TonB Energy Transduction Systems of Riemerella anatipestifer Are Required for Iron and Hemin Utilization

Abstract

Riemerella anatipestifer (R. anatipestifer) is one of the most important pathogens in ducks. The bacteria causes acute or chronic septicemia characterized by fibrinous pericarditis and meningitis. The R. anatipestifer genome encodes multiple iron/hemin-uptake systems that facilitate adaptation to iron-limited host environments. These systems include several TonB-dependent transporters and three TonB proteins responsible for energy transduction. These three tonB genes are present in all the R. anatipestifer genomes sequenced so far. Two of these genes are contained within the exbB-exbD-tonB1 and exbB-exbD-exbD-tonB2 operons. The third, tonB3, forms a monocistronic transcription unit. The inability to recover derivatives deleted for this gene suggests its product is essential for R. anatipestifer growth. Here, we show that deletion of tonB1 had no effect on hemin uptake of R. anatipestifer, though disruption of tonB2 strongly decreases hemin uptake, and disruption of both tonB1 and tonB2 abolishes the transport of exogenously added hemin. The ability of R. anatipestifer to grow on iron-depleted medium is decreased by tonB2 but not tonB1 disruption. When expressed in an E. coli model strain, the TonB1 complex, TonB2 complex, and TonB3 protein from R. anatipestifer cannot energize heterologous hemin transporters. Further, only the TonB1 complex can energize a R. anatipestifer hemin transporter when co-expressed in an E. coli model strain.

Fig 1. Genetic maps of R. anatipestifer ATCC11845 TonB systems.

Shown are the locations of the three tonB loci in the ATCC11845 chromosome (A) and the genetic organization within the ATCC11845 DNA regions, including the tonB1 (B), tonB2 (C) and tonB3 (D) genes and nearby related genes. DNA positions in the chromosome are given by the number at the top of each panel. Each horizontal arrow indicates the location of the coding region, and the direction of transcription for each tonB gene. The name of each gene is shown above or below the arrow.

Fig 2. Phylogenetic tree of TonB C-terminal domains.

The tree was created using a Neighbor-Joining Bootstrap method with 1000 bootstrap trials in ClustalW derived from the MSA shown in S2 Fig. The trees were drawn and visualized with MEGA v6.05. The clusters (1A–C, 2A–C, 3A–B and 4 shown on the right) were generated by previous research [27]. TonB proteins from R. anatipestifer are shown with overstriking typeface.

Fig 3. The effect of R. anatipestifer TonB knockouts on hemin and iron utilization.

R. anatipestifer strains (clockwise from the top left) ATCC11845, tonB1 mutant, tonB2 mutant and tonB1tonB2 double-mutant were spread on LB plates containing 5% bovine serum (A), LB-only plates (B), LB plates containing 20 μM hemin (C), and LB plates containing 5% bovine serum and 40 μM Dip (D). Growth was assessed by appearance of bacterial colonies on the plates. Pictures were taken after 48 hours of growth at 37°C under 5% CO₂ atmosphere. All the experiments were repeated three times. A representative result is presented.

Fig 4. Functional complementation of the E. coli tonB mutant impaired in hemin uptake.

E. coli strains (clockwise from the top left) C600ΔhemA tonB::Tn10 pAM238::OMhemR _RA harboring (1) pBAD24, (2) pBAD24::tonB1 and (3) pBAD24::exbB1-exbD1-tonB1 were inoculated on LB plates containing 50 μg/ml δ-ala (A), LB-only plates (B) and LB plate containing 20 μM hemin (C). Fig 4D shows the diameter of the different E. coli colonies on these plates. Three asterisks indicate significant differences (P<0.001). Error bars represent standard errors from measurements of ten bacteria colonies. All the experiments were repeated three times. A representative result is presented.

Fig 5. Functional complementation of the tonB mutant E. coli impaired in iron uptake.

E. coli strains (clockwise from the top left) including (1) C600ΔhemA tonB::Tn10 pBAD24, (2) C600ΔhemA pBAD24, (3) C600ΔhemA tonB::Tn10 pBAD24 exbB-exbD-tonB1 and (4) C600ΔhemA tonB::Tn10 pBAD24::tonB1 were grown on LB plates containing δ-ala (50 μg/ml), Amp (100 μg/ml) and 0 μM Dip (A) or 150 μM Dip (B). Part C shows the diameter of the E. coli colonies on the plates. Three asterisks indicate significant differences (P<0.001). Error bars represent standard errors from measurements of ten bacteria colonies. All the experiments were repeated three times. A representative result is presented.