Identification Of Novel Teichoic Acid Hydrolase Genes In Bacillussubtilis

Teichoic acid(TA)is a unique component of the cell wall of Gram-positive bacteria.It is a kind of anionic polymer formed by the linkage of repeating polyol-phosphate units with phosphate diester bond.TA has important physiological functions for microbes.The enzyme that breaks the skeleton of TA is defined as Teichoic Acid Hydrolase.teichoic acid hydrolase is essentially phosphatase,which has the activity of phosphomonoesterase or phosphodiesterase.Previous studies have shown that Teichoic Acid Hydrolase activity has been detected in fungi(Aspergillus niger),bacteria(mainly Bacillus)and viruses(mainly phages).Using the Amino acid sequence information of GP12 which is considered to be teichoic acid hydrolase as a reference,we found two new teichoic acid hydrolase genes,yobO and bsp,in the genome of B.subtilis.In this study,we constructed the candidate gene into pET vector and expressed it in E.coli.As a result,BSP was expressed well in soluble form,but YobO was always expressed in the form of inclusion body even after a series of optimization of induction conditions.Therefore,we constructed yobO into pHT43 vector and transformed it into B.subtilis for expression.Finally,we got the soluble expression of YobO.YobO and BSP were affinity purified to homogeneity using Nickel-charged IDA resins.Both YobO and BSP showed hydrolysis activity of bis-pNPP,pNPP and WTA.The enzymatic properties of YobO and BSP were determined with bis-pNPP as substrates.In Tris-HCl buffer,the optimal temperature for hydrolysis of bis-pNPP by BSP is 85?,and the optimal pH is 8.4.At 37 ?,pH8.0,the kcat values of YobO and BSP were 2.68±0.09 min-1 and 6.99±0.23 min-1,respectively.The catalytic activity of divalent ions to YobO and BSP is not necessary,but the catalytic activity of most divalent ions to BSP is promoted.Among them,the promotion effect of manganese and cobalt ions is the most obvious,with an increase of 414%and 331%respectively.The catalytic activity of divalent ions to YobO was not significantly improved.We further determined the effect of yobO on the growth of B.subtilis 168 by using the yobO deletion mutant,and speculated its possible physiological function.We found that the lack of yobO made B.subtilis enter the stage of spore production earlier.The spore yield of wild-type strain was 3.83%and that of mutant was 47.12%after 24 hours culture in sporulation medium.When cultured to 36 hours,the spore yield of wild type was 25.93%,and that of mutant was 75.04%.The absence of yobO also affects the autolysis ability of B.subtilis.In the presence of Triton X-100,there was no significant difference in autolysis between wild-type strains and mutants,but in the absence of Triton X-100,the mutants shows higher autolysis rate than the wild-type strains.