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Abstract
Introduction
Protocol
Results
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Acknowledgements
Materials
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Genetics

Konjugering Parring Analyser for Sekvens-specifik analyse af Transfer proteiner involveret i konjugation

Published: January 04, 2017
doi:
10.3791/54854
, , , , ,
1Department of Chemistry,York University, 2The Centre for Research on Biomolecular Interactions,York University

Summary

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

Abstract

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Introduction

Overførsel af gener og proteiner i mikro-organismal niveau spiller en central rolle i bakteriel overlevelse og evolution samt infektion processer. Udveksling af DNA mellem bakterier eller mellem en bakterie og en celle kan opnås ved transformation, konjugation eller vektor transduktion. 1,2 Konjugering er unik i forhold til transformation og transduktion ved, at under konjugering mellem gram-negative bakterier, såsom Escherichia coli, overførsel af DNA sker i en donor-styret måde, hvorved en kompleks makromolekylær system forbinder donor- og recipientceller. Konjugation er også den mest direkte måde, hvorpå bakterieceller interagerer med værtsceller at injicere gener, proteiner eller kemikalier i at værtssystemer. 3 Ofte overførsel af midler har bemærkelsesværdige virkninger på værten, der spænder fra patogenese og carcinogenese at være vært evolution og tilpasning. Det er blevet påvist, at konjugerende recombination Forhøjer tilpasningen 3 gange i bakterier med høje mutationsrater under forhold af miljømæssig stress. 4 Desuden konjugering er langt den mest almindelige vej, hvorigennem antibiotikaresistensgener i bakteriestammer er spredt. 5,6

Mikroorganismer har udviklet specialiserede sekretion systemer til at støtte overførsel af makromolekyler tværs cellemembraner; der i øjeblikket 9 typer af sekretion systemer (TSSs) i gram-negative bakterier, der er blevet beskrevet: T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS, samt Sec (sekretion) og Tat (to-arginin translokation) pathways. 7,8 Hver type sekretionssystem er yderligere opdelt i forskellige undertyper, en nødvendighed på grund af mangfoldigheden af proteiner og særpræg involverede veje, i forskellige bakteriestammer. For eksempel i type IV sekretionssystem (T4SS), Ti og Cag befordrer effektor transport hvorimod F-plasmid, R27 and pKM101 T4SSs lette overførsel af en konjugerende plasmid. 7,9,10 En detaljeret forståelse af de mekanismer, som organismer samler deres respektive sekretion systemer fra deres komponent proteiner og deler cellulære indhold med en modtager eller deres omgivende miljø er en vigtig faktor i udviklingen af målrettede strategier til bekæmpelse af sygdomsfremkaldende mikroorganismer og processer cellulær infektion.

Efter den indledende identifikation konjugation i E. coli ved Lederberg & Tatum, 11 er blevet identificeret og karakteriseret et stort antal mobile og konjugering plasmider. 12 Sådanne mobile plasmider viser betydelig rækkevidde er størrelse (fra 1 til over 200 kilobaser (kb)), men alle mobile plasmider indeholder en relaxase, som anerkender oprindelsen af overførsel (oriT) hvorved transmissionen af plasmidet. Konjugering plasmider yderligere koder gener for samling af en funktionel T4SS samt en typeIV kobling protein. 12 For eksempel 100 kb F plasmid af E. coli koder alle de konjugering gener i en 33,3 kB overførsel (tra) region. 13 Generne i tra region af F-plasmid koder for alle proteiner, der fremmer pilus-dannelse, parring pair formation (MPF), DNA-overførsel og eksklusionskriterier funktioner under konjugativ plasmid overførsel. 10,14,15 En væsentlig mængde af viden er tilgængelig for konjugativ T4SSs dog detaljerede strukturelle undersøgelser af konjugering proteiner og komplekser kun for nylig bliver tilgængelige. 16 – 28

For at samle et overblik over den konjugative proces en kobling af detaljerede strukturelle studier at mutationelle analyser af konjugering transfer proteiner påkrævet. Dette kan opnås gennem konjugering parring assays. For F-plasmid, hvert protein kodet inden for tra region spiller en rolle i F-medieret conjugation; derfor vil knockout / deletion af en overførsel gen afskaffe konjugative kapacitet af cellen (figur 1). Mens mindre mobile plasmider er mere befordrende for standard sletningsprocedurer, for større konjugering plasmider, såsom F, er gen-knockouts mere let opnås via homolog rekombination, hvor målgenet er erstattet med én formidle et distinkt antibiotikaresistensgen. I den nuværende protokol, beskæftiger vi homolog rekombination til at erstatte en overførsel gen af ​​interesse med chloramphenicolacetyltransferase (CAT) i 55 kb F plasmid derivat pOX38-Tc; 29,30 den resulterende knockout plasmid, pOX38-Tc Δgene :: Cm, letter modstand mod chloramphenicol (Cm) i vækstmediet. Donorceller huser pOX38-Tc Δgene :: Cm er ude af stand til at påvirke konjugative DNA-overførsel / parring som observeret ved anvendelse af en dertil passende assay; parring effektiviteten af ​​en pOX38-Tc Δgene :: Cm donor celle og en normal Recipient vil falde eller, oftere, afskaffes. Konjugative overførsel af pOX38-Tc Δgene :: Cm plasmid kan gendannes via en lille opsving plasmid, det målrettede overførsel genet. Dette opsving plasmid kan være en, der giver konstitutiv ekspression, såsom plasmid pK184 (pK184-genet), 31 eller en, der giver inducerbar ekspression så længe denne plasmidet korrekt målretter gen til den korrekte placering i cellen (cytoplasma eller periplasmaet). Følgelig i hinanden passende assays mellem denne nye donor (huser pOX38-Tc Δgene :: Cm + pK184-gen-plasmider) og en modtager celle, forventes parring effektivitet til at gendanne til næsten en normal donor-recipient parring assay. Dette system gør det muligt at undersøge funktionen af ​​det bankede ud genet ved produktion af en række pK184-genkonstruktioner (deletioner eller punktmutationer) og teste hver konstruktion evne til at genoprette den sammenpassende kapacitet pOX38-Tc Δgene :: Cm skjult donorcelles.

Protocol

1. Dannelse af DNA-konstruktioner Designe Oligomerer for homolog rekombination af målgenet Design en enkelt 55-72 bp fremad oligomer som følger: (a) vælge en 19-32 bp lang nukleotidsekvens, der er homolog med en DNA-sekvens i regionen 10-100 bp opstrøms for 5'-startstedet for chloramphenicolacetyltransferase-gen i den kommercielle pBAD33 plasmid, 32 og (b) vælge en 36-54 bp lang nukleotidsekvens homolog med en region 10-150 bp nedstrøms for 5'startstedet af m…

Representative Results

Processen med F-plasmid-drevet konjugation er en koordineret proces, der involverer overførsel proteiner i tra region af F-plasmid, der samler en T4SS at lette pilus syntese og konjugative DNA-overførsel. Proteinet Traf (GenBank # BAA97961; UniProt ID P14497) er påkrævet for konjugativ F-pilus formation. 10,14,35 – 37 Proteinet indeholder en C-terminal thioredoxin-lignende domæne, selvom det ikke har den katalytiske CXXC motiv. 35,38</s…

Discussion

Konjugation fremgangsmåde tilvejebringer et middel, hvormed bakterier kan sprede gener tilvejebringe en evolutionær fordel for vækst i udfordrende miljøer, såsom spredning af antibiotikaresistensmarkører. Fordi mange af de konjugering plasmider er så store, 12 funktionelle undersøgelser af involveret i montering af overførsel apparatet gennem mutation af målgener på selve konjugative plasmid proteiner er uhåndterlig. Protokollerne beskrevet heri tilvejebringer et middel, hvormed man kan lettere vu…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Denne forskning blev understøttet af en Discovery Grant fra Natural Sciences & Engineering Council of Canada (NSERC).

Materials

GeneJet Plasmid Mini-Prep Kit Fisher Scientific K0503
GeneJet Gel Extraction Kit Fisher Scientific K0692
GeneJet PCR Purification Kit Fisher Scientific K0702
Q5 Site-Directed Mutagenesis Kit New England Biolabs E0554S
Broad Range DNA Ladder New England Biolabs N0303A
Petri Dishes Fisher Scientific FB0875713
Electroporator Eppendorf 4309000027
Electroporation cuvettes Fisher Scientific FB101 Cuvettes have a 1 mm gap.
Enzymes
AvaI New England Biolabs R0152S
EcoRI New England Biolabs R0101S
HindIII New England Biolabs R0104L
NdeI New England Biolabs R0111S
Phusion DNA Polymerase New England Biolabs M0530L
T4 DNA Ligase New England Biolabs M0202S
DpnI New England Biolabs R0176S
Antibiotics Final Concentrations
Chloramphenicol (Cm) Fisher Scientific BP904-100 20 µg/mL
Kanamycin (Km) BioBasic Inc. DB0286 50 µg/mL
Nalidixic acid (Nal) Sigma-Aldrich N8878-25G 10 µg/mL
Rifampicin (Rif) Calbiochem 557303 20 µg/mL
Tetracycline (Tc) Fisher Scientific BP912-100 10 µg/mL
Streptomycin (Sm) Fisher Scientific BP910-50 50 µg/mL
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Tags

Conjugative Mating AssaysTransfer ProteinsBacterial ConjugationType Four Secretion ComplexHomologous RecombinationPBAD33 PlasmidCat CassettePCR AmplificationElectrocompetent DY330R CellsPOX38-Tc Plasmid
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Erdogan, F., Lento, C., Yaseen, A., Nowroozi-Dayeni, R., Kheyson, S., Audette, G. F. Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation. J. Vis. Exp. (119), e54854, doi:10.3791/54854 (2017).
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