Characterization of the Riemerella anatipestifer M949_RS00050 gene

Based on its causing ever-increasing heavy economic losses, Riemerella anatipestifer has been viewed as an important bacterial pathogen in the duck industry worldwide. However, the molecular mechanisms regarding its pathogenicity are poorly understood. In our previous study, we have built a random mutagenesis library of Riemerella anatipestifer CH3 using transposon Tn4351. In this study, we screened the library by determining bacterial median lethal dose in ducklings. A mutant strain showed about 376-fold attenuated virulence in comparison with the wild-type strain CH3 was obtained. Subsequently, the Tn4351 inserted gene was identified as M949_RS00050, which encodes a putative protein containing an outer membrane protein beta-barrel domain by genome walking and sequence analyses. Southern blot analysis indicated a single Tn4351 insertion in the CH3 chromosomal DNA. Inactivation of M949_RS00050 gene did not affect bacterial metabolic activity and the silver stained lipopolysaccharide pattern. However, the bacterial sensitivity to normal duck sera killing and bacterial hydrophobicity were dramatically enhanced in the M949_RS00050 gene inactivated mutant strain, compared to its wild-type strain CH3. Moreover, bacterial adherence and invasion abilities, bacterial capsular polysaccharide quantity, biofilm formation capacity and the bacterial virulence of the mutant strain were obviously decreased, compared to the wild-type strain CH3. Thus, our finding demonstrates that the M949_RS00050 gene functions on multiple bacterial biological properties and virulence in Riemerella anatipestifer.