, antifungal and anti-trypanosomal, respectively.     

Isolation,Classification and Antagonistic Properties of Alkalitolerant Actinobacteria from Algerian Saharan Soils

Abstract

The Sahara, one of the most extreme environments on Earth, constitutes an unexplored source of alkalitolerant actinobacteria. In this work, we studied the diversity of alkalitolerant actinobacteria in various soils collected from different regions of the Algerian Sahara. A total of 29 alkalitolerant actinobacterial strains were isolated by using a complex agar medium. The diversity of these actinobacteria was evaluated using a polyphasic approach, which included morphological, chemotaxonomic, physiological (numerical taxonomy) and 16S rRNA gene analyses. The isolates which were assigned to the genus Nocardiopsis, shared relatively low 16S rRNA gene sequences similarities compared to closely related species suggesting that they belonged to putatively new species. All of the strains were tested for antibiotic activity against a broad range of microorganisms and screened for genes encoding polyketide synthases and non-ribosomal peptide synthetases and found to have the potential to produce secondary metabolites. Consequently, the study supports the view that extreme environments contain many novel actinobacteria, which represent an unexplored source for the discovery of biologically active compounds.     

Research on marine actinobacteria in India

Marine actinobacteriology is one of the major emerging areas of research in tropics. Marine actinobacteria occur on the sediments and in water and also other biomass (mangrove) and substrates (animal). These organisms are gaining importance not only for their taxonomic and ecological perspectives, but also for their unique metabolites and enzymes. Many earlier studies on these organisms were confined only to the temperate regions. In tropical environment, investigations on them have gained importance only in the last two decades. So far, from the Indian peninsula, 41 species of actinobacteria belonging to 8 genera have been recorded. The genus, Streptomyces of marine origin has been more frequently recorded. Of 9 maritime states of India, only 4 have been extensively covered for the study of marine actinobacteria. Most of the studies conducted pertain to isolation, identification and maintenance of these organisms in different culture media. Further, attention has been focused on studying their antagonistic properties against different pathogens. Their biotechnological potentials are yet to be fully explored.     

Genetic background affects pathogenicity island function and pathogen emergence in Streptomyces

With few exceptions, thaxtomin A (ThxA), a nitrated diketopiperazine, is the pathogenicity determinant for plant‐pathogenic Streptomyces species. In Streptomyces scabiei (syn. S. scabies), the ThxA biosynthetic cluster is located within a 177‐kb mobile pathogenicity island (PAI), called the toxicogenic region (TR). In S. turgidiscabies, the ThxA biosynthetic cluster is located within a 674‐kb pathogenicity island (PAIst). The emergence of new plant pathogens occurs in this genus, but not frequently. This raises the question of whether the mobilization of these pathogenicity regions, through mating, is widespread and whether TR and PAIst can confer plant pathogenicity. We showed that ThxA biosynthetic clusters on TR and PAIst were transferred into strains from five non‐pathogenic Streptomyces species through mating with S. scabiei and S. turgidiscabies. However, not all of the transconjugants produced ThxA and exhibited the virulence phenotype, indicating that the genetic background of the recipient strains affects the functionality of the ThxA biosynthetic cluster and therefore would be expected to affect the emergence of novel pathogenic Streptomyces species. Thxs have been patented as natural herbicides, but have yet to be commercialized. Our results also demonstrated the potential of the heterologous production of ThxA as a natural and biodegradable herbicide in non‐pathogenic Streptomyces species.     

稀有放线菌Lechevalieria rhizosphaerae NEAU-A2的次级代谢产物研究及遗传操作系统的建立

【背景】菌株Lechevalieria rhizosphaerae NEAU-A2是一株新的稀有放线菌,其基因组中包含多个与次级代谢产物生物合成相关的基因簇,具有产生丰富代谢产物的潜力。【目的】对稀有放线菌L. rhizosphaerae NEAU-A2的次级代谢产物进行研究,以期发现结构新颖或生物活性独特的化合物,并建立其遗传操作系统。【方法】应用硅胶柱色谱和高效液相色谱等方法对菌株NEAU-A2的次级代谢产物进行分离和纯化,整合质谱分析、核磁共振等方法进行结构解析。在对该菌株全基因组测序的基础上,以基因组序列中编码杂合的聚酮合酶-非核糖体肽合成酶(PolyketideSynthase-Nonribosomal Peptide Synthetase,PKS-NRPSs)基因1609为目标基因,利用PCR-Targeting介导的基因置换技术构建重组质粒,通过接合转移的方式导入野生菌株。【结果】从菌株NEAU-A2中分离鉴定了2个新的吲哚二聚化合物(1,2)和5个已知化合物N-乙酰色胺(3)、4-((2-(1H-Indol-3-Yl)Ethyl)Amino)-4-Oxobutanoic Acid (4)、Brevianamide F (5)、4S,7R-Germacra-1(10)E,5E-Diene-11-Ol (6)、1H-吡咯-2-羧酸(7)。接合子通过培养2代即可获得双交换突变菌株,PCR分析结果显示PKS-NRPS基因被成功中断。【结论】从稀有放线菌L. rhizosphaerae NEAU-A2中分离鉴定出7个化合物,并成功建立了该菌的遗传操作系统。     

Red Soils Harbor Diverse Culturable Actinomycetes That Are Promising Sources of Novel Secondary Metabolites

Red soils, which are widely distributed in tropical and subtropical regions of southern China, are characterized by low organic carbon, high content of iron oxides, and acidity and, hence, are likely to be ideal habitats for acidophilic actinomycetes. However, the diversity and biosynthetic potential of actinomycetes in such habitats are underexplored. Here, a total of 600 actinomycete strains were isolated from red soils collected in Jiangxi Province in southeast China. 16S rRNA gene sequence analysis revealed a high diversity of the isolates, which were distributed into 26 genera, 10 families, and 7 orders within the class Actinobacteria; these taxa contained at least 49 phylotypes that are likely to represent new species within 15 genera. The isolates showed good physiological potentials for biosynthesis and biocontrol. Chemical screening of 107 semirandomly selected isolates spanning 20 genera revealed the presence of at least 193 secondary metabolites from 52 isolates, of which 125 compounds from 39 isolates of 12 genera were putatively novel. Macrolides, polyethers, diketopiperazines, and siderophores accounted for most of the known compounds. The structures of six novel compounds were elucidated, two of which had a unique skeleton and represented characteristic secondary metabolites of a putative novel Streptomyces phylotype. These results demonstrate that red soils are rich reservoirs for diverse culturable actinomycetes, notably members of the families Streptomycetaceae, Pseudonocardiaceae, and Streptosporangiaceae, with the capacity to synthesize novel bioactive compounds.