ARDRA Analysis, Antagonistic Activity Of Siderophore Analysis And Fur Gene Cloning Of Siderophore-Producing Bacteria Of Tobacco Rhizosphere

The siderophore-producing bacteria of tobacco rhizosphere were studied in this research. Firstly, the genetic diversity of the siderophore-producing bacteria was studied by ARDRA analysis. Secondly, siderophore-producing Pseudomonas mediterranea G-229-21T was identified and the antagonistic activity of its siderophore was studied. Thirdly, the ferric uptake regulation protein gene was cloned. Lastly, a siderophore-deletion mutant was obtained by Tn5-1063a mutagenesis. The conclusions were as follows:1. The genetic diversity of siderophore-producing bacteria of tobacco rhizosphere was studied by amplified ribosomal DNA restriction analysis (ARDRA), 16S rRNA sequence homology and phylogenetics analysis methods. Studies demonstrated that 85% of the total 354 isolates produced siderophores in iron limited liquid medium. A total of 28 ARDRA patterns were identified among the 299 siderophore-producing bacterial isolates. The 28 ARDRA patterns represented bacteria of 14 different genera belonging to six bacterial divisions, namelyβ-,γ,α-Proteobacteria, Sphingobacteria, Bacilli, and Actinobacteria. Especially,γ-Proteobacteria consisting of Pseudomonas, Enterobacter, Serratia, Pantoea, Erwinia and Stenotrophomonas genus encountered 18 different ARDRA groups. Results also showed a greater siderophore-producing bacterial diversity than previous researches. For example, Sphingobacterium (isolates G-2-21-1 and G-2-27-2), Pseudomonas poae (isolate G-2-1-1), Enterobacter endosymbiont (isolates G-2-10-2 and N-5-10), Delftia acidovorans (isolate G-1-15), and Achromobacter xylosoxidans (isolates N-46-11HH and N-5-20) were reported to be able to produce siderophores under low-iron conditions for the first time. Gram-negative isolates were more frequently encountered, with more than 95% total frequency. For Gram-positive bacteria, the Bacillus subtillus and Rhodococcus erythropolis were the only two genera, with less than 5% total frequency. Furthermore, the members of the genera Pseudomonas and Enterobacter were dominant in this environment, with more than 50% total frequency. It was also found that 75 percent of the isolates that had the high percentages of siderophore units (%) between 40 and 60 belonged to Pseudomonas.2. The antagonistic bacteria named G-229-21T were screened by dual culture with P. parasitica var. nicotianae (Breda de Hann) Tucker on low iron (2μmol/L FeCl3) Sucrose-L-Asparagine (SA) plate. The strain produced high-affinity siderophore under low iron conditions. The strain was identified as Pseudomonas mediterranea by using morphological, biochemical and physiological characteristics, 16S rRNA sequence homology, phylogenetics and specific species molecular analysis. The siderophore was isolated by column chromatography on Amberlite XAD-2 and analyzed by spectrophotometer. This strain produced carboxylate-type siderophore against P. parasitica var. nicotianae (Breda de Hann) Tucker. The suppression ratio was up to 92.3% under low iron conditions (0.16μmol/L~10μmol/L FeCl3). However, it was only 2.0% under Fe-replete conditions (100μmol/L FeCl3).3. The whole gene sequences that code the ferric uptake regulation protein (Fur) was obtained by using degenerate primer and TAIL-PCR(Thermal asymmetric interlaced PCR)methods. According to the result of BLAST analysis, the gene was made up of 405 nucleotides and was identical to fur of Pseudomonas fluorescens PfO-1 at the level of 82%. The fur gene encoded a protein of 104 amino acids and was identical to Fur of P. fluorescens PfO-1 at the level of 86%. Crystal structure of P. mediterranea G-229-21T Fur was simulated and analyzed by Swiss-Model.4. Tested by the plate of nutrition agar with antibiotics, G-229-21T was sensitive to kanamycin, but could tolerate 20μg/mL chloromycetin. The Tn5-1063a was used to mutagenize G-229-21T. One siderophore-deletion mutant named G-229-21TA was obtained. The specific-fragment of the mutant G- 229-21TA was amplified with primers PC5/1 and PC5/2. The amplification result approved that G-229-21TA was a mutant of G-229-21T. Designing primers according to luxA sequences from Tn5-1063a, the DNA of the wild strain, pRL1063a and the mutant as the templates, the sequences of luxA was obtained by PCR. The luxA of the mutant is identical to that of Tn5-1063a at 100% level, it indicates that Tn5-1063a was inserted directly into the genome of G-229-21T.