Acid Stress Response Of Bifi-dobacterium Longum Subsp.longum BBMN68m Involves In Biofilm

Bifidobacterium longum subsp.longum BBMN68 is a novel train isolated from the feces of a centenarian living in Bama Guangxi,China.Previous studies reported that strain BBMN68 plays vital roles in maintaining digestive function,improving constipation,and increasing the immunologic function of the host This study aims to improve the acid resistance of BBMN68 through adaptive evolution.The differences in the phenotype and genome between the wildtype?WT?and variant strains were compared to determine the acid tolerance mechanisms.The conclusions were summarized as follow:?1?The highest acid-resistant mutant strain?BBMN68m?is isolation from BBMN68 lines which were adapted to acid stress?pH 2.5?,through acid stress adaptive evolution.The variant strain showed a significant increase?4 log CFU/mL?in acid tolerance under conditions of pH 2.5 for 2 h compared with the wild type strain.The acid resistant of BBMN68m is genetic stability.?2?Sequencing is performed using an Illumina Hiseq2000,more than 2.3G raw data generated for BBMN68m.Genome comparison identified 16 single nucleotide polymorphisms?SNPs?in the mutant strain when compared with the WT strain.Six genes showed changes in amino acid sequences,of which three showed positional changes of amino acid within the predicted protein functional domains.The enzyme activity was detected,and found that of CpsD?BBMN68₁012?is discrepancy.Functional prediction analysis of eps-clusters proteins indicates that,BBMN68₁002 codes a polysaccharide repeat unit transport protein.Proteins are to participate in polymerization of repeat unit,which are coded by BBMN168₁003,BBMN68₁006,BBMN68₁007 and BBMN68₁011.BBMN68₁004 and BBMN68₁008 code proteins may areglycosyl-transferases.BBMN68₁012 encodes the key enzyme-priming glycosyltransferase?CpsD?in the synthesis of exopolysaccharide,which starts the first step of the exopolysaccharide synthesis.And also the protein of BBMN68₁009 synthetizes c-di-GMP,and the protein of BBMN68₁012 decomposes it.Functional characterization of promoters in eps-c luster genes that contributes to pH,carbon sources and ions response The promoters include DeoR,CpxR,OxyR,Lrp,FruR,RpoD,RpoS and Fur.?3?After 48 h culture,the mutant variant contains 191 mg/L EPS,whereas the wild type contains 332 mg/L.TEM shows that both strains produce an outer cell surface layer,presumes to bea capsule consisting of surface EPS,the mutant cell has a smaller/slimmer capsule and the EPS content is less.Structural analysis by NMR spectroscopy reveals that Wbl has a repeating unit with the following structure:?2-?-Glc-1?3-?-Glc-1?[3-?-Man-1]4?;and Mb1 with the following structure:?2-?-Glc-1?3-?-Glc-1?[3-?-Man-1]8?.The EPS-Mb1 may contain the helix structure.?4?Bioinformatics analysis and overexpression of the gene?coding for exopolysaccharide?were conducted to show that the synthesis of exopolysaccharide may control the biofilm formation of bifidobacteria.The CpsD,1007,1009 and 1011 genes are required for biofilm formation of bifidobacteria.?5?The matrix of BBMN68 biofilm contains water,EPS,proteins,DNA,and bacteria.The survival rate of mutant strain decreases 1 unit of log CFU/mL by adding the WT-EPS?900 ?g/L final concetration?;the live cell can not detect in the WT strain with the mutant-EPS.Cellulase breaks down the EPS thereby undermining biofilm formation and declining the acid tolerance.Analysis of the effects on biofilm is EPS,Mg2+,Mn2+,pH and carbon source.The CpsD,1007,1009 and 1011 genes are required for biofilm formation of bifidobacteria.