Optimization Of Actinomycete DNA-DNA Hybridization In Micro-wells And Identification Of Strains HA11090and HA11110

Actinomycetes are one of the most widely distributed groups of natural microorganisms; their unique and complicated metabolic processes have become a hot spot of people’s research.DNA-DNA hybridization is an effective method of analyzing DNA-DNA homology, and also a necessary step of defining novel taxon when the16S rRNA gene sequence similarity value is high enough. Type strains Isoptericola chiayiensis06182M-1T and Isoptericola halotolerans YIM70177T were chosen to improve the micro-well-format DNA hybridization method. Process of DNA extraction, conditions of ultrasonic shear, time of drying fixed DNA on microdilution plates, and the concentration of photo-activatable biotin for DNA labeling were improved in this study. DNA was extracted by SDS-CTAB method and ultrasonic sheared into fragments with conditions of amplitude45μm, pulse width2s, lasted for1min; fixed DNA was dried at15min in microdilution plates;500μg/mL DNA was labeled with0.5mg/ml photo-activatable biotin. After confirming the stability of the method, DNA of strains HA1021、HA11015、HA11090、HA11097. HA11110、HA11164and HA11166,each with their type strain was performed with homologous analysis.In accordance with polyphasic taxonomy, characteristics of morphological, physiological and biochemical, chemical and molecular were combined to confirm the taxon of strains HA11090and HA11110.Strain HA11090is Gram-positive. Growth occurs at14-43℃and pH6-10,1-5%(w/v) NaCl. Starch, coagulate and peptonize milk are degraded, but not cellulose and gelatin, does not reduce nitrate. Produce melanim, but not H2S. The major fatty acids are iso-C16:0, iso-C17:0and C15:0. The predominant polar lipid profile are DPG, PE, PG, PI and GL. The DNA G+C content is74.5%. The homology with Saccharopolyspora jiangxiensis W12T and Saccharopolyspora hirsuta subsp. Kobensis CGMCC4.1319T are both99.3%based on16S rRNA gene sequence alignment analysis. The DNA-DNA relatedness values of strain HA11090with Saccharopolyspora jiangxiensis W12T and Saccharopolyspora hirsuta subsp. Kobensis CGMCC4.1319T are82.3%and33.5%, respectively. Thus, strain HA11090is defined as Saccharopolyspora jiangxiensis.Strain HA11110is Gram-positive. Growth occurs at28-43℃, pH6-9, and1-5%(w/v) NaCl. Starch and cellulose are degraded and nitrate-reduction. Melanim and H2S are not produced; gelatin liquidation, milk coagulation and peptonization are negative. The major fatty acids are i-C16:0, ai-C15:0and C14:0. The predominant polar lipid profiles are DPG, PE, PG, PIM, PC and PI. The DNA G+C content is76.0%. The highest homology with Streptomyces sanyensis GIMN4.003T is99.1%, and followed by Streptomyces nanhaiensis SCSIO01248and Streptomyces radiopugnans R97T, both with98.8%based on16S rRNA gene sequence analysis. The DNA-DNA hybridization value of strain HA11110with Streptomyces sanyensis GIMN4.003T and Streptomyces nanhaiensis SCSIO01248T is58.4%,49.7%, and47.2%respectively. Strain HA11110represents a novel species of genus Streptomyces, the name proposed for this novel taxon is Streptomyces mangrovei sp.nov.