Bacterial Diversity Of Chinese Traditional Sourdough,Isolation Bacteriocin-producing Lactic Acid Bacteria,and Mechanism Of Activating Transcription The Operon Of RtcBA By RtcR Protein

With the increasing prevalence of more drug-resistant bacteria,the research on bacteriocin,an alternative to antibiotics,has become the focus of the current scientific field.The reason that restricts the wide application of bacteriocins is that the bacteriostatic spectrum of most bacteriocins is narrow.This problem can be solved in two ways:firstly,through exploring new types of bacteriocins with broad-inhibitory spectrum,enrich the bacteriocins species;secondly,by investigating the mechanism of bacteriocin-resistant pathogenic bacteria,so as to provide research basis for preventing or reduce drug resistance.For the first aspect,screening bacteriocin-producing LAB could provide abundant species of bacteria with broad-inhibitory spectrum.Sourdough is rich in LAB,which is a good source of isolating bacteriocin-producing LAB.The analysis of bacterial diversity of sourdough can lay the foundation for the isolation of LAB.As for the second aspect,selecting the representative bacteriocin and exploring the mechanism of bacteriocin-resistant of pathogenic bacteria,would provide clue for the overall bacteriocin-resistant among different pathogenic bacteria.Colicin,a representative bacteriocin,was selected to study the resistant mechanism of pathogenic bacteria to Colicin.Although Colicin E3,E4,E5,E6 and D can inhibit protein synthesis by cutting off the t-RNA and 16S rRNA of pathogenic bacteria,ultimately killing pathogenic bacteria,the pathogenic bacteria containing rtcBA operon,such as Escherichia coli MG1655,can tolerate Colicin's bactericidal action.This is because the transcription of rtcBA operon can up-regulate the expression of RtcB protein?ligase?and RtcA protein?cyclase?,both of which can repair damaged RNA.As a result,pathogenic bacteria become resistant.However,the transcription of rtcBA operon requires RtcR protein to activate E ?⁵⁴whole enzyme(complex of RNA polymerase and ?⁵⁴ actor)to transcript rtcB and rtcA genes.At present,the mechanism of activation transcription of RtcR protein and the structure of RtcR protein remains unclear.Investigation of activating rtcBA operon transcription mechanism of RtcR will lay the foundation of how bacteria behave resistant to Colicin.All in all,those studies can supply the clues of designing new measures to solve the problems of bacterial pathogen resistance to bacteriocin,and broaden the application of bacteriocin on food preservation and clinical application.The main findings of this study listed below.1.Firmicutes is the main phylum of all sourdoughs.Pediococcus and Clostridium are the dominant microorganisms in the west sourdoughs,while Lactobacillus in the South and part of sourdoughs in the North.The West group was significantly different from the North and South groups,whereas there is no significant difference among samples between the North and South groups.Fermentation quotient,11 genera such as Lactobacillus,metabolism of terpenoids and polyketides?MTP?and xenobiotics biodegradation and metabolism?XBM?were the main factors that caused these differences among all sourdoughs.This is the first report of the genus Clostridium and metabolic pathways related to MTP and XBM in sourdoughs.On the aspect of metabolic point,it is suggested that sourdough involves bacteriocin-producing LAB.2.Fifty-five strains of LAB were isolated from 11 sourdough collection points.Among them,22 strains had bacteriostatic effect on Escherichia coli ATCC 25922 or Staphylococcus aureus ATCC 25923,while 11 strains had bacteriostatic effect on both indicators.It was found that all bacteriocins with the best inhibitory effect was obtained at 80%ammonium sulfate saturation.The bacteriocin from 80%ammonium sulfate saturation precipitation of ZZ4 strain had the best bacteriostasis effect on Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 compared with other isolations,and it was identified as Lactobacillus rhamnosus by 16S rRNA.Bacteriocin produced by this bacterium can inhibit many common pathogenic bacteria and drug-resistant bacteria.3.The full length RtcR and?CARF-RtcR protein were successfully expressed through gene cloning.The best induction temperature was 16?,16?and 37?for full length RtcR,?CARF-RtcR and CARF-RtcR respectively.Affinity chromatography,ion exchange chromatography and gel filtration chromatography are employed to purify full length RtcR,?CARF-RtcR,IHF and ?⁵⁴ factor.The purity of these proteins was all more than 95%.The protein yields are full length RtcR 0.6 mg/L,?CARF-RtcR 0.75 mg/L,CARF 0.1 mg/L,IHF 2.0 mg/L, ?⁵44 factor 8.0 mg/L respectively.4.In vitro transcription assays showed that only when the concentration of?CARF-RtcR protein was higher than 100 nM,it could activate the transcription of rtcBA operon.The N-terminal regulatory domain of RtcR protein inhibited the activation of?CARF-RtcR and full length RtcR.Standard curve method and SEC-MALS method confirmed that both?CARF-RtcR protein and full length RtcR protein existed as a dimer.Using nondenaturing polyacrylamide gel electrophoresis,the affinity constant of?CARF-RtcR protein to 194 bp rtcBA promoter DNA was 550 nM,while that to the 32 bp rtcBA promoter DNA was 1730 nM.The affinity constant of IHF to the 194 bp rtcBA promoter DNA was 38 nM,and these three proteins were bound to rtcBA promoter DNA as a dimer.The hydrolysis rate of basic ATPase was determined by fluorescence spectrophotometry.With the help of 194 bp and 32 bp rtcBA promoter DNA,the hydrolysis rate of basic ATPase of?CARF-RtcR protein was 106 times and 20 times higher than that without rtcBA promoter DNA,respectively.Transmission electron microscopy revealed that the 194 bp rtcBA promoter DNA could assist hexamerization of?CARF-RtcR protein with ADP·AlF_x.Finally,gel filtration chromatography confirmed that the?CARF-RtcR protein formed a complex with ?⁵⁴ factor in the form of hexamer.5.The natural CARF-RtcR protein crystals and selenomethionine CARF-RtcR protein crystals were optimized by sitting-drop and hanging-drop method.Using UV light and running?CARF-RtcR crystals on SDS-PAGE,verify that?CARF-RtcR were crystallized.The resolution of natural CARF-RtcR protein crystals and selenomethionine CARF-RtcR protein crystals obtained by X-ray diffraction were 2.9 and 3.1,respectively.AAA⁺domain of?CARF-RtcR was completely built while DNA-binding domain was not,due to its poor density.The three-dimensional structure of?CARF-RtcR protein AAA⁺domain is mainly composed of N-terminal alpha helix-beta folding sub-domain and C-terminal alpha-helix sub-domain.GAFTGA ring was found in this domain,which is rarely seen in other homologous proteins.?CARF-RtcR existed as a dimer in an asymmetric unit cell,while crystallographic symmetry expanding further generated a hexamer ring which probably represent its functional form.