Isolation, Characterization, And Soil Application Of A Gram Positive Atrazine Degrading Bacterium

Through a long term enrichment, a Gram positive bacterium was isolated from a stable atrazine degrading consortium, named BTAH1, which could form a clearing zone on atrazine agar. This strain could use atrazine as sole carbon and nitrogen source, and degrade 1.000 mg/L atrazine within 126 hr completely. It could grow on most common carbon and nitrogen source, but it was an auxotroph strain of thiamine, and was strongly inhibited by high concentration of peptone. The optimum temperature and pH for BTAH1 growth was 30 C and 7.5, respectively. However, the bacterium was alkalophilic and could grow at a wide pH range, i.e., pH 6.5-11.5. Phylogenetic tree based on 16SrDNA sequence data showed that BTAH1 was closed to Exiguobacterium sp.We studied The differences in morphology, physiology, and genetics between BTAH1 and four type strains of Exiguobacterium sp., Exiguobacterium aurantiacum DSMZ 6208. Exiguobacterium antarcticum DSMZ 14480, Exiguobacterium unda DSMZ 14481, and Exiguobacterium acetylicum DSMZ 20416. The results showed that the bacterium size, clone shape, and glycolytic profile of BTAH1 were very different from those of four type strains. Especially. BTAH1 had no flagellum, but all four type strains had peritrichous flagella. 16S rDNA sequence analysis revealed 98.1% similarity between the 16S rDNA sequence of BTAH1 and Exiguobacterium aurantiacum DSMZ 6208, 94.0% between BTAH1 and Exiguobacterium antarcticum DSMZ 14480, 93.8% between BTAH1 and Exiguobacterium unda DSMZ 14481, and 94.5% between BTAH1 and Exiguobacterium acetylicum DSMZ 20416. DNA-DNA reassociation values indicated that BTAH1 was in a new genomic cluster of Exiguobacterium sp., and the DNA of BTAH1 and strain 6208 showed only 26.25% similarity, BTAH1 and strain 14480 24.67%, BTAH1 and strain 14481 48.11%, BTAH1 and strain 20416 51.22%, respectively. Based on genomic destinctiveness and the clear difference in chemotaxonomy, BTAH1 is proposed to be a strain in new specie of Exiguobacterium.Strain BTAH1 could grow in liquid medium with atrazine as sole carbon and nitrogensource, but extra carbon source in the medium would repress the degradation of atrazine by strain BTAHl. When BTAHl degraded atrazine, it could release all five nitrogen atoms from the chemical structure of atrazine as NH3, which led to a great increasing of solution pH. Therefore extra nitrogen source in the medium would greatly inhibit the growth of strain because of the high pH in solution. In lab conditions, strain BTAH1 could degrade atrazine very efficiently at 25-30C and at pH 7-9. During the course of atrazine degradation by BTAHl, it hydrolyzed the chlorine atom in the structure at first, and produced hydroxyatrazine. Furthermore, strain BTAHl did not cumulate cyanuric acid in the medium, which differed from most Gram positive atrazine degrading strains. Substrate range experiments showed that strain BTAHl could use chlorine-substituted s-trizine herbicide, but not use methyltyio-substitude and other structure s-trizine herbicide. The narrow substrate specificity of BTAHl suggested that strain BTAHl might has the same degradation pathway as Pseudomonas sp. strain ADP, but not most Gram positive strains.PCR amplification and Southern blotting were used to study the conservation of atrazine degrading genes in strain BTAHl. Results showed that the conserved fragments sequences of three atrazine degrading genes {atzA, atzB, and atzC) in strain BTAH1 shared 99% identity with those in strain ADP, which indicated that BTAHl had the same degrading pathway as strain ADP, and was rare in Gram positive strains. Strain BTAHl had two big plasmids, named pBTAHll and pBTAH12. The location experiment by Southern blotting showed that the first degrading gene, atzA, was located on chromosome DNA, and the other two on plasmid pBTAHl 1.To confirm the resource of atrazine degrading genes in strain BTAHl, an 8 kb PstI fragment was cloned into E. coli by clone hybridization, which contain atzB gene of BTAHl. Sequencing analysis of the fragment showed that it was 8581 bp, had 5 ORFs, and atzB...