Functional Identification Of TonB Proteins In Riemerella Anatipestifer

Riemerella anatipestifer, belonging to gram-negative bacterium, is one of the most harmful pathogens to the duck industry around the world. R. anatipestifer has natural resistance to several antibiotics and can produce antibiotic resistance to sensitive antibiotics. Moreover, there is almost no cross-protection between different serotypes. Thus, it's difficult to prevent and treat it by vaccines and antibiotics. Previous researches show that "Nutrition immunity" is usful way in the prevention of pathogen infection by limiting nutritions availability. Though Iron and hemin are essential nutrients for R. anatipestifer, the processes of their transportation are still poorly understood. So, we plan to research TonB firstly because it is the key protein in hemin and iron uptake. Our studies are as follows:1. Sequence analysis of tonB genesGenome analysis revealed the there are three hypothetical tonB genes in different locations in the R. anatipestifer ATCC 11845 genome (RA ATCC). We termed them tonB1, tonB2 and tonB3, respectively. tonBl gene is contained within the exb1B-exbD1-tonB1 operon and tonB2 gene is contained within the exbB2-exbD21-exbD22-tonB2 operon. However, the tonB3 is present as a monocistronic copy. In protein sequence, there is only approximately 10.00% identity between TonB1 and TonB2,10.65% identity between TonB1 and TonB3, and 34.71% identity between TonB2 and TonB3. All three TonBs lack the highly conserved "YP" motif and "SSG" motif.2. Expression, purification and polyclonal antibody preparation of TonBThe three tonB genes of RA ATCC were recombined on the expression plasmid pBAD24 by molecular method. These recombinant plasmids were transformed in E.coli JP313 to prokaryotic expression. The TonB proteins containing His-tag were purified by affinity chromatography. There purified TonB proteins were inoculated three times into KunMing mice. Two weeks after the last inoculation, blood samples were collected. Serum was obtained from blood samples by centrifuged. These serum are polyclonal antibodies for each TonB proteins.3. Construction of tonB deletion mutants in R. anatipestiferLeft flanking sequence of tonB1, ErmR cassette and the right flanking sequence of tonB 1 were ligated using the overlap PCR method. Left flanking sequence of tonB2, SpcR cassette and the right flanking sequence of tonB1 were ligated using Enzyme digestion-Ligase-PCR method. The fusion DNA fragments were recombined on the suicide plasmid pEX18Gm. The recombinant suicide plasmid was transformed in donor strain E.coli S17-1. Then suicide plasmid in donor strain S17-1 was transformed into RA ATCC by conjugational transfer methord. The transconjugants were selected on blood agar plates containing corresponding antibiotics. Finally, single-gene mutant strains RA ATCC ?tonB1::ermR, RA ATCC ?tonB2::spcR and double-gene mutant strain RA ATCC ?tonB1::ermR ?tonB2::spcR were constructed. Then, there mutant strains were identified in protein level by Western-Blot method. RT-PCR method also was used to prove that there is no polar effect in these mutant strains.4. The function research of tonBRA ATCC wild type and tonBl mutant can grow on LB plates containing 20?M Hemin while tonB2 mutant grow very slowly and tonB12 double mutant have no growth on this plate. It indicates that tonBl and tonB2 are function in hemin uptake process and tonB2 is dominated. RA ATCC wild type and tonBl mutant can grow on LB plates containing 5% serum and 40?M Dip while tonB2 mutant and tonB12 double mutant have no growth on this plate. This result shows that tonB2 play an important role in iron uptake process. The size of the ranking of the growth ring around hemoglobin on LB plate is:RA ATCC wild type>tonBl mutant>tonB2 mutant>tonB12 double mutant. It suggestes that tonBl and tonB2 are function in hemoglobin utilization process and tonB2 is dominated. RA ATCC wild type has the ability to produce biofilm obviously while all the tonBl mutant, tonB2 mutant and tonB12 double mutant almost cannot produce biofilm. It demonstrates that tonB gene deletions affect the capability of biofilm formation by some way. The mRNA levels of these three tonB genes have no significant difference when RA ATCC grow in LB broth and LB broth containing 50?M Hemin or TSB and TSB containing 80?M Dip. It reveals that the tonB genes of RA ATCC don't be regulated by their substrate Hemin or iron.