Identification And Function Analysis Of Genes Associated With Riemerella Anatipestifer Biotin Synthesis

Riemerella anatipestifer(R.anatipestifer)is a Gram-negative,nonmotile,nonspore forming,rodshaped bacterium.It belongs to the family Flavobacteriaceae in r RNA super-family V based on 16 s r RNA gene sequence analyses.R.anatipestifer causes the anatipestifer syndrome in ducks,characterized by diarrhea,lethargy,and respiratory and nervous symptoms,which can lead to high mortality.R.anatipestifer infections cause major economic losses in the duck industry through high mortality rates,poor feed conversion,increased condemnations,and high treatment costs.Up to now,21 serotypes have been identified in the world,and there is poor cross-protection among these serotypes.Various antibiotics are currently used to prevent and control R.anatipestifer infection in ducks,but they accelerate the emergence of drug-resistant strains.Once infection sets within a duck flock,the bacterium can become endemic with repeated infectious episodes possible making eradication difficult.In order to effectively prevent and control R.anatipestifer infection,researchers have conducted indepth research on the molecular mechanism of R.anatipestifer infection in recent years.A variety of virulence factors of R.anatipestifer have been found,which are related to outer membrane protein,lipopolysaccharide synthesis,iron metabolism,biofilm formation and so on.In previous study,we identified the virulence of AS87?RS05355 gene deletion mutant strain Yb2?bio B was attenuated by2040000 times.The AS87?RS05355 gene encodes a putative biotin synthesis associated Bio B protein,we identified its function.First,we determined that Yb2?bio B could not grow on biotin-deficient medium,the expression of biotin-dependent enzymes was reduced,and the content of acetyl-Co A was decreased,which proved that AS87?RS05355 gene was related to biotin-synthesis.The biological characteristics of the mutant Yb2?bio B were determined.Compared with the wild-type strain Yb2,the permeability of the cell wall of the mutant Yb2?bio B was changed and its ability to resist disinfectant were decreased.It suggested that the changes in cell wall lipids might lead to increased sensitivity of the cells to disinfectant.Non-targeted metabonomics determination of differential metabolites showed that the mutant strain Yb2?bio B mainly caused the change of amino acids of the metabolites.After the infection of the mutant strain Yb2?bio B,the blood bacterial load began to decrease at 12 hpi,and was completely cleared by the host at 24 h,which was consistent with the results of reduced resistance to serum determined in vitro,and both proved that the mutant strain Yb2?bio B could not establish infection against the host.The results showed that the deletion of AS87?RS05355 gene destroyed the synthesis of biotin in vivo,and then caused changes in the metabolism of amino acids and lipids,and significantly reduced the virulence,indicating that the deletion of AS87?RS05355 gene was crucial for the establishment of R.antipestifer systemic infection.In order to investigate the regulatory mechanism of biotin synthesis in vivo,we identified two genes involved in the regulation of biotin synthesis.AS87?RS05840 encodes Bir A protein that lacks the Nterminal wing helix-turn-helix DNA binding domain.Through sequence alignment analysis,it is found that Bir A protein belongs to Group ? biotin protein ligase,which only has the function of catalytic biotin binding to the receptor protein,but does not have the function of regulating biotin synthesis.Enzymatic reactions demonstrated that Bir A could catalyse the binding of biotins to the receptor protein Acc B in vitro,and EMSA assay further demonstrated that Bir A could not bind to the biotin-biosynthesis operon to play a transcriptional inhibitory role in regulating biotin-biosynthesis.Further investigation revealed that AS87?RS09325 gene encoded a xenobiotic response element family(XRE),named Bio X.EMSA assay proved that Bio X could bind to the biotin-biosynthesis operon,and q PCR results showed that Bio X gene could regulate the expression of biotin-biosynthesis related genes.At the same time,our results also showed that the virulence of the mutant strain Yb2?bio X was attenuated by 500 times.In this study,a novel regulatory mechanism was defined,in which the XRE family factor Bio X acts as an inhibitor to regulate biotin synthesis.In order to study the mechanism of the effect of downstream products on virulence caused by impaired biotin synthesis,we found the down-regulated protein AS87?RS06700 encoding acyl-coenzyme A thioesterase from the transcriptome sequencing results of the mutant Yb2?bio F and the wild strain Yb2 previously studied.The virulence of mutant strain Yb2?AS87?RS06700 was determinanted.Compared with the wild-type strain Yb2,the virulence of the mutant strain Yb2?AS87?RS06700 was not significantly decreased,indicating that the impaired biotin synthesis attenuated virulence is not associated with the down-regulationof the AS87?RS06700 gene.The function of AS87?RS06700 gene in vivo remains to be further explored.