Functional Characterization Of C-di-GMP-related Genes And The Study Of C-di-gmp Downstream Signaling Pathways In Lactobacillus Acidophilus

Background and objectivec-di-GMP,synthesized from 2 molecules of GTP,is an universal bacterial second messenger regulating different physiology processes.Opposing activities of diguanylate cyclases?DGCs?containing GGDEF domain and phosphodiesterases?PDEs?containing EAL or HD-GYP domain control c-di-GMP homeostasis in the cell.The gram-positive Lactobacillus acidophilus,producing acid through fermenting sugars into lactic acid and growing readily at rather low pH values,is considered to be one of the major bacteria relation with caries disease.In silico analysis show that the genome of Lactobacillus acidophilus contain a diguanylate cyclases encoded gene?NH13₀7045?,and two phosphodiesterases encoded genes?NH13₀7050 and NH13 07055?.In this study,the c-di-GMP signal pathway was studied in Lactobacillus acidophilus ATCC4356 for the first time.It is aim to explore the signal mechanism of c-di-GMP regulating the physiological activity of Lactobacillus acidophilus,and provide new evidence for the prevention and treatment of dental caries.MethodsFirst,the three genes involved in c-di-GMP upstream metabolic pathways in Lactobacillus acidophilus were analyzed by bioinformatics.DNA fragment of NH13₀7045-GGDEF,NH13₀7050 and NH13₀7055 from Lactobacillus acidophilus ATCC4356 were cloned,expressed and purified using expression vector pMAL-c2-His to obtain fusion proteins for enzymatic activity in vitro,and the reaction products were analyzed by High Performance Liquid Chromatography?HPLC?.The full length of NH13 07045 gene was cloned into expression vector pBAD-Myc-His and expressed in E.coli MG1655 and BL21.The phenotypic changes of E.coli were observed by motility assay and Congo red binding assay to identify whether DgcA had DGC activity.The expression of PdeB-EAL domain was purified using pMAL-c2-His and the target fusion protein was obtained.Then,DraCALA and UV cross-linking assay were performed with Fluo-c-di-GMP and Biotin-c-di-GMP respectively.The Biotin-c-di-GMP conjugated magnetic beads were used to perform the affinity pull down assay in Lactoacillus acidophilus lysates.After SDS-PAGE and silver staining,Protein identification was carried out by in-gel proteolysis and the resultant peptides were sequenced by liquid chromatography-tandem mass spectrometry?LC-MS/MS?.The total RNA of Lactobacillus acidophilus ATCC4356 was extracted and reverse transcribed into cDNA as a template for PCR reaction.The genes between NH13₀7045 to NH13₀7065 were analyzed for operon.The NH13₀7060 gene was cloned and expressed in E.coli,and the function was analyzed by Congo red binding assay and biofilm formation assay.ResultsThe bioinformatics analysis of the c-di-GMP metabolic related genes and the coding proteins were carried out,which provided the theoretical basis for further gene cloning,expression and purification,and enzyme activity analysis.After recombinant vectors pMAL-7045-GGDEF,pMAL-7050 and pMAL-7055 successfully constructed,the fusion proteins were obtained by expression and purification.In vitro enzyme activity assay showed that the EAL-containing cytoplasmic protein PdeA possesses PDE activity and can specifically hydrolyze c-di-GMP.The recombinant plasmid pBAD-7045 was successfully constructed.Under the induction conditions,E.coli MG 1655 and BL21 exhibited a high intracellular c-di-GMP phenotype,indicating that DgcA protein has DGC activity and can synthesize c-di-GMP in vivo.The expression vector pMAL-7055-EAL was successfully constructed.After induction and purification,the target fusion protein was obtained.The results of DRaCALA and UV cross-linking assay showed that c-di-GMP could bind PdeB specifically.27 putative proteins were identified to directly or indirectly bound to c-di-GMP in Lactobacillus acidophilus by high performance liquid chromatography-mass spectrometry?LC-MS?.The PCR analysis of cDNA showed that NH13₀7045 and NH13₀7050 form an operon,NH13₀7055,NH13₀7060 and NH13₀7065 form another operon.The pBAD-7060 plasmid was successfully constructed and expressed in E.coli BL21 and MG 1655,the redness of colonies and the biofilm formation were increased.It suggested that the glycosyltransferase encoded by NH13₀7060 might be related to the synthesis of exopolysaccharide.ConclusionsFor the first time,c-di-GMP signaling pathway was found in Lactobacillus acidophilus,and DgcA and PdeA had the activity to synthesize and degrade c-di-GMP.The CrpA?PdeB?,containing a degenerate EAL domain,serves as a c-di-GMP specifical receptor,but its biological function has not been clarified.The 27 potential receptor of c-di-GMP in Lactobacillus acidophilus were screened.It was validated that DgcA and PdeA were encoded by the same operon to regulate the concentration of c-di-GMP in Lactobacillus acidophilus,and the c-di-GMP receptor CrpA was associated with the unknown function of the glycosyltransferase and the hypothetical transmembrane protein under another operon regulation.It was confirmed that NH13 07060 was involved in the synthesis of exopolysaccharide and biofilm.This suggests that c-di-GMP may regulate the formation of polysaccharides and biofilm in Lactobacillus acidophilus,thereby affecting the caries capacity of Lactobacillus acidophilus.It laid a foundation for further clarifying the regulation mechanism of c-di-GMP in Lactobacillus acidophilus.