Isolation Of Micromonospora From Mangrove Rhizhosphere Soil In Wenchang And Identification Of Strain 211018

Micromonospora is a genus of actinomycete under the family of Micromonosporaceae. In order to selectively isolate Micromonospora and to exploit their new resources in mangrove soil,23 samples of rhizosphere soil of different mangrove plants were collected from Touyuan, Wenchang, China.One gram of soil was heated in electric-heated thermostatic water bath at 55℃for 20 min, then 100μl of the 10-1 to 10-3 dilutions of the resultant suspensions were inoculated on the selective agar plates of 1/10 ATCC172 medium supplemented with potassium bichromate, cycloheximide, nystatin and novobiocin. One hundred and forty five actinomycetes including 96 Micromonospora-like isolates and 49 non-Micromonospora-like isolates were isolated according to their morphological characteristics on agar plates. Forty nine non-Micromonospora-like isolates were mainly Streptomyces. These strains only isolated from 7 out of 23 mangrove plants namely Acrostichum speciosum, Bruguiera sexangula, Acanthus ebracteatus, Excoecaria agallocha, Heritiera littoralis, Cerbera manghas and Hibiscus tiliscus. The number of Micromonospora-like isolates from the soil sample of Hibiscus tiliscus rhizosphere was the most. Furthermore, the 16S rRNA gene sequence analysis of 30 of 96 Micromonospora-like isolates indicated that the 30 isolates should be assigned to the genus Micromonospora.One hundred and twenty-six of 145 isolates were fermented for 7 days, and the bioactivities detection of their metabolites revealed that 4 isolates showed the activity of anti-human colon tumor (HCT)-116 cell line and 5 isolates showed the activity of anti-PTPlB (protein tyrosine phosphatase 1B, a kind of protein related with diabetes). Two of 4 isolates showed stronger activity of anti-HCT-116 and their IC50 reached 0.523±0.088 and 3.227±0.522μg/mL, respectively; one of 5 isolates showed the strongest activity of anti-PTP1B and its IC50 reached 3.41±0.58μg/mL. The largest number of bioactive isolates were from the soil sample of Bruguiera sexangula rhizosphere.A polyphasic study was carried out to establish the taxonomic position of strain 211018T, which was selected from these isolates together with our lab former collections. Strain 211018Twas isolated from mangrove Excocaria agallocha rhizosphere soil. The 16S rRNA gene sequence analysis showed the highest similarity to the type strains of Micromonospora olivasterospora DSM 43868T(=JCM 7348T,98.6%) and Micromonospora pattaloongensis TJ2-2T(=JCM 12833T,98.4%). The gyrB gene sequence analysis also indicated that 211018T should be assigned to the genus Micromonospora. The characteristic whole-cell sugars are xylose, mannose and arabinose. The predominant menaquinone is MK-9(H4) and the major fatty acids are iso-C15:0 (27.5%),10-Methyl C17:0(14.2%), C17:1ω8c (12.8%), iso-C16:0 (12.6 %), anteiso-C15:0(6.1%), iso-17:0 (4.1%) and anteiso-C17:0 (4.0%). The phospholipid profile comprises phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides. The DNA G+C content is 70.8 mol%. The chemotaxonomic data of the strain coincided with those of the genus Micromonospora. Furthermore, a combination of DNA-DNA hybridization results and some physiological and biochemical properties indicated that the novel strain could be readily distinguished from the closest phylogenetic relatives. On the basis of these phenotypic and genotypic data, strain 211018T represents a novel species of the genus Micromonospora, for which the name Micromonospora rhizosphaerae sp. nov. is proposed. The type strain is 211018T(= CGMCC 4.5599T= DSM 45431T). The GenBank/EMBL/DDJB accession number for the 16S rRNA gene and gyrB gene sequence of Micromonospora rhizosphaerae 211018T are FJ261956 and GU126413, respectively.