Regulation Mechanism Of The Two-component System TcsR2/K2 In Lactococcus Lactis Under Acid Stress

Lactococcus lactis is a gram-positive bacterium with important economic value,which is widely used in the field of food fermentation.Moreover,it has the probiotic characteristics,no inclusion body and endotoxin,and a series of genetic engineering tools have been developed.In recent years,its application has gradually expanded from the food field to the microbial cell factory,leading to an important application prospect in the pharmaceutical industry.In the growth process,it faces a variety of environmental stresses,especially acid stress,which becomes a limiting factor for the yield of products and biomass accumulation.Therefore,it is of great value to study the stress response and regulation mechanism of L.lactis.To further study the regulation mechanism of L.lactis under stress,this study took L.lactis F44 as an example and studied the cooperative regulation mechanism of the two-component system(TCS)TcsR2/K2,phage shock protein(Psp)system Yth A,small RNA C277,and global regulator CodY,mainly from the four aspects:1.Firstly,quantitative proteome technology was performed to identify the acid stress response mechanism.Except for the typical acid-resistant pathways,the expression levels of fine genes encoding histidine kinases involved in TCSs and the Psp C family regulator Yth A were activated.Secondly,five TCSs overexpressed strains were constructed.The acid tolerance of tcs R2/K2 overexpressed strain was 7.4±1.2 times higher than that of the control.Then,we performed the transcriptome and Ch IP-seq technique to further investigate its regulatory mechanism.It was showed that TcsR2 could directly bind to the promoter regions of 9 genes,including yth A,and the potential Motif of TcsR2 binding to its targets was identified.2.To investigate the cooperative response mechanism between TCS TcsR2 and Psp system Yth A,we performed quantitative real-time RT-PCR,founding that TcsR2 could increase the transcriptional level of Yth A.Moreover,we constructed tcs R2 and yth A mutants,based on phenotypic analysis,finding that deleting tcs R2 significantly influenced the ability of Yth A regulating the acid resistance and biofilm formation.Besides,GST-pull down and surface plasmon resonance was conducted,showing that TcsR2 could bind to Yth A in vitro and the KD value was 46.4 ?M.Overall,these results suggest that TcsR2 and Yth A have a synergistic effect on acid stress.Not only the transcription of Yth A is activated by TcsR2,but they may interact with each other,which could affect the growth,tolerance,and biofilm formation of L.lactis.3.Crosstalk mechanisms between sRNAs and transcriptional regulators are widespread in bacteria.Our previous study found that acid-induced sRNA C277 suppressed the translation of yth A by binding its m RNA.In this study,bioinformatics prediction found C277 could also interact with tcs R2 m RNA by complementary base pairing.We demonstrated C277 bound to the region including RBS of tcs R2 m RNA to inhibit its translation using fluorescence hybrid system and mutation analysis.Furthermore,the RNA EMSA experiment confirmed that C277 could directly bind to both yth A and tcs R2 m RNA.By phenotypic analysis,it was further confirmed that sRNA C277 could affect the acid and Nisin tolerance of strain by repressing the expression of yth A and tcs R2.In general,we suggest that sRNA C277 can directly inhibit the expression of yth A and tcs R2 by interacting with their RBS regions to avoid excessive stress response.4.The global transcriptional regulator CodY is responsible for the regulation of multiple pathways,enabling bacteria to quickly adapt to environmental changes.Based on the previous proteome analysis,we speculated that CodY could participate in the acid stress response by reducing the inhibition of nitrogen metabolism.To further investigate the function of CodY and its direct targets,we performed the quantitative proteome and Ch IP-seq analysis,showing that it could serve as an activator or inhibitor.Except for widely inhibiting nitrogen metabolism,it regulated the genes involved in cell wall synthesis,Nisin synthesis,transcriptional regulation,and it could also bind to the encoding region of its gene.Through screening and EMSA,23 genes were verified to be directly bound by CodY.Further study confirmed that CodY could promote the synthesis and immunity of Nisin by directly regulating the transcription of Nisin related genes,and had a positive regulatory effect on the cell wall synthesis genes.Also,the binding regions were identified through the DNase I footprint analysis,and two potential DNA binding Motifs were predicted.Here,the crosstalk mechanisms among two-component system TcsR2 / K2,Psp system Yth A,and sRNA C277 under acid stress and the regulation of the global regulator CodY were investigated,which provides theoretical guidance for the comprehensive analysis of the regulation of L.lactis under acid stress and the construction of the regulatory network,and gives new ideas for the construction of robust industrial strains.