| Small non-coding RNA(sRNA)is an important kind of regulator that is widely present in microorganisms and is involved in the regulation of many physiological processes and substances.Putrescine is the main polyamine compound in bacteria.It not only interacts with biomolecules on the cell membrane,but also participates in various physiological functions.It can also be metabolized by microorganisms as a non-primary carbon and nitrogen source under certain conditions.Escherichia coli can degrade the putrescine into succinic acid through the glutamine-based putrescine pathway composed of PuuDRCBE for cell growth and resistance to environmental stress.However,it is unclear whether small RNAs are involved in the metabolic regulation of putrescine.In this study,E.coli was used as the research object to construct a knockout mutant library of E.coli small RNAs.Screened and predicted potential targets and pathways for different small RNAs,identified Spot 42 can regulate the pathway of putrescine metabolism.Moreover,the target gene of Spot 42 was identified and its molecular mechanism was resolved.Through bioinformatics prediction,we determined the sequence of 49 small RNAs contained in E.coli DY329 strain and predicted their possible target genes.The homologous recombination system was used to knock out the E.coli small RNAs and acquired a single knockout mutant library containing 28 sRNAs knockout strains.Screening experiments were carried out to determine the regulation of sRNA on target genes by temperature tolerance,biofilm formation,oxidative stress tolerance and Real-Time quantitative PCR.Ultimately,multiple sRNAs were identified to regulate different traits.sRNA Spot 42 in E.coli DY329 strain,is encoded by spf gene with a length of 109 bases.Sequence alignment and secondary structure prediction show that Spot 42 is highly conserved and specific for γ-proteobacteria,contains 5 loop areas.CopraRNA software predicted that Spot 42 had a base pairing with the upstream non-coding region of the gene puuE in the glutamine-based putrescine metabolic pathway of Escherichia coli,and may be involved in the regulation of expression of puuE.RT-qPCR showed that glucose could induce transcription of spf gene,and glucose deficiency would inhibit its transcription;putrescine induced transcription of puuDRCBE gene and spf gene.Knockout and complementary experiments showed that PuuR,as the main transcriptional regulator,could directly regulate the transcription of genes such as puuDCBE,but could not regulate the transcription of spf gene;Spot 42 could not regulate puuE at the transcriptional level.We constructed a series of Spot 42 mutants for possible binding sites of spf and puuE.The results of the fluorescent protein reporter system showed that excessive transcription of Spot 42 significantly inhibited the expression level of puuE;mutations at 50-57 nts(UAAUCGGA)of Spot 42 abolished this inhibition,suggesting that this site might be a key site for direct base pairing between Spot 42 and the 5’ UTR of puuE.Through this antisense regulation mechanism,Spot 42 can regulate the expression of puuE,achieve the regulation of putrescine metabolism and switch primary and secondary carbon sources.In summary,this work constructed a sRNA knockout mutant library of E.coli,clarified that Spot 42 could inhibit the translation of PuuE through base pairing and directly participate in the regulation of putrescine metabolism.This work not only built a technical platform for E.coli sRNA research,but also improved the metabolic regulation network of putrescine,clarified the important role of Spot 42 in primary and secondary carbon source utilization and carbon and nitrogen source switching. |
PDF Full Text Request