Functional Identification Of HmuY-like Protein In Riemerella Anatipestifer

Riemerella anatipestifer is a Gram-negative bacteria,that can cause infectious serositis in ducks and other birds.It's a very important pathogenic bacteria,which discourage the development of aviculture.Studies have shown that R.anatipestifer can use hemin and hemoglobin as nutriment.So far,the utilization mechanism of hemin and hemoglobin in R.anatipestifer has not been studied.We found that R.anatipestifer CH-1 B739-1417 gene encoding a hypothetical protein similar to Porphyromonas gingivalis HmuY.It's belong to the HmuY-like protein superfamily.We named it hmuYRA,and the protein is HmuYRA.We plan to study the function of HmuYRA,and whether the protein is involved in the utilization of heme and hemglobin.Our results are below:1 Cloning,expression and preparation polyclonal antibody of R.anatipestifer hmuYRAAccording to sequence analysis,We found hmuYRA was cotranscription with the upstream gene B739-1416,which encodeed a TonB-dependent outer membrane receptor.We used the genome of RA CH-1 as template,amplified the hmuYRA fragment.Ligated the fragment to the expression vector pBAD24,and then we used arabinose to induce the expression of HmuYRA.the purified HmuYRA protein was inoculated to KunMing mice,We collected the immune serum,that is polyclonal antibody of HmuYRA.and the antibody could recognize the HmuYRA protein in R.anatipestifer CH-1.2 Consturction of hmuYRA deletion mutant and complementation strain of R.anatipestifer CH-1We inserted the chloramphenicol resistance fragment(CmpR cassette)into the hmuYRA gene result in the hmuYRA disruption.We amplified the left and right flanking sequence of hmuYRA gene from thegenome of CH-1.and we amplified the CmpR cassette from the genome of CH-2.Subsequently,the three PCR fragments were ligated using the overlap PCR.The fused fragments were ligated with the suicide plasmid pEX18GM.We transformed the suicide plasmid into RA CH-1 byconjugational transfer method.Then we selected the mutant strain RA CH-1 AhmuYRA.The entire hmuYRA gene was amplifed from RA CH-1 genome.the hmuYRA fragment was ligated into the plasmid pLMF03.We also transformed the plasmid pLMF03::hmuYRA into RA CH-1 ?hmuYRA.Then we successfully selected the complementaion strain RA CH-1 ?hmuYRA pLMF03::hmuYRA.3 Functional study of HmuYRA proteinHemin binding experiment showed that HmuYRA protein can bind hemin with 1:1 stoichiometric ratio.The HmuYRA protein were mainly located at outer membrane,and when the conditions was iron-limited,It was also found in cultured supernatant.The transcriptional level of hmuYRA and the expression of HmuYRA protein were regulated by iron but not hemin.The deletion of hmuYRA could seriously damage hemin utilization from hemoglobin in RA CH-1.The HmuYRA was able to inhibited the utilization of hemin in C600?hemApAM238:;hasR,but did not have an obvious effect on the utilization of hemin from hemoglobin.Hemoglobin binding experiments showed that HmuYRA can bind hemoglobin,but can not extract hemin from hemoglobin.