Study On The Transcriptional Regulation Mechanism Of Catablite Control Protein A On The Metabolism Of Fructooligosaccharides By Lactobacillus Plantarum

As one of the prefer prebiotics,although the selective stimulation of fructooligosaccharides(FOS)to lactic acid bacteria in the gut has been proven both in vivo and in vitro,study on the pathways and regulatory mechanism for FOS metabolism is still in its infancy.In our previous study,two clusters(sac PTS1 and sac PTS26)were found and involved in the metabolism process.The global regulator catabolite control protein A(CcpA)may be involved in the regulation.To confirm the hypothsis,this study focused on the regulation relationships of CcpA,the specific binding was verified by electrophoretic mobility shift assays in vitro and chromatin immunoprecipitation in vivo.Knockout of ccpA gene and RNA-seq studied the CCR of CcpA-dependent.Furthermore,the manner of CcpA with target genes were studied by Ch IP-seq.The main conclusions of this paper are as bellows:(1)The sac PTS1 cluster is transcribed in two polycistronic units: sac K and pts1 are transcribed together and sac A,sac R,and agl2 are divergently oriented and cotranscribed as an operon.The sac PTS26 constitutes a single operon.A conserved common cre consensus motif within the genome of L.plantarum ST-III were searched based on the Reg Precise database by bioinformatics software.Using the generated PFM to search in the sac PTS1 and sac PTS26 clusters,six potential the catabolite responsive element(cre)sites were found,and that calculated the score values for each potential cre site.To verify CcpA towards target genes and the binding effects to the cre site,CcpA was expressed heterologously in E.coil,and the purified His6-CcpA protein was used to perform an EMSA,demonstrated the specific binding of CcpA to the six sites.Ch IP assay was performed to specifically enrich the DNA fragment bound with CcpA,the regions of promoter were remarkably enriched by CcpA protein,suggesting that CcpA interacts specifically with those promoter regions in vivo.(2)A mutant ccpA deletion strain was constructed using the Cre-lox-based mutagenesis system to study the differential gene expression and the possible regulatory way of CcpA for mutant strain compared with the wild-type strain using fructooligosaccharides(FOS)or glucose as the sole carbon source.The inactivation of ccpA significantly affected the growth and production of metabolites under both carbon sources.About 15% and 12% of the total genes were significantly altered between wild-type and ccpA strains grown on glucose and FOS,respectively,while only 7% were obviously changed due to the loss of CcpA when comparing strains grown on glucose and FOS.After the deletion of ccpA,the largest proportion of upregulated genes is involved in carbohydrate metabolism,and the genes of significantly downregulated involved fatty acid biosynthesis.Although most of the differentially expressed genes mediated by CcpA are glucose dependent,FOS can also induce carbon catabolite repression(CCR)through the CcpA pathway.Moreover,the inactivation of ccpA led to a transformation from homolactic fermentation to mixed fermentation under aerobic conditions.CcpA can control genes directly,indirectly t,or have a double effect via direct and indirect regulation.(3)To analyze the direct binding site of CcpA in L.plantarum ST-III,a genome-wide scan of the transcription factor binding sites contained in L.plantarum ST-III was performed using the Ch IP-seq assay.The results showed that a total of 507 different encrichment peaks were captured.Firstly,5 motifs were identified using bioiniformatics software,and then 159 genes were found on the L.plantarum ST-III genome based on these 5 motifs.39 genes with motifs were significant change combining RNA-seq data,suggesting that these genes containg motif and significant changes were directily regulated by CcpA.Finally,a global regulatory network model is constructed to analyze the regulatory mechanism based on CcpA.