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.
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.
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 interact with host cells to inject genes, proteins or chemicals in to host systems.3 Quite often, the transfer of such agents has remarkable effects on the host, ranging from pathogenesis and carcinogenesis to host evolution and adaptation. It has been shown that conjugative recombination increases the rate of adaptation 3-fold in bacteria with high mutation rates under conditions of environmental stress.4 Moreover, conjugation is by far the most common route through which antibiotic resistance genes in bacterial strains are spread.5,6
Microorganisms have evolved specialized secretion systems to support the transfer of macromolecules across cellular membranes; there are currently 9 types of secretion systems (TSSs) in gram negative bacteria that have been described: T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS, as well as the Sec (secretion) and Tat (two-arginine translocation) pathways.7,8 Each type of secretion system is further divided into different subtypes, a necessity due to diversity of proteins and the distinctiveness of pathways involved, in different bacterial strains. For example, in the type IV secretion system (T4SS), the Ti and Cag systems facilitate effector transport whereas the F-plasmid, R27 and pKM101 T4SSs facilitate transfer of a conjugative plasmid.7,9,10 A detailed understanding of the mechanisms by which organisms assemble their respective secretion systems from their component proteins and share cellular contents with a recipient or their surrounding environment is an important factor in development of targeted strategies to combat pathogenic microorganisms and processes of cellular infection.
Following the initial identification bacterial conjugation in E. coli by Lederberg & Tatum,11 a large number of mobile and conjugative plasmids have been identified and characterized.12 Such mobile plasmids show considerable range is size (from 1 to over 200 kilobases (kb)), however all mobile plasmids contain a relaxase, which recognizes the origin of transfer (oriT) thereby enabling transmission of the plasmid. Conjugative plasmids further encode genes for assembly of a functional T4SS as well as a type IV coupling protein.12 For example the 100 kb F plasmid of E. coli encodes all the conjugative genes within a 33.3 kB transfer (tra) region.13 The genes in the tra region of the F plasmid encode all proteins that facilitate pilus formation, mating pair formation (Mpf), DNA transfer and exclusion functions during conjugative plasmid transfer.10,14,15 A significant body of knowledge is available for conjugative T4SSs, however detailed structural studies of the conjugative proteins and complexes are only more recently becoming available.16–28
In order to assemble a comprehensive view of the conjugative process, a coupling of detailed structural studies to mutational analyses of conjugative transfer proteins is required. This can be achieved through conjugative mating assays. For the F plasmid, each protein encoded within the tra region plays a role in the F-mediated conjugation; therefore, the knockout/deletion of a transfer gene will abolish the conjugative capacity of the cell (Figure 1). While smaller mobile plasmids are more conducive to standard deletion procedures, for larger conjugative plasmids such as F, gene knockouts are more readily achieved via homologous recombination where the target gene is replaced with one conveying a distinct antibiotic resistance gene. In the current protocol, we employ homologous recombination to replace a transfer gene of interest with chloramphenicol acetyltransferase (CAT) in the 55 kb F plasmid derivative pOX38-Tc;29,30 the resultant knockout plasmid, pOX38-Tc Δgene::Cm, facilitates resistance to the presence of chloramphenicol (Cm) in the growth media. Donor cells harboring pOX38-Tc Δgene::Cm are unable to affect conjugative DNA transfer/mating as observed through the use of a mating assay; the mating efficiency of a pOX38-Tc Δgene::Cm donor cell and a normal recipient will decrease or, more often, be abolished. Conjugative transfer of the pOX38-Tc Δgene::Cm plasmid can be restored via a small recovery plasmid harboring the targeted transfer gene. This recovery plasmid can be one that provides constitutive expression, such as plasmid pK184 (pK184-gene),31 or one that provides inducible expression so long as that plasmid properly targets the gene to the correct location within the cell (cytoplasm or periplasm). Consequently, in mating assays between this new donor (harboring pOX38-Tc Δgene::Cm + pK184-gene plasmids) and a recipient cell, the mating efficiency is expected to restore to nearly that of a normal donor-recipient mating assay. This system enables one to probe the function of the knocked out gene through the generation of a series of pK184-gene constructs (deletions or point mutations) and testing each construct's ability to restore the mating capacity of the pOX38-Tc Δgene::Cm harboring donor cells.
1. Generation of DNA Constructs
2. Generation of pOX38-Tc Δgene::Cm Strains
3. Conjugative Mating Assays
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 catalytic CXXC motif.35,38 Although it has been predicted to interact with the TraH protein through its N-terminal domain,39 a region shown to be more dynamic than its C-terminal domain,37 not much else is known about the protein's structural features. To assess the functional aspects of the TraF protein in conjunction with structural studies, we first knocked out the traF gene on the pOX38-Tc via homologous recombination, generating the pOX38-Tc ΔtraF::Cm plasmid (Table 1) in E. coli DY330R cells.33,34 Also generated was the pK184-TraF plasmid from traF-specific primers (Table 2) to provide recovery of conjugation and enable probing the protein's sequence. The transfer of the pOX38-Tc ΔtraF::Cm plasmid into XK1200 cells40 from DY330R cells when pK184-TraF was provided in trans (transconjugants grown on 10 μg/mL Nal, 10 μg/mL Tc and 20 μg/mL Cm) indicates that (a) the traF knockout in pOx38-Tc ΔtraF::Cm provides an in-frame CAT cassette, and (b) that pK184-TraF can restore conjugative function.
A series of TraF mutants were generated for analysis using the conjugative mating assay37 using XK1200 pOX38-Tc ΔtraF::Cm + pK184-TraFΔX cells and MC4100 cells41 as donors and recipients, respectively. One representative mutant is TraF55-247 (Table 1), an N-terminal deletion mutant that removes the region of the protein predicted to interact with TraH. When the full-length TraF protein is provided to the donor cells, conjugative transfer is restored (Figure 2), while providing the empty plasmid pK184 does not (Table 3). Similarly, conjugative function in the XK1200 donor cells is not restored when provided with plasmid pK184-TraF55-247 (Table 3). This indicates that the truncated region of the protein is important for TraF's function within the conjugative apparatus, likely through interaction with TraH, and provides a region of the protein to target for further mutational analysis.
Figure 1: Schematic representation of the conjugative mating assay. In Step 1, target genes are knocked out by homologous recombination in DY330R cells, an efficient recombination strain, using a PCR generated CAT cassette with overhangs homologous for the target gene. The resultant DY330R clone harbors the plasmid pOX38-Tc Δgene::Cm, which is unable to facilitate conjugation unless the knocked out gene is provided in trans via a recovery plasmid (pK184-gene). The resultant pOX38-Tc Δgene::Cm plasmid is transferred to a XK1200 strain (Step 2) for further assessment of the gene using mating assays with a MC4100 recipient (Step 3). Please click here to view a larger version of this figure.
Figure 2: Mating assay to assess the target gene's function in conjugation. Donor and recipient cells were E. coli XK1200 pOX38-Tc ΔtraF::Cm transformed with pK184-TraF and MC4100, respectively. The resulting transconjugants (MC4100 pOX38-Tc ΔtraF::Cm) grow on plates containing Sm and Cm, indicating successful recovery of conjugative function. The experiment is done in duplicate on a single agar plate, and serial dilutions are used in order calculate the mating efficiency of restorative gene mutants (Table 2). Please click here to view a larger version of this figure.
Bacterial strain/Plasmid | Relevant Characteristics | Selective Marker(s)† | Reference |
Bacterial Strains | |||
DY330R | W3110 ΔlacU169 gal490 λc1857 Δ(cro-bioA) RifR | Rif | 33,34 |
XK1200 | F– lacΔU124 Δ(nadA aroG gal attλ bio gyrA) | Nal | 40 |
MC4100 | araD139 Δ(argF-lac)U169 rpsL150 relA1 flbB3501 deoC1 ptsF25 rbsR | Sm | 41 |
Vectors and Constructs | |||
pBAD33 | Plasmid for expression under from ParaBAD | Cm | 32 |
pK184 | 2.4 kb cloning vector, p15a replicon | Km | 31 |
pK184-TraF‡ | F TraF from pOX38 in pK184 | Km | 35 |
pK184-TraF56-247 | F TraF aa 56-247 from pK184-TraF | Km | |
Conjugative Plasmids | |||
pOX38-Tc | IncFI, Tra+, RepFIA+, f1 HindIII fragment of F, mini-Tn | Tc | 29,30 |
pOX38-Tc ΔtraF::Cm | pOX38-Tc with CAT inserted in traF | Tc Cm | 35 |
†Cm, chloramphenicol; Km, kanamycin; Nal, nalidixic acid; Rif, rifampicin; Sm, streptomycin; Tc, tetracycline | |||
‡All TraF constructs contain the 19-residue leader sequence to ensure localization to the periplasmic space. |
Table 1: E. coli strains and plasmids used in this study.
Construct | Primer* |
TraF-Cm-For | 5’- GATCGAGGCTGGCAGTGGTATAACGAGAAAATAAATCCGAAGGA – CTGTGACG GAAGATCACTTC -3’ |
TraF-Cm-Rev | 5’- TCTTCAGAAACGTTCAGGAACTGTTTTGCCAGGTCGTCCT – CTTATTCAGGCGT AGCACCAG -3’ |
pK184-TraF-For | 5’- TTTTTTGAATTCTATGAATAAAGCATTACTGCCAC -3’ |
pK184-TraF-Rev | 5’- TTTTTTAAGCTTTAAAAATTGGGTTTAAAATCTTCAGAAA -3’ |
pK184-TraF55-247-For | 5’- TACGCATATGATGGCCGCACTGCAGACGG -3’ |
pK184-TraF55-247-Rev | 5’- TACGCATATGTCCTGACGCCGGAAAAATAAAGCAGCAGAGTAA -3’ |
†Table adapted from Lento et al. 201635 with permission | |
*TraF overhanging regions are italicized. Restriction enzyme sites are underlined (HindIII: AAGCTT, EcoRI: GAATTC, NdeI: CATATG) |
Table 2: A list of primers used in this study.
Donor Plasmid† | Recovery Plasmid | Transconjugants‡§ (cells mL-1) | Mating Efficiency§|| |
pOX38-Tc ΔtraF::Cm | None | 0 | 0 |
pOX38-Tc ΔtraF::Cm | pK184 | 0 | 0 |
pOX38-Tc ΔtraF::Cm | pK184-TraF | 5 x 103 | 0.0167 |
pOX38-Tc ΔtraF::Cm | pK184-TraF55-247 | 0 | 0 |
*Table adapted from Lento et al. 201635 with permission | |||
†Donor cells were E. coli XK1200, with an average concentration of 3 x 107 cells mL-1 | |||
‡Recipient cells were E. coli MC4100. The number of transconjugants for the positive control is from a 10-5 dilution. 0 indicates no transconjugants from a 10-2 dilution. | |||
§An average of two to four mating experiments were performed for each construct. | |||
||Mating efficiency is defined as transconjugants per 100 donor cells. 0 mating efficiency indicates no transconjugants from a 10-2 dilution |
Table 3: Abolished mating efficiency by TraF deletion constructs.
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 the target gene of interest through the use of smaller, more manageable expression plasmids (Figure 1). We employ the F plasmid derivative pOX38-Tc (Table 1) to study F plasmid-mediated conjugation; other conjugative plasmids can be studied using the protocols detailed here and appropriate derivative plasmids. The mating assays outlined have been adapted from Frost and colleagues,42 with some modifications. In previous studies,8,33,42 the creation of the pOX38-Tc Δgene::Cm construct was achieved by cleaving the gene of interest with the appropriate restriction enzymes and inserting the amplified CAT cassette into pOX38-Tc.42 In the current method, we employ homologous recombination in the recombineering E. coli strain DY330R33,34 to knockout the target gene and replace it with the CAT cassette. This has an advantage of allowing the resultant DY330R strain harboring the pOX38-Tc Δgene::Cm construct to act as a control for the gene-specific knockout out F-T4SS mediated conjugative transfer via the recovery of transfer using the pK184-gene recovery plasmid. While it may be possible to generate knockouts using a CRISPER-Cas9 methodology,43 we have not at this time explored this possibility.
The process begins with the generation of a gene knockout in the F derivative plasmid pOX38-Tc (Figure 1). This is achieved via homologous recombination in DY330R cells (Table 1), other strains with similar features such as DY329, DY331 and DY378 can also be used. Primers are initially designed to PCR amplify the CAT cassette from the pBAD33 plasmid32 and contain overhanging bases that are specific for the target gene (Table 2); the PCR product is then electroporated into DY330R cells harboring pOX38-Tc. Homologous recombination generates the knock-out plasmid pOX38-Tc Δgene::Cm where the CAT cassette is inserted in-frame within the target gene, effectively disrupting T4SS assembly and conjugation while providing Cm resistance. At the same time, the target gene is PCR amplified from pOX38-Tc and inserted into a small expression plasmid; in this protocol we use pK184 for constitutive expression, however one could chose a plasmid with inducible expression of the target gene if desired. The pK184-gene plasmid is then transformed into the DY330R pOX38-Tc Δgene::Cm cells, and these cells are then used as donors to transfer the pOX38-Tc Δgene::Cm construct via conjugation into XK1200 cells for mating assays. In the mating assays, the donor and recipient cells are XK1200 pOX38-Tc Δgene::Cm and MC4100, respectively. The pK184-gene recovery plasmid, as well as the series mutants of the target gene (deletions or point mutations), is provided to the donor cells to assess their ability to restore mating, and determine its efficiency, with MC4100 recipient cells (Figure 2; Table 3).
Critical to the procedure is the use of appropriate donor and recipient strains (Table 1), and the design of the primers used. For each primer designed, there are general guidelines that should be followed. While we strictly try to abide with 40-60% GC content, this may not always be possible. In such cases, it is the experimenter's discretion to test a primer with GC content slightly below or above this range. The melting temperature (Tm) of the forward and reverse primer must be similar, and the annealing temperature (Ta) value should always be lower than the Tm by 2-5 °C for PCR. The free energy available for allowing a hairpin to develop should be much lower than the Ta, while the homo- and hetero- dimerization Tm's must be very low (less than -10 kJ and 30 °C). Primers can be designed to probe deletions, insertions or point mutations as desired. It is of course critical that the resultant gene construct be amplified and ligated into the recovery plasmid in-frame such that it is properly expressed. Transformation of competent cells can be done via electroporation or heat shock using electrocompetent or chemically competent cells, respectively. We find that electroporation is more efficient for larger constructs such as pOX38-Tc and the homologous recombination oligonucleotide, while the smaller expression plasmids such as pK184-TraF can be readily transformed into cells using heat shock methods. Lastly, it is important to remember that there will be multiple antibiotics in use throughout the protocol, as both donor and recipient strains require different resistances that are different from the ones employed on the conjugative and recovery plasmids.
Aside from the mating assays techniques described here, there are other methods that are used to study bacterial conjugation, varying slightly in their approach. Horizontal gene transfer is a process where bacteria transfer a plasmid to a recipient cell, including interspecies recipients.44 A study by Dahlberg and colleagues44 for instance used bacterial conjugation to determine the extent of interspecies horizontal gene transfer. They utilized the incorporation of green fluorescent protein (GFP) into a plasmid cloned into KT2442 cells; the chromosomal lac Iq gene in the KT2442 cells repress GFP expression. When the plasmid carrying GFP is transferred to a species without the lac Iq gene, fluorescence is observed.44 Despite its limitation to provide protein specific function in various species, the interspecies conjugation experiment could possibly be coupled with the protocols presented here to make evolutionary predictions for protein-protein interactions between different species.
The authors have nothing to disclose.
This research was supported by a Discovery Grant from the Natural Sciences & Engineering Council of Canada (NSERC).
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 |