| Lactococcus lactis produce lactic acid through glycolysis during the growth process,and the accumulation of lactic acid will acidify the environment,which is harmful to the growth of bacteria.In this case,L.lactis formed a series of acid-resistant mechanisms,such as producing alkaline to neutralize H~+,draining H~+by proton pumps,and the protection and repair of macromolecular.These intricate mechanisms require the regulation of regulatory systems.Transcriptional regulators play an important role in the regulation of acid resistance of L.lactis.Our previous research found that the function-unknown regulator ATY87832.1 is related to acid-resistantance,and bioinformatics analysis found that it is also a type of xylose transcriptional regulator,which may play an important role in the utilization of xylose.We renamed it to Xyl R2.The purpose of this study is to further explore the regulatory mechanism of Xyl R2 in acid tolerance and xylose utilization.First,we overexpressed xyl R2 in F44,cultivated it in the medium with xylose as the only carbon source,and compared it with the thoroughly studied xylose transcriptional regulator Xyl R.Through OD,p H and residual xylose analysis we found overexpression of xyl R2 could significantly increase the growth rate in xylose medium,but it is still lower than the xyl R overexpressing strain,which suggests that Xyl R2 is beneficial to improve xylose utilization ability of F44,but it is not as effective as Xyl R.In order to further explore the reasons why Xyl R2 can improve acid tolerance and xylose utilization and investgate its target genes,transcriptome analysis was performed on Fxyl R2,showing that genes with significant changes in transcripts mainly involved in amino acid transport metabolism,such as genes pro C,leu A,ysf E,trp A,pct A,arc C1,gad B,pyr R,etc.;carbon transport and metabolism,such as genes kdg A-kdg K-uxac,ica B,dexb,gnd,etc.;natural competence systems,such as genes com GC,com GD,com GE,and com GF.In addition,Sig X is an ECF?factor,which is related to the cell envelope stress response.It was significantly increased by 1.98 times in the transcriptome;further verification by constructing the overexpressing sig X and pyr R strains shows that the strains increased the acid resistance by 3.0 and 2.8 times compared with the control,respectively.At the same time,Ch IP-seq technique was used to investigate the direct regulation of target genes by Xyl R2,showing that the genes that bind to Xyl R2 were mainly focused on the carbon metabolism and DNA repair.Combining transcriptome and Ch IP-seq results,we selected genes fbp,uvrb,gnd,glg C,arg B,rec D,pyr R,com GC and sig X for EMSA experiments to further verification.Finally,we identified three genes,pyr R,com GC and gnd was bound by Xyl R2 in vitro,indicating that Xyl R2 directly regulated these genes.Meanwhile,sequence mutation was performed according to the binding site of Xyl R2 to DNA predicted by MEME analysis.Combined with EMSA,we identified TGYCDGMMTBKCTS was the binding site of Xyl R2.In this study,the regulation mechanism of Xyl R2 on acid tolerance and xylose utilization was deeply explored,and the target genes directly regulated by Xyl R2 at a genome-scale level were identified,which has laid s the foundation for a comprehensive analysis of the acid resistance mechanism of L.lactis,and provideds new insights into the regulatory mechanism of xylose utilization. |
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