The Identification And Function Study Of Acid-tolerant SRNA042 In Lactococuus Lactis

In the industrial fermentation process,Lactococcus lactis can produce lactic acid,the accumulation of which can result in the acidification of the fermentation broth.Even worse,this self-posed acid stress will negatively affect the bacterial growth and concomitant nisin production.Non-coding sRNA(sRNA),a global transcriptional regulatory factor,is acknowledged to play important roles in response to environmental stresses in bacteria.In order to explore the relations between sRNA and acid resistance,we performed High throughput Seq and RT-PCR to screen acid-associated sRNAs.Furthermore,these acid-associated sRNAs were overexpressed and verified by acid resistance test.Here,a series of physiological and bioinformatics methods were carried out on the basis of the verificated acid-associatedd sRNA 042.In accordance with the RT-PCR results,northern blot analysis showed that the transcription level of sRNA042 at pH5.0 is higer than that at pH7.0,which is indicated that the transcription level of sRNA042 is upregulated in acidic conditions.To further investigate its function,sRNA042-negative strain(F44-?sRNA042)was constructed by homologous recombination method,The phenotypic analysis results showed that the ability of acid tolerance of the mutanted strain decreased significantly compared with that of the wild-type F44.Interestingly,the absence of sRNA 042 did not affect the growth and energy of the strain in the fermentation medium.Traditionally,sRNA exerts their wide range of regulatory functions in bacterial cell by base pairing with target mRNAs,which leads to mRNA protection or translational repression.On this basis,the specific mechanism of 042 in regulating acid-ressitance capacity of L.lactis needs to be further studied.With the prediction of TarRNA IntaRNA and sTarpiker and the analysis of its secondary structure and interaction with m RNA,four targets(ArgR?AccD?Cbio and ribF)were selected and verified by EGFP fusion protein,the results showed that sRNA042 can activate the translation process of argR and accDm RNA,and increase the transcription level of target gene.This study verified that sRNA042 were acid-associated,whose overexpression contribute a higher acid-resistance capacity.We also substantiated that the specific base-pairing mechanisms between sRNA042 and its target genes and showed that sRNA had positively effects in regulating the acid resistance of strain.