Regulation And Functional Analysis TonB Genes In Riemerella Anatipestifer

Riemerella anatipestifer(R.anatipestifer)is one of the most important pathogens for ducks.The disease caused by R.anatipestifer was characterized by fibrinous pericarditis and meninitis.It has been reported R.anatipestifer have three ton B genes,ton B1,ton B2 and Tbf A(ton B3).Although ton B1 and ton B2 were shown involved in iron and heme utilization,Tbf A was not participate these activity.This study aimed at the transcriptional regulation and compensatory development of three ton B genes.The main results are as follows:1.Identification of ton B1,ton B2 and tbf A gene locusAccording Sequence analysis showed that Riemerella anatipestifer ATCC 11845 contain three ton B genes locus.the study was performed by reverse transcriptase PCR(RT-PCR)to amplify the intergenic regions.The resulting amplicons shown that ton B1-exb B1-exb D1,ton B2-exb B2-exb D21-exb D22 are cotranscribed.However,the ton B3 is present as a monocistronic copy.2 Transcriptional regulation of ton B genes of Riemerella anatipestiferQuantitative reverse transcriptase-PCR(q RT-PCR)shown that all three ton B genes are expressed under normal culture conditions,with ton B2 and tbf A transcripts being the most abundant and the rarest,respectively.All three ton B genes neither regulated by iron nor heme.Moreover,all three ton B genes are expressed under different temperatures conditions,it was shown that the transcription of tbf A was up-regulated more than 2-folds under 42°C(the temperature of duck),compare to the transcription at 37°C.whereas the ton B1 and ton B2 genes are remain unchanged at the same temperature.3 The function research of ton B genesKnockout one of Tonbs did not alter transcript levels for the other ton B members,suggesting that compensatory expression did not occur.we are successfully constructed several complementation strains.Growth assays of the strains confirmed that ton B1 was not able to be substitued by ton B2 and ton B1 can't be replace by tbf A,whereas tbf A probably perform ton B2 function.Wild type,ton B1 or ton B2 mutant strains and complemented strains were treated with streptomycin and hydrogen peroxide,respectively.These result suggest that wild-type strain and compensation strains are dramatically inhibited,whereas the ton B mutant strains are slightly inhibitation.It also indirectly proves that inactivation of ton B1 and ton B2 reduced the intracellular iron pool;In contrast,the tbf A mutant strain had no significant effect on the resistance to streptomycin and hydrogen peroxide.