Study On Acid-resistant SRNA C277 In Lactococcus Lactis F44 And Construction Of High-yield Strains

Bacterial small noncoding RNAs(sRNAs)play significant roles in stress response,but few studies have explored on the functions of sRNA in Lactococcus lactis.Previous studies have shown that 58 sRNAs that are found by the deep sequencing are highly expressed under acidic condition pH 5.0.It has been verified that these sRNAs are related to the acid resistance of the strains and the regulatory mechanism of them need to be further investigated.After screening,we found that c277 had a higher transcription level under acid stress through sRNA deep-sequencing,which was further confirmed by Northern blot and qRT-PCR.The total length of c277 was determined by rapid-amplification of cDNA ends.The c277 deletion and overexpression strains(L.lactis F44-?277 and L.lactis F44-p277)were constructed.The acid resistance of L.lactis F44-?277 was improved.Overexpression of c277 led to a weaker survivability and mutants lacking c277 enhanced the viability compared with the wild type L.lactis F44.To further explore the regulation mechanism of c277,proteomics analysis of F44-?277 was performed.We identified a set of genes encoding transcriptional regulators and amino acid synthesis affected by c277 in response to acidic stress.Meanwhile,we used online software to predict the potential targets of c277.Combined with proteomics and online software prediction results,we selected seven targets to perform report fusion and mutation experiments.The results confirmed that c277 inhibited the expression of three aminoacyl-tRNA synthetases,Alas,Thrs,and Tyrs,and a transcriptional regulator YthA through direct base pairing to the ribosome binding site in a conserved region(GCCTTTC).Moreover,we previously found that YthA increased several amino acid synthesis pathways,especially arginine,and therefore it was indirectly confirmed that c277 could impact arginine synthesis via YthA.Taken together,we modeled a regulatory network of c277 and revealed the regulation mechanism in response to acid stress.In addition,we constructed an artificial sRNA library and screened out artificial sRNAs that inhibited the expression of glnR.We constructed an overexpression strain,which showed better acid resistance over than the wild type,and improved the nisin yield effectively,which provided a new strategy for the further construction of high-yield strains.