Uncovering the potential of actinobacterium BLH 1-22 isolated from marine sediment as a producer of antibiotics

Actinobacteria have been known as producers of many bioactive compounds. The present study examines ten marine Actinobacterial isolates, aiming to investigate their potential as producers of antimicrobial compounds. The secondary metabolites were extracted from these Actinobacteria using ethyl acetate, and the crude extracts were tested for their bioactivity against Escherichia coli, Bacillus subtilis, and Micrococcus luteus. The antibacterial screening showed that the crude extracts of these Actinobacteria inhibit the growth of indicator strains. The extracts of isolate BLH 1-22 were further analysed using high-performance liquid chromatography (HPLC), which showed potential compounds with peak and retention time similar to the antibiotic standards (i.e., erythromycin, ampicillin, tetracycline, and penicillin). In addition to the HPLC profile, molecular identification showed that the isolate BLH 1-22 was similar to Micromonospora chalcea (99.6%). Further genome characterization of the strain, as well as purification and fractionation of the metabolite extracts, are important to obtain a comprehensive study on the potential of isolate BLH 1-22 as antibiotic compound producers. This study reported the potential of Micromonospora BLH 1-22 isolated from marine sediment. Hence, it also highlighted the potential of Actinobacteria isolated from Indonesian environments for bioprospecting studies.

Micromonospora maritima sp. nov., isolated from mangrove soil

Strain D10-9-5T was isolated from mangrove soil in Samut Sakhon province, Thailand. A polyphasic approach was used to determine the taxonomic position of the strain. The strain presented single rough spores on substrate mycelium and no aerial mycelium. Chemotaxonomic data supported the assignment of strain D10-9-5T to the genus Micromonospora based on the presence of meso-diaminopimelic acid and glycolyl muramic acid in the peptidoglycan, ribose, mannose, galactose, xylose and glucose as whole-cell sugars, MK-10(H4) (14.8 %), MK-10(H6) (46.7 %) and MK-10(H8) (27.5 %) as the predominant isoprenoid quinones, iso-C15 : 0 (17.9 %), anteiso-C17 : 0 (14.6 %), iso-C17 : 0 (9.6 %), C17 : 0 (8.0 %), iso-C16 : 0 (7.7 %) and C17 : 1ω8c (7.0 %) as the major cellular fatty acids, and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and phosphatidylethanolamine as the predominant phospholipids in the cell wall. The 16S rRNA gene sequence and phylogenetic analysis showed that strain D10-9-5 was closely related to Micromonospora marina JCM 12870T (99.6 %), Micromonospora coxensis JCM 13248 T (99.4 %), Micromonospora aurantiaca JCM 10878T (99.3 %), Micromonospora humi JCM15292T (99.3 %), Micromonospora halophytica JCM 3125T (99.1%) and Micromonospora chalcea JCM 3031T (99.1 %). Strain D10-9-5T could be clearly distinguished from related members of the genus Micromonospora by its physiological and biochemical characteristics as well as its phylogenetic position and level of DNA–DNA relatedness. Therefore, the strain represents a novel species for which the name Micromonospora maritima sp. nov. is proposed; the type strain is D10-9-5T ( = JCM 17013T = NBRC 108767T = PCU 322T = TISTR 2000T).

Micromonospora tulbaghiae sp. nov., isolated from the leaves of wild garlic, Tulbaghia violacea

A novel actinomycete, strain TVU1T, was isolated from leaves of the indigenous South African plant Tulbaghia violacea. Applying a polyphasic approach, the isolate was identified as a member of the genus Micromonospora. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain TVU1T was most closely related to Micromonospora echinospora DSM 43816T. However, phylogenetic analysis based on gyrB gene sequences showed that strain TVU1T was most closely related to the type strains of Micromonospora aurantiaca and Micromonospora chalcea. DNA–DNA relatedness values between strain TVU1T and the type strains of M. echinospora, M. aurantiaca and M. chalcea were 7.6±4.5, 45.9±2.0 and 60.9±4.5 %, respectively. Strain TVU1T could be distinguished from the type strains of all three of these species by several physiological characteristics, such as colony colour, NaCl tolerance, growth temperature range and sole carbon source utilization pattern. Strain TVU1T (=DSM 45142T=NRRL B-24576T) therefore represents a novel species for which the name Micromonospora tulbaghiae sp. nov. is proposed.

Micromonospora marina sp. nov., isolated from sea sand

Two actinomycete strains, JSM1-1T and JSM1-3, were isolated from sea sand collected in Thailand. Their taxonomic position was determined using a polyphasic approach. The chemotaxonomic characteristics of these strains coincided with those of the genus Micromonospora, i.e. the presence of meso-diaminopimelic acid and N-glycolyl muramic acid in the peptidoglycan, whole cell sugar pattern D, phospholipids type II, and cellular fatty acid type 3b. Phylogenetic analysis of 16S rRNA gene sequences revealed a close relationship between strains JSM1-1T and JSM1-3 (99.8 %), and between JSM1-1T and Micromonospora aurantiaca JCM 10878T (99.3 %), Micromonospora chalcea JCM 3031T (99.0 %), and Micromonospora coxensis JCM 13248 T (99.0 %). However, strains JSM1-1T and JSM1-3 could be clearly distinguished from these type strains by a low DNA–DNA relatedness and by phenotypic differences. On the basis of the data presented, a new species, Micromonospora marina sp. nov., is proposed. The type strain is JSM1-1T (=JCM 12870T =PCU 269T =TISTR 1566T).

Cloning and Characterization of the Tetrocarcin A Gene Cluster from Micromonospora chalcea NRRL 11289 Reveals a Highly Conserved Strategy for Tetronate Biosynthesis in Spirotetronate Antibiotics

ABSTRACT Tetrocarcin A (TCA), produced by Micromonospora chalcea NRRL 11289, is a spirotetronate antibiotic with potent antitumor activity and versatile modes of action. In this study, the biosynthetic gene cluster of TCA was cloned and localized to a 108-kb contiguous DNA region. In silico sequence analysis revealed 36 putative genes that constitute this cluster (including 11 for unusual sugar biosynthesis, 13 for aglycone formation, and 4 for glycosylations) and allowed us to propose the biosynthetic pathway of TCA. The formation of d-tetronitrose, l-amicetose, and l-digitoxose may begin with d-glucose-1-phosphate, share early enzymatic steps, and branch into different pathways by competitive actions of specific enzymes. Tetronolide biosynthesis involves the incorporation of a 3-C unit with a polyketide intermediate to form the characteristic spirotetronate moiety and trans-decalin system. Further substitution of tetronolide with five deoxysugars (one being a deoxynitrosugar) was likely due to the activities of four glycosyltransferases. In vitro characterization of the first enzymatic step by utilization of 1,3-biphosphoglycerate as the substrate and in vivo cross-complementation of the bifunctional fused gene tcaD3 (with the functions of chlD3 and chlD4) to ΔchlD3 and ΔchlD4 in chlorothricin biosynthesis supported the highly conserved tetronate biosynthetic strategy in the spirotetronate family. Deletion of a large DNA fragment encoding polyketide synthases resulted in a non-TCA-producing strain, providing a clear background for the identification of novel analogs. These findings provide insights into spirotetronate biosynthesis and demonstrate that combinatorial-biosynthesis methods can be applied to the TCA biosynthetic machinery to generate structural diversity.

Rhizosphere-competent isolates of streptomycete and non-streptomycete actinomycetes capable of producing cell-wall-degrading enzymes to controlPythium aphanidermatumdamping-off disease of cucumber

Fifty-eight streptomycete and 35 non-streptomycete actinomycetes were isolated from cucumber rhizosphere soil. These isolates were screened for the production of cell-wall-degrading enzymes using mycelial ( Pythium aphanidermatum (Edson) Fitzp.) fragment agar. Eighteen promising isolates were screened for their competence as root colonizers. Eight isolates showing exceptional rhizosphere competence significantly inhibited, in vitro, P. aphanidermatum, the causal agent of postemergence damping-off of cucumber ( Cucumis sativus L.) seedlings. The four most inhibitory isolates ( Actinoplanes philippinensis Couch, Microbispora rosea Nonomura and Ohara, Micromonospora chalcea (Foulerton) Ørskov, and Streptomyces griseoloalbus (Kudrina) Pridham et al.) produced in vitro β-1,3-, β-1,4-, and β-1,6-glucanases and caused lysis of P. aphanidermatum hyphae. None of these produced volatile inhibitors or siderophores. Only S. griseoloalbus produced diffusible inhibitory metabolites, whilst A. philippinensis and Micromonospora chalcea parasitized the oospores of P. aphanidermatum. These four isolates were subsequently tested in the greenhouse, individually or as a mixture, for their ability to suppress damping-off of cucumber seedlings in soil with or without cellulose amendment. The treatment, which included all four isolates in soil amended with cellulose, was significantly superior to all other treatments in suppressing damping-off and was nearly as good as the metalaxyl treatment. Results show that there is a potential to use a mixture of antagonistic rhizosphere-competent actinomycetes along with cellulose amendment rather than fungicides for the field management of this disease. This is the first study that has involved the screening of rhizosphere-competent non-streptomycete actinomycetes capable of producing cell-wall-degrading enzymes, for the management of Pythium diseases.