Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

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

The transfer of genes and proteins at the micro-organismal level plays a central role in bacterial survival and evolution as well as infection processes. The exchange of DNA between bacteria or between a bacterium and a cell can be achieved through transformation, conjugation or vector transduction.^1,2 Conjugation is unique in comparison to transformation and transduction in that during conjugation between gram-negative bacteria such as Escherichia coli, the transfer of DNA occurs in a donor-controlled fashion whereby a complex macromolecular system connects donor and recipient cells. Conjugation is also the most direct way in which bacterial cells....

Protocol

1. Generation of DNA Constructs

1. Designing Oligomers for Homologous Recombination of the Target Gene
   1. Design a single 55-72 bp forward oligomer as follows: (a) pick a 19-32 bp long nucleotide sequence that is homologous to a DNA sequence in the region 10-100 bp upstream of the 5' start site of the chloramphenicol acetyltransferase gene in the commercial pBAD33 plasmid,³² and (b) select a 36-54 bp long nucleotide sequence homologous to a region 10-150 bp downstream of the 5' start site of the target gene of interest.
      1. Join the 3' end of nucleotide sequence picked in (b) to the ....

Results

The process of F plasmid-driven bacterial conjugation is a coordinated process that involves transfer proteins within the tra region of the F-plasmid that assembles a T4SS to facilitate pilus synthesis and conjugative DNA transfer. The protein TraF (GenBank accession # BAA97961; UniProt ID P14497) is required for conjugative F-pilus formation.^10,14,35-37 The protein contains a C-terminal thioredoxin-like domain, though it does not have the catalyt.......

Discussion

Bacterial conjugation process provides a means by which bacteria can spread genes providing an evolutionary advantage for growth in challenging environments, such as the spread of antibiotic resistance markers. Because many of the conjugative plasmids are so large,¹² functional studies on the proteins involved in assembly of the transfer apparatus through mutation of target genes on the conjugative plasmid itself are unwieldy. The protocols detailed herein provide a means by which one can more readily assess t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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