The Functional Identification Of B739?RS00825 In Heme Tolerance Of Riemerella Anatipestifer CH-1

The gram-negative bacteria Riemerella anatipestifer(RA)cause great economic losses in duck industry.Heme,as an essential nutrient for most bacterium,plays a key role in RA reproduction in vitro and invading host.But excess heme is toxic to RA,so there may be some genes with the function of resisting to heme toxicity in RA.The result of transcriptome sequencing shown that the transcript of B739_RS00825 gene was upregulated by 3.5 times in RA CH-1?hemH mutant,compared to that of RA CH-1.HemH is a chelatase can link protoporphyrin IX and ferric iron to form heme.The protoporphyrin IX and ferric iron will accumulate with hemH deletion and be toxic to bacteria.So,the function of B739_RS00825 gene is thought to be associated with the changes of heme concentration in bacteria.The mutant RA CH-1?B739_RS00825 and RA CH-1?fur?B739_RS00825 were constructed in this study.The function of B739_RS00825 gene in anti heme toxicity,resistance to oxidative stress was determined in this study.Here are the results:1 B739_RS00825 is involved in resistance to heme toxicity in Riemerella anatipestiferThe growth rate between RA CH-1 and RA CH-1?B739_RS00825 were similar in GCB medium.In GCB containing 35 ?M/L heme medium,both RA CH-1 and RA CH-1?B739_RS00825 grew slowly than that in GCB medium,the growth rate between the two strains without significantly difference.Compared to that of RA CH-1 ?fur,the growth of RA CH-1?fur?B739_RS00825 was slower in the logarithmic phase in GCB medium.Addition of 10 ?M/L heme in CGB medium had no effect on RA CH-1?fur growth.However,the growth of RA CH-1?fur?B739_RS00825 was significantly inhibited in GCB+10 ?M/L heme medium compared with GCB medium.Taken together,it was suggested that B739_RS00825 gene has the function in resisting to heme toxicity in RA CH-1.2 B739_RS00825 is involved in resistance to oxidative stress in Riemerella anatipestiferThe survival rate of RA CH-1,RA CH-1?B739_RS00825,RA CH-1?fur and RA CH-1?fur?B739_RS00825 was determined when incubation with 5 mmol/L H2O2 for 30 mins,respectively.The survival rate of RA CH-1?B739_RS00825 was the same as RA CH-1 after incubation with 5 mmol/L H2O2.But the survival rate of RA CH-1?fur?B739_RS008255 was reduced by 4 times compared to RA CH-1?fur after incubation with 5 mmol/L H2O2.The results suggested that B739_RS00825 gene plays a role in resisting to oxidative stress in RA CH-1,and the function is more obvious in RA CH-1?fur mutant.3 The transcription of B739_RS00825 gene is regulated by FurThe total RNA of RA CH-1,RA CH-1?fur and RA CH-1?OxyR under different culture conditions were collected and transferred into cDNA through reverse transcription.The transcription level of B739_RS00825 gene was determined by quantitative real-time PCR in these templates.The transcription level of B739_RS00825 gene was higher in the logarithmic phase than in the stationary phase.In contrast to RA CH-1,the transcription level of B739_RS00825 gene was upregulated in RA CH-1?fur but not changed in RA CH-1?OxyR.The transcription level of B739_RS00825 gene was the same with or without H2O2 stimulation.The results suggested that the B739_RS00825 gene may play a role in the logarithmic phase and its transcription is regulated by Fur.4 Determination of heme binding capacity of the conserved domain of B739_RS00825 proteinFull length B739_RS00825 protein cannot be expressed in E.coli.So,the conserved domain of B739_RS00825 was chosen to be truncated expressed in E.coli strain Rosetta.The binding capacity of truncated B739_RS00825 protein was determined by E.coli C600?hemA pAM238::hemR.The result shows that the truncated B739_RS00825 protein cannot bind heme in vitro.5 B739_RS00825 deletion has no effect on pathogenicity of ducklingsThe ducks were challenged by 109 CFU RA CH-1 and RA CH-1?B739_RS00825 respectively at the age of 3 days.After 24 h and 48 h,the bacteria were isolated from blood,liver,spleen and brain and then counted to compare the colonization between RA CH-1 and RA CH-1?B739_RS00825.No matter at 24 h or 48 h,the bacteria load of RA CH-1 and RA CH-1?B739_RS00825 were same in every organ of ducks.It was suggested that B739_RS00825 deletion has no effect on pathogenicity of RA CH-1.