International Streptomyces Project Research Articles

Unraveling growth-promoting potential of plant beneficial actinobacteria on tropical bryophytes

Bryophytes are non-vascular plants with dominant gametophyte stage that play vital ecological roles in natural ecosystems. Unfortunately, their populations are currently in decline due to habitat destruction and various anthropogenic activities. The conservation efforts for bryophytes are hampered by their slow growth rates. This study aims to investigate the potential of actinobacteria to promote the growth of bryophytes. In this study, three plant growth-promoting actinobacteria, Dermacoccus abyssi MT1.1T, Micromonospora chalcea CMU55-4 and Streptomyces thermocarboxydus S3 were cultured in International Streptomyces Project medium 2 (ISP2) broth to obtain culture filtrates containing bioactive compounds for enhancing the growth of two bryophyte species, Physcomotrium sphaericum (C. Ludw.) Fürnr and Sphagnum cuspidatulum C. Müll. Interestingly, the incorporation of actinobacterial culture filtrates into 1/16 Murashige and Skoog (MS) medium yielded superior growth performance of P. sphaericum (C. Ludw.) Fürnr and S. cuspidatulum C. Müll, as observed from the thallus height, fresh weight, total chlorophyll contents, and total carotenoid contents compared to control groups. In addition, the inoculation of M. chalcea CMU55-4 on S. cuspidatulum C. Müll grown in sterile peat moss demonstrated the highest values for thallus height, fresh weight, dry weight, total chlorophyll content, and total carotenoid content. All actinobacteria successfully colonized the moss seedlings without any observable negative impacts, indicating beneficial interactions between actinobacteria and bryophytes. This research sheds light on the potential of harnessing plant beneficial actinobacteria to enhance the growth of bryophytes for conservation purposes.

Identification of Endophytic Actinomycetes Isolated From Hopea ferrea and Its Antibacterial Activity Against Cariogenic Bacterium

Dental caries affects around 36% of the world's population and results in the loss of primary teeth in about 530 million youngsters. It is described as the loss of the enamel layer of the tooth by acids generated by the activity of cariogenic bacterium such as Streptococcus mutans on carbohydrates. This study aims to identify bioactive endophytic actinomycetes as an antibacterial agent against S. mutans DSM 20523 and to identify the bioactive endophytic actinomycetes using the 16S rRNA sequencing approach. To determine the percentage zone of inhibition, three endophytes at 10-day-old culture have been streaked onto ISP2. Then, S. mutans was streaked perpendicular to the endophytes. Crude extracts from three endophytes have been prepared by using four different types of media namely Tryptic Soy broth, Kings B, International Streptomyces Project Medium No.1 (ISP1) and Starch Casein broth. These crude extracts proceeded to undergo disk diffusion and MIC assays against S. mutans. All three endophytes from streaked plates with the highest percentage zone of inhibition were identified using the 16S rRNA gene molecular approach for characterisation. Endophytic actinomycetes which were isolated from Hopea ferrea showed potent antibacterial activity against S. mutans. All endophytic actinomycetes isolates from streak plates showed good inhibitory activity against the S. mutans. The highest percentage of inhibition was shown by the PT9-13S2 isolate followed by PT9-8S2 and PT9-13W2. Furthermore, crude extracts of PT9-8S2 and PT9-13W2 (the highest ZOI) from TSB media showed inhibitory activities against S. mutans, from disk diffusion as well as minimum inhibitory assays. From the 16S rRNA molecular approach, endophytic actinomycetes PT9-8S2 was closely related to Streptomyces collinus while PT9-13S2 and PT9-13W2 were closely related to Streptomyces malachitospinus. From this study, endophytic actinomycetes showed a promising source as antibacterial agents against cariogenic bacterium S. mutans which is the main causative agent of dental caries.

Streptomyces albipurpureus sp. nov., a novel actinobacterium with antimicrobial activity isolated from the rhizospheric soil of Fritillaria cirrhosa D. Don.

A novel actinobacterium, designated strain CWNU-1T, was isolated from the rhizospheric soil of Fritillaria cirrhosa D. Don and examined using a polyphasic taxonomic approach. The organism developed pale blue aerial mycelia that was simply branched and terminated in open or closed coils of three or more volutions on International Streptomyces Project 3 agar. Spores were ellipsoidal to cylindrical with wrinkled surfaces. The strain showed high 16S rRNA gene sequence similarity to Streptomyces kurssanovii NBRC 13192T (98.8 %), Streptomyces xantholiticus NBRC 13354T (98.7 %) and Streptomyces peucetius JCM 9920T (98.6 %). The phylogenetic result based on 16S rRNA gene and genome sequences clearly demonstrated that strain CWNU-1T formed an independent phylogenetic lineage. On the basis of orthologous average nucleotide identity, CWNU-1T was most closely related to Streptomyces inusitatus NBRC 13601T with 79.3 % identity. The results of the digital DNA-DNA hybridization analysis also indicated low levels of relatedness with other species, as the highest value was observed with S. inusitatus NBRC 13601T (25.3 %). With reference to phenotypic characteristics, phylogenetic data, orthologous average nucleotide identity and digital DNA-DNA hybridization results, strain CWNU-1T was readily distinguished from its most closely related strains and classified as representing a novel species, for which the name Streptomyces albipurpureus sp. nov. is proposed. The type strain is CWNU-1T (=CGMCC 4.7758T=MCCC 1K07402T=JCM 35391T).

Antibacterial activity of Streptomyces spp. crude extract on multidrug resistant Psedomonas aeruginosa isolated from clinical samples

Abstract Background: Actinomycetes are the heterogeneous group of microorganisms, which have thread-like filaments, isolated from the soil. Objectives: Isolation, identification, and partial purification of crude bioactive agents of the Actinomycetes isolates and isolation and identification of Pseudomonas aeruginosa from clinical samples, and to study antibiotics susceptibility for these isolates. Materials and Methods: Forty soil samples were collected from different agriculture locations in Hilla city. Soil samples were air dried and pre-treated with calcium carbonate (1%). The best selective medium for growth Actinomycetes is yeast malt extract agar medium (International Streptomyces project—ISP2) with 1 mg/mL Nystatin as antifungal agent. Results: A total of 68 actinomycetes species were isolated and 23 Streptomyces spp. isolates were recovered after molecular identification. By cross- streaking method , the antibacterial activity for these isolated were tested against 30 clinical isolates of P. aeruginosa that showed resistance to 17 antibiotic types of streptomyces isolates demonstrated potential antibacterial activity were mass cultured and secondary metabolites were extracted using ethyl acetate. Among the 23 isolated strains, seven isolates (St4, St5, St6, St8, St11, St12, and St14) demonstrated a significant antibacterial activity with the zone of inhibition ranging from 0 to 22 mm against P. aeruginosa, according to the primary and secondary screening. Conclusion: The isolates St6 and St12 showed the higher antimicrobial metabolites activity, so these isolates were elected for sequencing.

Phenotypic and Genomic Characterization of Streptomyces pakalii sp. nov., a Novel Species with Anti-Biofilm and Anti-Quorum Sensing Activity in ESKAPE Bacteria.

The increasing number of infections caused by antimicrobial multi-resistant microorganisms has led to the search for new microorganisms capable of producing novel antibiotics. This work proposes Streptomyces pakalii sp. nov. as a new member of the Streptomycetaceae family. The strain ENCB-J15 was isolated from the jungle soil in Palenque National Park, Chiapas, Mexico. The strain formed pale brown, dry, tough, and buried colonies in the agar with no diffusible pigment in GAE (glucose-asparagine-yeast extract) medium. Scanning electron micrographs showed typical mycelium with long chains of smooth and oval-shaped spores (3-10 m). The strain grew in all of the International Streptomyces Project (ISP)'s media at 28-37 °C with a pH of 6-9 and 0-10% NaCl. S. pakalii ENCB-J15 assimilated diverse carbon as well as organic and inorganic nitrogen sources. The strain also exhibited significant inhibitory activity against the prodigiosin synthesis of Serratia marcescens and the inhibition of the formation and destruction of biofilms of ESKAPE strains of Acinetobacter baumannii and Klebsiella pneumoniae. The draft genome sequencing of ENCB-J15 revealed a 7.6 Mb genome with a high G + C content (71.6%), 6833 total genes, and 6746 genes encoding putative proteins. A total of 26 accessory clusters of proteins associated with carbon sources and amino acid catabolism, DNA modification, and the antibiotic biosynthetic process were annotated. The 16S rRNA gene phylogeny, core-proteome phylogenomic tree, and virtual genome fingerprints support that S. pakalii ENCB-J15 is a new species related to Streptomyces badius and Streptomyces globisporus. Similarly, its average nucleotide identity (ANI) (96.4%), average amino acid identity (AAI) (96.06%), and virtual DNA-DNA hybridization (67.3%) provide evidence to recognize it as a new species. Comparative genomics revealed that S. pakalli and its closest related species maintain a well-conserved genomic synteny. This work proposes Streptomyces pakalii sp. nov. as a novel species that expresses anti-biofilm and anti-quorum sensing activities.

Streptomyces fuscus sp. nov., a brown-black pigment producing actinomycete isolated from dry mudflat sand.

A novel Gram-stain-positive, aerobic actinobacterial strain, designated GXMU-J15T, was isolated from dry mudflat sand. A polyphasic approach was employed for its taxonomic characterization. The strain developed extensively branched yellowish white to light yellow substrate mycelia and white aerial mycelia, and produced smooth cylindrical spores in a loose straight spore chain on International Streptomyces Project 2-7 agar media. Strain GXMU-J15T grew at 20-50 °C (optimum, 35 °C), at pH 5.0-8.0 (optimum, pH 7.0) and in the presence of 0-8 % (w/v) NaCl. Analysis of 16S rRNA gene sequences indicated that strain GXMU-J15T represents a member of the genus Streptomyces. Strain GXMU-J15T showed the highest 16S rRNA gene sequence similarity to Streptomyces lusitanus CGMCC 4.1745T (99.1 %) and Streptomyces thermocarboxydus CGMCC 4.1883T (98.8 %). Phylogenetic tree analysis based on multilocus sequence analysis (MLSA) and whole genome sequence construction revealed that strain GXMU-J15T was most closely related to Streptomyces cupreus PSKA01T, Streptomyces cinnabarinus DSM 40467T and Streptomyces davaonensis JCM 4913T. The MLSA and genome-to-genome distances between strain GXMU-J15T and its relatives were 0.0418, 0.0443 and 0.0485 and 0.1237, 0.1188 and 0.1179, respectively. The results of orthologous average nucleotide identity and digital DNA-DNA hybridization analysis corroborated the results of the MLSA and whole genome sequence evolution analysis, indicating that the novel isolate represents a distinct species of the genus Streptomyces. The whole-cell sugars of strain GXMU-J15T were xylose, glucose and galactose. The characteristic diamino acid in the cell-wall hydrolysate was ll-diaminopimelic acid. The lipids contained diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylglycerides, phosphatidylcholine, two phospholipids of an unknown structure containing glucosamine, one unknown phospholipid and two unknown lipids. The major cellular fatty acid components were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The main respiratory quinone types were MK-9(H6) and MK-9(H8). The whole genome size of strain GXMU-J15T was 8.68 Mbp, with 71.23 mol% G+C content. Genomic analysis indicated that strain GXMU-J15T has the potential to synthesize polyketides, terpenes and a series of important antibiotics besides the gene cluster for melanin synthesis. Based on these genotypic and phenotypic data, strain GXMU-J15T is proposed to represent a new species of the genus Streptomyces named Streptomyces fuscus sp. nov. The type strain is GXMU-J15T (=MCCC 1K08211T=JCM 35917T).

Biological characteristics and ability of Streptomyces rochei BT02 to inhibit the Ralstonia solanacearum causing bacterial wilt disease

In the agricultural field, actinomycetes are outstanding biological control agents to inhibit the growth of pathogenic microorganisms. Based on the manual of bacterial classification of the International Streptomyces Project (ISP) and the 16S-rRNA gene sequence, the actinomycete BT02 strain was named Streptomyces rochei BT02. This strain contains two polyketide synthase type I (pks-I) and polyketide synthase type II (pks-II) genes related to antibiotic synthesis. The result showed that the actinomycetes S. rochei BT02 grow well at 31οC, 1% salt, pH 7.0, and assimilate different carbon and nitrogen sources. The S. rochei BT02 showed antibacterial activity against three tested strains of Ralstonia solanacearum (Minimum inhibitory concentration =15 mg/ml). In greenhouse conditions, this strain reduced 43.5% the wilt disease index. That demonstrated that S. rochei BT02 could be a promising source for the production of bioproducts.

Streptomyces zingiberis sp. nov., an endophytic actinobacterium isolated from the root tissue of Zingiber montanum.

An endophytic actinobacterium, designated strain PLAI 1-29T, was isolated from the root tissue of Zingiber montanum collected from Pathum Thani province, Thailand. Strain PLAI 1-29T was characterized using a polyphasic taxonomic approach. It typically exhibited morphological and chemotaxonomic properties of the genus Streptomyces. Strain PLAI 1-29T produced a spiral spore chain on aerial mycelium and grew at 15-40 °C, pH 6-10 on International Streptomyces Project 2 agar. The maximum NaCl concentration for growth was 9 % (w/v). Cells of strain PLAI 1-29T presented ll-diaminopimelic acid, arabinose, galactose and ribose. The detected phospholipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. The major menaquinones were MK-9(H6) and MK-9(H8). The major cellular fatty acids were iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. The genome-based taxonomic details revealed the assignment of strain PLAI 1-29T to the genus Streptomyces and exhibited low threshold values for the delineation of a novel species by average nucleotide identity-blast (84.0%), average amino acid identity (80.0%) and digital DNA-DNA hybridization (27.6%) with its closest type strain, Streptomyces xinghaiensis S187T. Furthermore, several differential physiological and biochemical characteristics were detected between strain PLAI 1-29T and the closest type strain. Based on the combined phenotypic and genomic features, strain PLAI 1-29T (=TBRC 7645T=NBRC 113170T) is considered to represent a new Streptomyces species, for which we propose the name Streptomyces zingiberis sp. nov.

Streptomyces salinarius sp. nov., an actinomycete isolated from solar saltern soil.

An actinobacterium, designated strain SS06011T, was isolated from solar saltern soil collected from Samut Sakhon province, Thailand. The taxonomic position of this strain was established using the polyphasic taxonomic approach. The strain produced grey aerial spore mass on International Streptomyces Project 2 seawater agar that differentiated into spiral spore chains with rugose-surfaced spores. Strain SS06011T was found to have ll-diaminopimelic acid in the cell peptidoglycan. Whole-cell hydrolysates contained galactose, glucose and ribose. MK-9(H6) and MK-9(H4) were major menaquinones. The major cellular fatty acids comprised iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were detected in cells. These characteristics were coincident with the typical morphological and chemotaxonomic properties of the genus Streptomyces. The taxonomic affiliation at the genus level of this strain could also be confirmed by its 16S rRNA gene sequence data. Strain SS06011T showed the highest 16S rRNA gene sequence similarity to Streptomyces ardesiacus NRRL B-1773T (99.1 %), Streptomyces coelicoflavus NBRC 15399T (99.1 %) and Streptomyces hyderabadensis OU-40T (99.1 %). Digital DNA-DNA hybridization (dDDH), average nucleotide identity-blast (ANIb) and average amino acid identity (AAI) values between strain SS06011T and its closely related type strains, S. ardesiacus NBRC 15402T, S. coelicoflavus NBRC 15399T and S. hyderabadensis JCM 17657T, were in the range of 45.4-48.4 % (for dDDH), 90.8-91.9 % (for ANIb) and 90.8-91.7 % (for AAI), respectively, which are lower than the cut-off criteria for species delineation. The DNA G+C content of genomic DNA was 71.9 mol%. With the differences in physiological, biochemical and genotypic data, strain SS06011T could be discriminated from its closest neighbours. Thus, strain SS06011T should be recognized as representing a novel species of the genus Streptomyces, for which the name Streptomyces salinarius sp. nov. is proposed. The type strain is SS06011T (=TBRC 9951T=NBRC 113998T).

Streptomyces longhuiensis sp. nov, a novel species isolated from soil of Lilium brownii in Hunan Province, PR China.

An actinobacterium strain, designated BH-MK-02T, was isolated from the soil of Lilium brownii. The taxonomic position was determined using a polyphasic approach. Strain BH-MK-02T grew well on International Streptomyces Project series media and formed well-developed, branched substrate hyphae and aerial mycelium that differentiated into straight spore chains with a wrinkled surface. The diagnostic diamino acid was ll-diaminopimelic acid. The major menaquinones were MK-9(H4), MK-9(H6) and MK-9(H8). The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannosides, phosphatidylglycerol and unidentified lipid spots. The predominant fatty acids were anteiso-C15 : 0, iso-C16 : 0, C16 : 0 and C16 : 1 ω7c/C16 : 1 ω6c. The phenotypic characteristics of strain BH-MK-02T indicated that it belonged to the genus Streptomyces. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain BH-MK-02T was most closely related to Streptomyces aureus CGMCC 4.1833T (99.7 %). However, the average nucleotide identity and digital DNA-DNA hybridization values between the whole-genome sequences of strain BH-MK-02T and S. aureus CGMCC 4.1833T were 78.1 and 23.2 %, respectively, below the 96.7 and 70 % cut-off points respectively recommended for delineating Streptomyces species. Furthermore, the novel isolate could be distinguished from S. aureus CGMCC 4.1833T by morphological, physiological and biochemical characteristics. Based on all these data, strain BH-MK-02T (=MCCC 1K06237T=JCM 34789T) clearly represents a novel species within the genus Streptomyces, for which the name Streptomyces longhuiensis sp. nov. is proposed.

Exploring the potency of Streptomyces koyangensis strain SHP 9-3 isolated from the soil of Enggano Island (Indonesia) as an antibacterial source

Abstract. Pahira DSJ, Damayanti E, Lisdiyanti P, Mustofa, Hertiani T. 2023. Exploring the potency of Streptomyces koyangensis strain SHP 9-3 isolated from the soil of Enggano Island (Indonesia) as an antibacterial source. Biodiversitas 24: 1460-1466. Streptomyces is a Gram-positive bacterium with the potential a produce beneficial antimicrobials. This study aimed to determine the antimicrobial activity, morphological characteristics, cytotoxicity, and chemical profile of S. koyangensis SHP 9-3. This bacterium was isolated from Enggano Island, Indonesia, and the morphological characteristics were determined using a Scanning Electron Microscope (SEM). Cultivation was conducted in the International Streptomyces Project (ISP-2) media using baffled flasks and a shaker incubator. The secondary metabolites were conducted on the extract against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans by a twofold dilution method. Concurrently, cytotoxicity testing was done on Vero cells. The extract’s chemical profile was analyzed using LC-MS/MS. The results showed a prominent antibacterial activity as the extract inhibited the growth of S. aureus and C. albicans at 7.81 µg/mL. However, a growth inhibition against E. coli was observed at 250 µg/mL and P. aeruginosa at 31.25 µg/mL. The cytotoxic test showed the IC50 value on Vero cells was 5,015 µg/mL, indicating that the extract was nontoxic against Vero cells. The chemical profile of the extract by LC-MS/MS revealed the presence of five major components, i.e., cyclo (Pro-Val) (C10H16N2O2), kuraramine (C12H18N2O2), 1,4-diphenyl-1-pentanone (C17H18O), (3S,4S)-4-amino-3-hydroxy-6-methyl heptanoic acid (C21H42N2O7), and 2-ethyl-8-methyl-2,8-diazaspiro (4,5) decane-1,3-dione (C11H18N2O2).

Antifungal Activity of Streptomyces spectabilis SP-O2 against Aflatoxin Producing Mold, Aspergillus flavus

Among soil isolated species of actinomycetes bacteria that affected the growth of aflatoxin producing mold; Aspergillus flavus TISTR 3041, the isolate SP-O2 showed a promising inhibitory activity on the dual culture assay. This isolated bacterium was characterized on various International Streptomyces Project media. The macroscopic morphological analysis showed mycelium, sporogenesis, and orangish pink of colonial pigment. The scanning electron microscope (SEM) photograph showed that the sporogenesis has occurred by shrinking of both sides of aerial mycelium. After the sporogenesis has been completed, the fragmentation of altered mycelium started from the tips of aerial mycelium to produce 0.8-1 micrometer fragmented spores. The 16S rRNA sequences analysis revealed that isolate SP-O2 shared a 99.93 % (1 mismatch/1436 nt) similarity with Streptomyces spectabilis NBRC 13424 AB184393. Co-culture liquid assay was analyzed for the aflatoxin produced by A. flavus in the presence of Streptomyces spectabilis SP-O2. The result showed that UV-visualized band of aflatoxin B1 was not detected by co-cultivation of Streptomyces spectabilis SP-O2 with A. flavus by thin layer chromatography compared to the control. Indeed, fungal growth of A. flavus was destroyed by the antifungal substance produced by Streptomyces spectabilis SP-O2 in the liquid culture. Furthermore, biocontrol of contaminated A. flavus was conducted on peanut kernels and the results showed that Streptomyces spectabilis SP-O2 has antifungal activity against the presence of A. flavus. Together, these results suggested that further investigation on the development of biocontrol use of Streptomyces spectabilis SP-O2 to overcome the contamination of aflatoxin producing mold in the feedstock and agricultural-based industry is needed. HIGHLIGHTS The isolated actinobacteria in this study was identified as Streptomyces spectabilis isolate SP-O2 based on its morphological characteristics, physiological properties and molecular identification SEM showed the sporogenesis of 0.8-1 micrometer fragmented spores Streptomyces spectabilis isolate SP-O2 exhibited the promising fungicidal activity against flavus in the dual culture agar assay, co-culture liquid assay and peanut kernels assay with consistency results The secreted metabolites play a crucial role on the fungal killing against Aspergillus flavus rather than the aflatoxin degradation GRAPHICAL ABSTRACT

Diversity and Bioactivity of Endophytic Actinobacteria Associated with Grapevines

Grapevine trunk diseases (GTDs) are a significant problem for New Zealand viticulture. Endophytic actinobacteria are of interest as potential biocontrol agents due to their ability to inhibit plant pathogens and improve plant growth. However, no studies have investigated the diversity of actinobacteria associated with grapevines in New Zealand vineyards and their bioactivity. Actinobacteria diversity in different ‘Sauvignon blanc’ vine tissues from three vineyards (conventional and organic management, and different vine ages) was assessed using different methods and media. Forty-six endophytic actinobacteria were isolated, with more isolates recovered from roots (n = 45) than leaves (n = 1) and shoot internodes (n = 0). More isolates were recovered from the organic (n = 21) than conventional (n = 8) vineyard, mature (25-year old; n = 21) than young (2-year old; n = 2) vines and using a tissue maceration technique (n = 40). Actinomycete Isolation Agar, International Streptomyces Project 2, and Starch Casein media were effective for actinobacteria isolation. Most of the isolates recovered belonged to Streptomyces, with one isolate identified as Mycolicibacterium. Forty isolates were assessed for antifungal activity and plant growth-promoting (PGP) characteristics. Of these, 13 isolates had antifungal activity against test GTD pathogens (Dactylonectria macrodidyma, Eutypa lata, Ilyonectria liriodendri, Neofusicoccum parvum, and N. luteum). Eighteen isolates exhibited more than one PGP trait; 25siderophore production (n = 25), phosphate solubilization (n = 6), and indole acetic acid production (n = 16). Two strains, Streptomyces sp. LUVPK-22 and Streptomyces sp. LUVPK-30, exhibited the best antifungal and PGP properties. This study revealed the diversity of culturable endophytic actinobacteria from grapevines in New Zealand vineyards and their biocontrol potential against GTD pathogens.

Antioxidant activities from melanin pigment produced by marine actinobacterium of Streptomyces species.

Melanin is macromolecules which have been developed while oxidative polymerization of phenolic compounds. It has been found that the majority of marine organisms produce melanin pigment. To obtain the melanin from marine actinobacteria and their biological properties are studied. Isolation and identification of marine actinobacteria were carried out using the media of ISP-1, ISP-7. Spore chain morphology, chemotaxonomic characteristics were also analyzed by the International Streptomyces Project. The antioxidant activities of DPPH, lipid peroxide using actinobacterial melanin were determined. The Streptomyces species have the capacity to produce melanin pigments and it shows potential antioxidant properties. When DPPH concentrations were compared with the ascorbic acid standard, the melanin of 150 μg/ml showed 93.47% of scavenging. The present study was concluded that melanin pigment obtained from marine actinobacterium of Streptomyces species has potential antioxidant activities and these components might be useful to pharmaceutical industries.

Paenibacillus hamazuiensis sp. nov., a bacterium isolated from Hamazui hot spring in Yunnan province, south-west China.

A bacterial strain, Gram-positive, aerobic, rod-shaped, motile, designated YIM B00624T which was isolated from a Hamazui hot spring in Tengchong, Yunnan province, south-west China. The strain grew well on International Streptomyces Project (ISP) 2 medium and colonies were creamy yellow, flat and circular. The results of 16S rRNA gene sequence similarity analysis showed that strain YIM B00624T was closely related to the type strain of Paenibacillus filicis S4T (95.9%). The main menaquinone of strain YIM B00624T was menaquinone-7 (MK-7) and major fatty acids were anteiso-C15:0, anteiso-C17:0 and C16:0. The isolate contained meso-diaminopimelic acid as the diagnostic diamino acid and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and four unidentified glycolipids. The DNA G+C content of strain YIM B00624T was 53.4mol%. Based on physiological, phenotypic and chemotaxonomic data, strain YIM B00624T belongs to a novel species of the genus Paenibacillus, for which the name Paenibacillus hamazuiensis sp. nov. is proposed. The type strain is YIM B00624T (= CGMCC 1.19245T = KCTC 43365T).

Antimicrobial Activity of Ten Local Actinobacterial Strains against ESKAPE, Bacillus subtilis and Pseudomonas baetica Pathogens

Aims: The study included the isolation, purification, cultural characteristics, antimicrobial activities and molecular identification of local actinobacterial strains isolated from different locations north of Iraq.
 Methodology: Oligotrophic medium supplemented with the antifungals cycloheximide (50 mg/l) and nystatin (30 mg/l) was used for preliminary isolation. ISP-3 medium was chosen as a potential medium for subsequent purification of actinobacterial strains. The cultural characteristics of all isolated actinobacterial strains were elucidated on International Streptomyces Project media (ISP2-ISP-7). 16S rRNA marker gene was used for molecular identification using 27F and 1492R universal primers.
 Results: Ten isolates were biologically active against tested ESKAPE, Bacillus subtilis and pseudomonas paetica pathogens when cultured on different ISPs media under the OSMAC approach. Six representative isolates that exhibited antimicrobial activity against all or almost tested bacteria were sequenced using 16S rRNA gene. The sequences were compared with those of other actinobacterial strains that are found in Genebank database to find the best similarity and the close reference strains to our isolates. Five of the sequenced strains have been identified as Streptomyces species; MT5, MT8, MT12, MT23 and MT26 and one was identified as a rare actinobacterial strain Lentzea sp.; MT4. Nucleotide sequences have been provided and deposited in the National Center for Biotechnology Information NCBI under the accession numbers ON514131, ON514133, ON514134, ON514135, ON514136 and ON514130 respectively.

Juvenile hormone antagonistic activity of secondary metabolites from Streptomyces lactacystinicus and their insecticidal activity against Plutella xylostella

• IMBL-1412 isolate showed high JHAN and insecticidal activity against P. xylostella . • IMBL-1823 isolate showed high JHAN and insecticidal activity against P. xylostella . • Both IMBL-1412 and IMBL-1823 were identified as Streptomyces lactacystinicus . • Ethyl acetate fraction of them showed high JHAN and insecticidal activities. Due to their specificity to target insects and low toxicity to non-target organisms, insect growth regulators (IGRs) have been promising alternatives to neurotoxic insecticides. Actinobacteria produce a wide range of secondary metabolites with insecticidal and insect growth regulatory activities. In this study, the culture media of 25 actinobacteria isolates showing high juvenile hormone antagonist (JHAN) activity were assessed for their insecticidal activity to identify novel IGR compounds toxic to Plutella xylostella . Among them, four isolates exhibited high insecticidal activity against 3rd instar larvae of P. xylostella . Two isolates of IMBL-1412 and IMBL-1823 showing relatively high insecticidal activities (greater than90% mortality) were identified as Streptomyces lactacystinicus based on colony color on various International Streptomyces Project (ISP) media and nucleotide sequences of the 16S rRNA gene. The ethyl acetate fractions of both isolates showed high JHAN and insecticidal activities against P. xylostella larvae at a concentration of 100 ppm when the culture media of these two isolates were extracted sequentially using hexane, ethyl acetate, and butyl alcohol. These results suggested that secondary metabolites of these actinobacterial isolates could be efficiently applied to develop novel IGR insecticides for the control of P. xylostella .

Streptomyces montanisoli sp. nov., exhibiting antimicrobial activity, isolated from mountain soil around a decaying tree.

A Gram-stain-positive, aerobic actinobacterial strain designated MMS17-BM035T isolated from mountain soil around a decaying tree was subjected to taxonomic characterization. The isolate developed extensively branched substrate mycelia and white aerial hyphae on International Streptomyces Project 2 agar. Strain MMS17-BM035T grew at 15-34 °C (optimum, 30 °C), at pH 5.0-8.0 (optimum, pH 7.0) and in the presence of 0-6 % NaCl (optimum, 0 %). Analysis of 16S rRNA gene sequences indicated that MMS17-BM035T fell into a phylogenetic cluster belonging to the genus Streptomyces. MMS17-BM035T shared the highest sequence similarity of 99.45 % with Streptomyces fuscigenes JBL-20T, and no higher than 98.7 % with other species of Streptomyces. Based on the orthologous average nucleotide identity, MMS17-BM035T was again mostly related to S. fuscigenes JBL-20T with 84.14 % identity, and less than 80 % with other species. The digital DNA-DNA hybridization analysis also indicated low levels of relatedness with related species, as the highest value was observed with S. fuscigenes JBL-20T (28.8 %). The major fatty acids of the strain were anteiso-C15 : 0, a summed feature (consisting of C18 : 1 ω7c/C18 : 1 ω6c), iso-C15 : 0, C16 : 0 and C20 : 0. The major respiratory quinones were MK-9(H4) and MK-9(H6). The diagnostic polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositolmannoside. The major cell-wall diamino acid was ll-diaminopimelic acid, and the characteristic whole-cell sugars were glucose and ribose. The DNA G+C content was 72.1 mol%. Strain MMS17-BM035T exhibited antimicrobial activity against several Gram-positive bacteria and yeasts. Based on both phenotypic and phylogenetic evidences, strain MMS17-BM035T should be classified as representing a novel species, for which the name Streptomyces montanisoli sp. nov. (type strain=MMS17-BM035T=KCTC 49544T=JCM 34528T) is proposed.

Streptomyces caniscabiei sp. nov., which causes potato common scab and is distributed across the world.

Fourteen strains of Streptomyces isolated from scab lesions on potato are described as members of a novel species based on genetic distance, morphological observation and biochemical analyses. Morphological and biochemical characteristics of these strains are distinct from other described phytopathogenic species. Strain NE06-02DT has white aerial mycelium and grey, cylindrical, smooth spores on rectus-flexibilis spore chains. Members of this species group can utilize most of the International Streptomyces Project sugars, utilize melibiose and trehalose, produce melanin, grow on 6-7 % NaCl and pH 5-5.5 media, and are susceptible to oleandomycin (100 µg ml-1), streptomycin (20 µg ml-1) and penicillin G (30 µg ml-1). Though the 16S rRNA gene sequences from several members of this novel species are identical to the Streptomyces bottropensis 16S rRNA gene sequence, whole-genome average nucleotide identity and multi-locus sequence analysis confirm that the strains are members of a novel species. Strains belonging to this novel species have been isolated from the United States, Egypt and China with the earliest known members being isolated in 1961 from common scab lesions of potato in both California, USA, and Maine, USA. The name Streptomyces caniscabiei sp. nov. is proposed for strain NE06-02DT (=DSM111602T=ATCC TSD-236T) and the other members of this novel species group.

Correct interpretation of actinomycete imagery using scanning electron microscopy

Antibiotic discovery was one of the most significant advances in therapeutic medicine following the advances in fermentation technology owing to Howard Florey and his associates. The ‘Golden era’ of antibiotics following the first discoveries in the laboratory of Waksman and his colleagues from a group of microorganisms known as actinomycetes lasted for 34 years (1940–1974). These fascinating microorganisms especially the members of the genus Streptomyces gave us the majority of the known antibiotics we use today, like streptomycin, kanamycin, neomycin, gentamicin, vancomycin and many more. To be able to produce these antibiotics in large-scale, the producer actinomycetes had to be selectively isolated. This resulted in a collaboration of over 40 laboratories from 18 different countries called ‘The International Streptomyces Project (ISP)’. The isolates generated in this project were studied in-depth including their morphologies together with their bioactivity. One of the components of these investigation was the correct interpretation of actinomycete morphology including the use of scanning electron microscopy. At the end of the first European Actinomycete Conference in Bradford University in England (1984), I had the opportunity to be trained by late Professor Cross on actinomycete growth morphologies. Thirty-eight years later when I witness the frequent difficulties students encounter in the interpretation of the actinomycete SEM images, I decided to write this paper and pass the skills given to me by late Professor Cross to the younger generation.