Study On The Function Of B739₀873 Gene In Riemerella Anatipestifer

Riemerella anatipestifer?RA?is an important pathogenic bacterium that infects ducks,geese and other poultry.The widespread use of antibiotics to treat RA infection has resulted in multidrug resistance in RA.The over-expression of the resistance nodulation division family?RND?efflux pump play an important role in producing multidrug resistance in Gram-negative bacteria.The purpose of this study is to search for the RND efflux pump in RA and research on its resistance mechanism.The results are as follows:1 B739₀873 mediated aminoglycosides and detergents resistance in RABioinformatics analysis was carried out on the genome of the sequenced strain RA-CH-1.The results showed that the encoding proteins of of B739₀781,B739₀873and B739₁785 had 11-12 transmembrane regions?TMD?,and two extremely large periplasmic loops around 300 aa between helix 1 and 2 and between helix 7 and 8,all of them located in the cytoplasmic membrane.Analysis of the adjacent regions showed that B739₀782,B739₀872 and B739₁783 encode membrane fusion proteins,B739₀780,B739₀871 and B739₁784 encode membrane channel proteins.RA-CH-1?B739₀873 and RA-CH-1?B739₀873 pLMF03::B739₀873 were constructed.The results showed that:?1?There was no significant difference in the growth rate among RA-CH-1,RA-CH-1?B739₀873 and RA-CH-1?B739₀873pLMF03::B739₀873,and the competition index?CI?of RA-CH-1 and RA-CH-1?B739₀873 was between 0.98-1.08;?2?Compared with RA-CH-1,the minimum inhibitory concentration?MIC?of streptomycin,gentamicin,kanamycin,SDS and Triton X-100 decreased from 128,32,256,640 and 160?g/mL to 16,2,32,40 and 40?g/mL?that is,the sensitivities increased by 4-16 fold?,and the RA-CH-1?B739₀873pLMF03::B739₀873 restored resistance to aminoglycosides and detergents;?3?The median lethal dose(LD₅₀)of RA-CH-1 and RA-CH-1?B739₀873 was similar on 1 day old ducklings,which were 5.62×10⁷ CFU/mL and 6.14×10⁷ CFU/mL respectively.Therefore,B739₀781,B739₀873 and B739₁785 have the basic structural characteristics of the RND efflux pump proteins;the deletion of the B739₀873 gene had no significant impact on the fitness and virulence of RA,and B739₀873 gene mediated aminoglycosides and detergents resistance in RA.2 Research on the energy source of B739₀873 of RAThe addition of CCCP?1?g/mL?decreased the MICs of streptomycin,gentamycin,kanamycin,SDS and Triton X-100 for RA-CH-1 and RA-CH-1?B739₀873 pLMF03::B739₀873?from 128,32,256,640,and 160?g/mL to 16,2,32,40,and 40?g/mL?.In contrast,CCCP addition did not modify the MICs of aminoglycosides and detergents for RA-CH-1?B739₀873.The gentamycin ELISA test kit was used to measure the gentamicin accumulation of RA-CH-1,RA-CH-1?B739₀873 and RA-CH-1?B739₀873 pLMF03::B739₀873.At the 24-min the accumulation level of gentamicin achieved a steady-state for all strains,RA-CH-1?13 pmol/mg?,RA-CH-1?B739₀873 pLMF03::B739₀873?14.2 pmol/mg?and RA-CH-1?B739₀873?86.7 pmol/mg?.After CCCP was added at 24-min,the accumulation level of gentamicin achieved a steady-state again at 56-min,the accumulation of gentamicin increased in RA-CH-1?B739₀873 pLMF03::B739₀873?54.5 pmol/mg?and RA-CH-1?61.6 pmol/mg?.In contrast,under our conditions,CCCP had no significant effect on the level of gentamicin accumulation in RA-CH-1?B739₀873?82.9 pmol/mg?.These data indicated that CCCP could inhibited B739₀873 activity,and B739₀873 pumped out gentamicin in an energy-dependent process,coupling to the PMF.3 Research on the functional sites of B739₀873 of RAThesite-directedmutagenesisstrainRA-CH-1?B739₀873pD400A,RA-CH-1?B739₀873 pD401A,RA-CH-1?B739₀873 pK929E,RA-CH-1?B739₀873pR959A and RA-CH-1?B739₀873 pT966A were constructed.The MICs of streptomycin,gentamycin,kanamycin,SDS,and Triton X-100 were 16,2,32,40,and 40?g/mL for these five mutant strains.Compared to RA-CH-1?B739₀873 pLMF03::B739₀873,none of these five mutant strains restored resistance to aminoglycosides and detergents.The western-blot analysis with antibody against the B739₀873 protein showed that all these B739₀873 mutant proteins expressed in RA-CH-1?B739₀873 were detectable and indistinguishable from the B739₀873 protein expressed in RA-CH-1?B739₀873,These results indicate that Asp 400,Asp 401,Lys 929,Arg 959,and Thr 966 in B739₀873 played an essential role in aminoglycoside and detergent transport.4 The function of B739₀873 requires the participation of B739₀872 in RAUsing the RA-CH-1 cDNA as template,PCR amplification results showed that B739₀871 and B739₀872 were co-transcribed.There were no MIC changes among RA-CH-1?B739₀873,RA-CH-1?B739₀872?B739₀873 pLMF03::B739₀873 and RA-CH-1?B739₀872?B739₀873 for all the aminoglycosides and detergents tested,these results indicated that B739₀872 is required for aminoglycoside and detergent resistance of B739₀873.The recombinant expression strain JP313 pBAD24::B739₀872-B739₀873,JP313pBAD24::B739₀872 and JP313 pBAD24::B739₀873 were constructed.The chemical cross-linking agent DSP was used to cross link the proteins in the recombinant expression bacteria after arabinose-inducible.The western-blot showed that the protein of 150 kDa in the expression product of JP313 pBAD24::B739₀872-B739₀873 was the complex protein of B739₀872 and B739₀873,indicating an interaction between B739₀872 and B739₀873.5 Research on the regulation effect of B739₀870 on B739₀873 in RAB739₀870 is located in the upstream of B739₀873 in chromosome.The bioinformatics analysis shows that B739₀870 encodes a TetR transcription regulatory protein.Using the RA-CH-1 cDNA as template,PCR amplification results showed that B739₀870 is co-transcribed with B739₀871 and B739₀872.RA-CH-1 pLMF03::B739₀870 was constructed.The RT-PCR results showed that the mRNA levels of B739₀871,B739₀872 and B739₀873 in RA-CH-1pLMF03::B739₀870 were about 5-fold lower than those of RA-CH-1.The MICs of streptomycin,gentamicin,kanamycin,SDS and Triton X-100 to RA-CH-1pLMF03::B739₀870 were 32,8,64,160 and 80 g/mL?compare with RA-CH-1,the sensitivity was up-regulated by 2-4 fold?.These results indicated that B739₀870 could inhibit the aminoglycoside and detergent efflux of B739₀873.In summary,the B739₀873 gene encodes a RND efflux pump that mediates the aminoglycoside and detergents resistance of RA.It shows that the expression of the RND efflux pump is one of the resistance mechanisms of RA.