Analysis On The Functional Domain And Key Amino Acids Of CodY-DNA Binding

The global transcriptional regulator CodY could regulate various processes,such as carbon metabolism,nitrogen metabolism,oligopeptide transport,amino acids degradation,virulence,and genetic transformation in Lactococcus lactis F44.However,with the lactic acid production,the pH of the medium is becoming lower and then the cell growth is inhibited or even dead.Cell wall,the first barrier of bacteria,plays a vital role in protecting cells and coping with various environmental stresses.The primary study found that global transcriptional regulator CodY regulates the expression of ddl gene that codes the D-alanine-D-alanine ligase Ddl participated in peptidoglycan synthesis through the DNA pull-down and electrophoretic mobility shift assays(EMSA).Based on that,there are many other genes include the similar DNA domain related to the peptidoglycan biosynthesis according to previous studies and bioinformatics analysis.To further explore whether the cell wall biosynthesis regulated by the CodY transcriptional regulator,we expressed and purified CodY protein and used the electrophoretic mobility shift assays(EMSA)to verify that CodY could bind with the promoter sequences of murC,murD and murG genes related to cell wall peptidoglycan biosynthesis.In addition,the results of EMSA demonstrated that branched-chain amino acid(BCAAs)as the only effect factor of CodY in L.lactis could enhance the stability of CodY protein binding with DNA and promote the CodY-DNA binding,especially murC and murD.Through the MEME analysis,we found the conservative DNA Motif that can be recognized by the CodY binding with DNA promotors.According to mutation analysis of Motif combined with EMSA,we found that CodY could regulate the transcription of peptidoglycan biosynthesis genes by binding with the 5'-CTGWCAG-3' Motif.Moreover,the molecular simulation was performed to predict the key amino acids in the wHTH structural domain combining with DNA sequences.PCR point mutation experiments were conducted to replace those important amino acids with the nonpolar alanine.Overall,the results showed that the 218 th arginine,227 th lysine,and 237 th arginine played critical roles in CodY-DNA binding.In conclusion,this study researched on the functional domains of the CodY transcriptional regulator and made a prediction of the key amino acids that could bind with DNA sequence.Moreover,CodY could bind with the promoters of cell wall synthesis genes murC,murD and murG,and the Motif and key amino acids of CodY binding with DNA were verified.The results gave new insights on the cell wall and regulatory mechanism of CodY in L.lactis F44.