Isolation, Purification, Taxonomy And Identification Of Myxobacteria

The soil and bark samples collected from Yunnan, Hebei and Shanxi provinces of China, and also from Vietnam, were used as the materials in this experiment. And dung pellets of rabbits or goats that had been autoclaved can be used to induce many kinds of fruiting bodies of the Myxobacteria in different colors, sizes, and shapes. We isolated and purified Myxobacteria strains with the improved method which was found by our laboratory. The using of tissue culture plates instead of traditional plates accelerated the purification process and enhanceed the purification efficiency. From 94 soil samples and 9 bark samples in these areas, 259 Myxobacteria strains were isolated and 140 strains of them were purified.These 259 Myxobacteria strains were primarily identified in genus or species level by their fruiting bodies, myxospores and vegetative cells on the base of Sergey's manual of determinative bacteriology and they belonged to 9 genera(Myxococcus, Corallococcm. Angiococcus. Archangium, Cystobacter. Stigmatella . Hapoangium. Nannocystis. Polyangium) of Myxococcales. The characteristic in liquid culture of the purified strains were described, and the relation between the characteristic and the taxonomic traits was researched for the first time. The two groups of Myxobacteria differentiated according to the shape character of the myxospores and vegetative cells had different characteristic in liquid culture. The strains belonged to the first group including Myxococcus, Corallococcus, Angiococcus, Archangium, Cystobacter, Melittangium and Stigmatella were usual pieces or floe in liquid culture, while strains of the other group including Hapoangium, Nannocystis, Polyangium, Sorangium and Chondromyces were spheriform mostly.The effect of three methods for extracting total DNA of Myxobacteria which were Acetone isolation method .Alkaline lysis method and innovative NaOH-CTAB method was compared. The NaOH-CTAB method can destroy cell wall of Myxobacteria more drastically and wipe off extracellular amylose more effectively than by Acetoneisolation method and Alkaline lysis method which offer total DNA of high quality for the study of molecular biology of Myxobacetia.Six strains(serial numbers were YN34, YN65, YN76, YN112, BD85, BD105) which had the inhibitory activity to the tumour belonged to Myxococcus according to their shapes and their total DNA were extracted by the NaOH-CTAB method. The 16S rDNA sequencing analyses of them was carried out. Complete sequences of them were obtained and phylogenesis analyses were carried out of Myxococcus, their phylogenetic position was determined. We did also G+Cmol%, physiological and biochemical characteristics tests. The results show that the 6 strains should be clustered into the genus Myxococcus; YN34, YN112 and BD105 had close relationship and their sequence similarity were 100%, the most closed strain was Myxococcus fulvus AJ233919; YN65 was similar to Myxococcus macrosporus; YN76 was closed to YN34, YN112 and BD105 and their sequence comparability were 99.86%; BD85 similar to Myxococcus xanthus. G+CmoI% of the 6 strains were in the range of 65% to 69%.The 16S rDNA sequences results were almost identical to the physiological and biochemical results, this conformed the importance of molecular, physiological and biochemical function in the taxonomy of Myxobacteria . While the physiological and biochemical function and other taxology methods were need to combined for the exist of some differences between them.