Mining Binding Sites And Functional Analysis Of Transcription Factors In Genus Corynebacterium

Corynebacterium glutamicum and Corynebacterium stationis are important strains of Corynebacterium,and are widely used in the production of amino acids,organic acids,and nucleotides.There are more than 140 highly diverse species in the genus Corynebacterium,but the genetic background and regulatory network of some strains with industrial potential are not clear and are somewhat limited in their application.As the core element of the metabolic regulatory network,transcription factors have the function of "multi-point regulation" and dynamic regulation,which can regulate metabolic pathways globally,efficiently,and specifically,and are of great significance for studying the metabolic regulatory network of Corynebacterium.Transcription factors are of great significance for the regulation and production of Corynebacterium.But there are few reports on the function of transcription factors in C.glutamicum non-model strain ATCC 14067 and C.stationis ATCC 6872 that used in the production of amino acids and nucleotides.Based on this,in this study,C.glutamicum non model strain ATCC 14067 and C.stationis ATCC 6872 were studied.Firstly,the complete genome of non-model strain ATCC 14067 was sequenced and its genome was preliminarily analyzed;Secondly,the chromatin immunoprecipitation and sequencing(ChIP-seq),electrophoretic mobility shift assays(EMSA)and real-time quantitative PCR(RT-q PCR)were used to study Amt R,Ytr A,Gab R in ATCC 14067 and Glx R,Mcb R,Amt R in ATCC 6872,revealing the binding sites of related transcription factors on the genome-wide and their regulatory functions on related genes,this study enriched the metabolic regulation network in Corynebacterium.The details and results are as follows:1)Complete genome sequencing and analysis of ATCC 14067The genome of ATCC 14067 was sequenced by the second-generation sequencing platform Illumina Hiseq and the third-generation sequencing platform Pacbio RSII.The results showed that the complete genome was 3,329,895 bp and the GC content was 54.14%,there were 3,222 genes in ATCC 14067.The analysis of genes related to horizontal transfer elements revealed that ATCC 14067 contained 33 putative GIs,30 ISs,and 3 Prophages.The transcriptional regulatory proteins in the genome of ATCC 14067 were searched and compared with ATCC 13032,there were 11 specific transcription factors and a specific two-component system in ATCC 14067.2)Binding sites and function of Amt R in the genome-wide ATCC 14067Firstly,we investigated the effects of microsphere nuclease(MNase)and non-contact ultrasonic disruptor fragmentation of chromatin on subsequent ChIP enrichment of DNA,the effects of microsphere nuclease(MNase)and non-contact ultrasonic fragmentation of chromatin on subsequently ChIP enrichment of DNA were studied,and the method of noncontact ultrasonic fragmentation for staining was determined.A total of 10 Amt R related DNA enrichment regions(peaks)were obtained by the ChIP-seq experiment,7 peaks were related to26 reported genes have been reported,and 2 peaks were related to 4 ATCC 14067 specific genes cre T,crn A,csh A and hyu B;EMSA identified that there were 5 Amt R binding sites in the promoter region of related genes.Protein sequence alignment analysis,gene knockout and phenotypic analysis showed that cre T,crn A,csh A and hyub were related to creatine metabolism,in which cre T encoded creatine transporter,creatine transporter was reported in bacteria for the first time.RT-q PCR and promoter activity analysis revealed that Amt R repressed the expression of cre T,crn A,csh A and hyub.3)Binding sites and function of Ytr A and Gab R in the genome-wide ATCC 14067Ytr A and Gab R are highly conserved in genus Corynebacterium.ChIP-seq was used to identify the genome-wide binding sites of Ytr A and Gab R in ATCC 14067.The results showed that Ytr A and Gab R have 5 and 1 binding sites in the genome,respectively.Secondly,EMSA,sequence alignment,and MEME analysis showed that the conserved sequence of Ytr A binding sites was GGTT-N16-AACC,3 of the Ytr A binding sites were located in the encoding region,and 2 were located in the promoter region.RT-q PCR analysis showed that only CEY17＿RS02205 showed a significantly decreasing,and the others were no significantly difference in knockout ytr A.DNase I footprinting and EMSA analysis showed that the conserved sequence of Gab R binding site in the genome was GGCGAA-N14-TTCGCC.RTq PCR and promoter activity analysis showed that Gab R could activate γ-aminobutyric acid metabolism-related enzymes.4)Binding sites and function of Glx R,Mcb R,and Amt R in the genome-wide ATCC 6872Combining ChIP-seq and EMSA was used to identify the conserved sequences of the binding sites of the global regulatory transcription factor Glx R,thiometabolism regulatory transcription factor Mcb R and nitrogen metabolism regulatory transcription factor Amt R in C.stationis ATCC 6872,which have 33,17 and 7 binding sites in the genome,respectively,the motif of Glx R,Mcb R,and Amt R were GTG-N8-CAC,TAGAC-N6-GTCTA,and CTAT-N6-ATAG,respectively.Compared with C.glutamicum,Glx R in ATCC 6872 repressed quinolinic acid synthesis,TRAP transport system and t RNA aminotransferase,activated the aminopeptidase N,histidine transporter,tartrate dehydrogenase,glycerate kinase and Amp nucleosidase;Mcb R repressed the expression of Tau E/Saf E family sulfite transport system,type II methionine synthase,DL-Methionine transporter and ABC nitrate/Sulfonate/aminoethanesulfonic acid/bicarbonate transport system;Amt R repressed the expression of CTP synthase and activated the expression of D-amino acid oxidase,it is reported for the first time that Amt R can activate gene expressionIn this study,the complete genome of non-model strain ATCC 14067 was sequenced and analyzed.The binding sites of transcription factors Amt R,Ytr A,Gab R in ATCC 14067 and Glx R,Mcb R,Amt R in ATCC 6872 and their regulatory functions on related genes were studied by ChIP-seq,EMSA and RT-q PCR,this study not only enriched the regulatory network in genus Corynebacterium,but also provided a theoretical basis for the construction and transformation of metabolic engineering strains.