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BioDesign Research / 2022 / Article

Research Article | Open Access

Volume 2022 |Article ID 9802168 | https://doi.org/10.34133/2022/9802168

Ryan R. Cochrane, Arina Shrestha, Mariana M. Severo de Almeida, Michelle Agyare-Tabbi, Stephanie L. Brumwell, Samir Hamadache, Jordyn S. Meaney, Daniel P. Nucifora, Henry Heng Say, Jehoshua Sharma, Maximillian P. M. Soltysiak, Cheryl Tong, Katherine Van Belois, Emma J. L. Walker, Marc-André Lachance, Gregory B. Gloor, David R. Edgell, Rebecca S. Shapiro, Bogumil J. Karas, "Superior Conjugative Plasmids Delivered by Bacteria to Diverse Fungi", BioDesign Research, vol. 2022, Article ID 9802168, 15 pages, 2022. https://doi.org/10.34133/2022/9802168

Superior Conjugative Plasmids Delivered by Bacteria to Diverse Fungi

Received11 Mar 2022
Accepted28 Jul 2022
Published01 Sep 2022

Abstract

Fungi are nature’s recyclers, allowing for ecological nutrient cycling and, in turn, the continuation of life on Earth. Some fungi inhabit the human microbiome where they can provide health benefits, while others are opportunistic pathogens that can cause disease. Yeasts, members of the fungal kingdom, have been domesticated by humans for the production of beer, bread, and, recently, medicine and chemicals. Still, the great untapped potential exists within the diverse fungal kingdom. However, many yeasts are intractable, preventing their use in biotechnology or in the development of novel treatments for pathogenic fungi. Therefore, as a first step for the domestication of new fungi, an efficient DNA delivery method needs to be developed. Here, we report the creation of superior conjugative plasmids and demonstrate their transfer via conjugation from bacteria to 7 diverse yeast species including the emerging pathogen Candida auris. To create our superior plasmids, derivatives of the 57 kb conjugative plasmid pTA-Mob 2.0 were built using designed gene deletions and insertions, as well as some unintentional mutations. Specifically, a cluster mutation in the promoter of the conjugative gene traJ had the most significant effect on improving conjugation to yeasts. In addition, we created Golden Gate assembly-compatible plasmid derivatives that allow for the generation of custom plasmids to enable the rapid insertion of designer genetic cassettes. Finally, we demonstrated that designer conjugative plasmids harboring engineered restriction endonucleases can be used as a novel antifungal agent, with important applications for the development of next-generation antifungal therapeutics.

1. Introduction

The fungal kingdom is exquisitely diverse and home to countless species with profound impacts on ecological nutrient cycling, industrial manufacturing, and health and disease in humans, animals, and plants [1, 2]. Yeast species are amongst the best-studied fungi and include the common yeast Saccharomyces cerevisiae, which is a primary fermenter of beer, wine, and bread, and an ubiquitous eukaryotic model system. S. cerevisiae is also an important synthetic biology chassis for the production of insulin, vaccine components, and other critical recombinant proteins [3]. The closely related Saccharomyces boulardii is a promising probiotic therapeutic, particularly in the context of obesity and type 2 diabetes [4, 5]. Yeasts are also critical components of the human microbiota, including Candida species associated with vaginal yeast infections and invasive candidiasis [6], as well as Malassezia species with notable associations to Crohn’s disease and pancreatic cancer [7, 8]. The skin-associated yeast Candida auris is an emerging fungal pathogen that can cause life-threatening infections and is highly refractory to antifungal drug treatment [911]. These diverse and critical roles in health, disease, and industrial manufacturing highlight the importance of studying and manipulating the biology of these key yeast species.

Given the diversity of yeast species and the breadth of niches they inhabit, there is a need to develop improved and innovative methods for DNA transformation in these organisms. Genetic transformation techniques enable the manipulation of genomes of industrially important yeasts, and further promote the ability to target, modify, or damage the genomes of fungal pathogens. Indeed, genetic-editing tools such as CRISPR have a promising role as novel antimicrobial agents due to their ability to specifically target pathogen-associated genes, leading to microbial death, growth inhibition, or targeted deletion of genes involved in antimicrobial resistance or virulence [1218]. However, laboratory-based transformation protocols typically rely on chemical strategies to promote DNA uptake, which is not broadly applicable for manipulating yeasts in their native environments, such as those inhabiting the microbiome. One innovative strategy to promote the uptake of genetic material in situ is to exploit bacterial conjugation as a viable mechanism to transfer plasmids from bacteria to a recipient microbe via the bacterial type IV secretion system. Previous work has demonstrated the utility of conjugation for transferring plasmids, including those encoding CRISPR-based antimicrobials, between bacterial species, both in vitro [19, 20] and in vivo in mouse microbiome models [2123]. While conjugation typically occurs between bacterial species, cross-kingdom conjugation from bacteria to yeast and algae has been demonstrated [2427]. Despite recently optimized protocols [28, 29], conjugation to yeast still suffers from relatively low conjugation frequency compared to prokaryotic recipients.

Thus, we sought to improve DNA transfer from bacteria to yeast by optimizing the genetic conjugation machinery of the pTA-Mob 2.0 plasmid [28]. This conjugative plasmid is composed of genetic elements required for plasmid maintenance and transfer [30, 31]. Two regions, Tra1 and Tra2, are responsible for the transfer of plasmid DNA. Tra1 harbors the relaxase (traH-J), primase (traA-G), and leader (traK-M) operons, which together coordinate mobilization of the plasmid to the recipient [31]. The relaxase and leader operon encode the relaxosome, a protein complex essential for initial DNA processing during conjugation. Assembly of the protein complex (TraH-J) is initiated by TraJ binding to the 19-bp inverted repeat sequence in the origin of transfer (oriT) [3234]. The interaction of TraI and TraJ, which is stabilized by TraH, then orients the relaxase toward the nic-site [34]. After formation of the relaxosome, TraI nicks and covalently binds to the plasmid DNA, ready for transfer to the recipient cell [35, 36]. The efficiency of the nicking reaction is attenuated by the binding of TraK to the oriT which orients the plasmid DNA into a more favorable position [33, 37]. TraC1, of the primase operon, is a DNA primase that co-transfers (along with single-stranded binding (SSB) proteins) with the DNA to the recipient cell where it is involved in the restoration of a double-stranded plasmid [38]. The primase operon also includes the TraG protein, which couples DNA processing by the relaxosome to DNA transfer by delivering the protein-DNA complex to the mating pair formation proteins [39, 40]. The Tra2 region contains proteins (TrbB-L and TraF) required for mating pair formation, many of which are associated with the cell membrane. TrbC encodes a peptide responsible for forming the pilus. This peptide undergoes maturation by proteolytic cleavage followed by cyclization by TraF resulting in rigid pili [41, 42]. The pilus allows initial contact between the two cells and enables the transfer of single-stranded plasmid DNA to the recipient cell.

Here, we develop and validate novel plasmids for improved conjugation efficiency between bacteria and diverse yeast species. We demonstrate that a cluster mutation in the relaxase operon, specifically in the traJ promoter, significantly improved DNA transfer from bacteria to S. cerevisiae and diverse yeasts, including the emerging pathogen C. auris. We generate improved, streamlined, and Golden Gate assembly-compatible plasmid derivatives of pTA-Mob 2.0 to enable facile insertion of custom genetic cassettes. Finally, we demonstrate that these designer conjugative plasmids can be used as a novel antifungal reagent, with important applications for the development of next-generation antifungal therapeutics.

2. Materials and Methods

2.1. Experimental Design

Experimental design is shown in Figure 1.

2.2. Microbial Strains and Growth Conditions

Saccharomyces cerevisiae VL6−48 (ATCC MYA-3666: MATα, his3-Δ200, trp1-Δ1, ura3-52, lys2, ade2-101, met14, psi + cir0) was grown in yeast media supplemented with ampicillin (100 μg mL-1; BioBasic, Cat #: AB0028, Canada) as previously described [43]; or grown with selection on either (1) yeast synthetic complete medium lacking histidine supplemented with adenine hemi-sulfate (Teknova, Inc., Cat #: C7112, USA), (2) yeast synthetic complete medium lacking tryptophan (Teknova, Inc., Cat #: C7131, USA), (3) 2 × YPDA supplemented with nourseothricin (100 μg mL-1; Jena BioScience, Cat #: AB-102XL, Germany), or (4) 2 × YPDA supplemented with zeocin (100 μg mL-1; Invivogen, Cat #: ant-zn-5p, USA). Solid yeast media contained 2% agar (BioShop Canada Inc., Cat # AGA001.500, Canada). All yeast spheroplast preparation and transformation were performed as previously described [44, 45]. Escherichia coli Epi300 (Lucigen Corp., Cat #: LGN-EC300110, USA) was grown as previously described [43] supplemented with appropriate antibiotics (gentamicin (40 μg mL-1; BioBasic, Cat #: GB0217, Canada) and chloramphenicol (15 μg mL-1; BioBasic, Cat #: CB0118, Canada). Solid media contained 1.5% agar. For transformation of E. coli, SOC medium (20 g L-1 tryptone, 5 g L-1 yeast extract, 0.5 g L-1 NaCl, 10 mL 250 mM KCl, 5 mL 2 M MgCl2, 20 mL 1 M glucose) was used during the recovery time. Diverse yeasts, Metschnikowia gruessii (H53), Metschnikowia pulcherrima (CBS 5833), Metschnikowia lunata (BS 5946), Metschnikowia borealis (SUB 99-207.1), C. auris, Candida tolerans (UWOPS 98-117.5), Candida bromeliacearum (UNESP 00-103), Candida pseudointermedia (UWOPS 11-105.1), Candida ubatubensis (UNESP 01-247R), and Candida aff. bentonensis (UWOPS 00-168.1) were grown at 30°C in 2 × YPDA (all diverse yeasts were obtained from Dr. Marc-Andre Lachance collection at Western University except C. auris came from https://wwwn.cdc.gov/arisolatebank/ accession number SAMN05379609). Sinorhizobium meliloti (Rm4123 R-; obtained from Dr. Finan Lab, McMaster University) was grown at 30°C in LBmc medium (10 g L-1 tryptone, 5 g L-1 yeast extract, 5 g L-1 NaCl, 0.301 g L-1 MgSO4, and 0.277 g L-1 anhydrous CaCl2) supplemented with appropriate antibiotics (gentamicin 40 μg mL-1 and streptomycin 100 μg mL-1; BioBasic, Cat #: SB0494, Canada). Solid media contained 1.5% agar.

2.3. Plasmid Construction
2.3.1. PCR Amplification

Plasmid fragments were amplified with GXL polymerase (Takara Bio Inc., Cat #: R050A, Japan) according to the manufacturer’s instructions using annealing temperatures between 50 and 60°C and 25–30 cycles.

2.3.2. Plasmid Assembly in Yeast

Plasmids were assembled in yeast as previously described [28]. Primers for deletion plasmids are listed in Supplemental Table S1 and all primers and templates used to generate other plasmids are listed in Supplemental Table S2: (1) Single-gene/fragment deletion plasmids: pTA-Mob 2.0 plasmid was used as a template. Each plasmid was created with nine standard fragments as previously described [28] and two additional fragments designed as shown in Supplemental Figure S1. (2) Minimized plasmids (M1–8): Eight minimized conjugative plasmids (M1–8) were designed based on the results obtained for the pTA-Mob 2.0 deletion plasmids. pTA-Mob 2.0 or M3C1 plasmid was used as a template for PCR fragments listed in Supplemental Table S2. (3) M3C1_F1 - F5 hybrid plasmids: These plasmids were assembled by swapping the fragments between M3C1 and M3C2. (4) pTA-Mob 2.0 Tp and pTA-Mob 2.0 To: Primer-mediated mutagenesis was performed to introduce each mutation (Tp- in traJ promoter and To- in traJ ORF) into pTA-Mob 2.0. (5) Superior conjugative plasmid (pSC5): The pSC5 plasmid was derived from M3C1 plasmid by the addition of two versions of Nourseothricin N-acetyl transferase (NAT) genes which provide resistance for nourseothricin antibiotic. The first version was amplified from a plasmid pTA-Mob-NAT (unpublished, Karas lab) allowing selection in diatoms and referred to as dNAT; and the version was amplified from pGMO1 (unpublished, Karas lab), which contained an alternative genetic code for selection in diverse yeasts as previously described [46] and referred to as yNAT. The remaining fragments of the pSC5 plasmid were amplified from M3C1 as previously described [28]. (6) pTA-Mob 2.1/pSC5.1: These two plasmids were created lacking the second copy of traJ located in the vector backbone. (7) Domesticated pSC5 (pSC5GGv1/v2): Two BsaI cut sites located within the fcpD promoter and traC1 ORF were removed from pSC5 using primer-mediated mutagenesis. An RFP landing pad, consisting of a monomeric Red Fluorescent Protein (mRFP) gene driven by an arabinose-inducible pBAD promoter and a terminator, was amplified from pAGE2.0-i (unpublished, Karas lab) using primers designed with new BsaI cut sites and homology either to directly downstream the native I-SceI restriction site (pSC5GGv1) or within the HIS3/CEN6/ARS4 element of the vector backbone (pSC5GGv2).

2.3.3. Golden Gate Assembly

(1) Golden Gate (GG) assembly. For GG assembly, 20 fmol of plasmid and insert were mixed in a 15 μL reaction with 1.0 μL T4 DNA ligase (New England BioLabs, Inc., Cat #: M0202L, USA) and 0.5 μL BsaI-HF V2 (New England BioLabs, Inc., Cat #: R3733S, USA), using the following conditions: 10 cycles of 37°C for 5 minutes and 16°C for 10 minutes followed by incubation at 37°C for 5 minutes, 80°C for 10 minutes, and infinite hold at 12°C. Primers are listed in Supplemental Table S2. (2) pSC5GGv1_ShBle. The zeocin (ShBle) resistance marker cassette was amplified with flanking BsaI cut sites from pRS32 (unpublished, Shapiro Lab) and GG assembly was performed with pSC5GGv1. The primers used are listed in Supplemental Table S2. (3) pSC5-toxic1, pSC5-toxic2, and pSC5-toxic3. Three versions of toxic plasmids to kill yeast cells were created, one with an Acholeplasma laidlawii toxic gene (ACL0117) [47] and two versions with the restriction enzyme HindII. The A. laidlawii and HindII cassettes both contained an ACT1 yeast intron, flanking BsaI cut sites, and either an A. laidlawii or HindII toxic gene. The A. laidlawii toxic gene cassette was amplified in three fragments: the ACT1 yeast intron from S. cerevisiaeVL6-48 gDNA and the toxic gene in two halves from A. laidlawii PG-8A gDNA with primers listed in Supplemental Table S2. The A. laidlawii toxic gene cassette was then constructed through a hierarchical GG assembly. First, the ACT1 yeast intron and the second half of the A. laidlawii toxic gene were assembled by GG assembly, and then 1 μL of the product was used as a template for PCR amplification of the joined fragments. Next, GG assembly was performed with 20 fmol of the PCR product with the first half of the toxic gene, and the complete toxic gene cassette was PCR amplified. The fully constructed cassette was then mixed with pSC5GGv1, and GG assembly was performed. The HindII cassette split by the ACT1 intron was flanked by the URA3 promoter/terminator and was synthesized (BioMatik, Canada), then PCR amplified and used in GG assembly with pSC5GGv1 or pSC5GGv2

2.4. Plasmid Analysis

Screening in yeast. Following yeast assembly of the pTA-Mob 2.0 deletion plasmids, 20 individual yeast colonies were passed twice on solid media lacking histidine, and DNA was isolated and screened by multiplex PCR using the Qiagen Multiplex Kit (Qiagen, Inc., Cat #: 206143, Germany) according to the Qiagen Multiplex PCR Handbook. For all other plasmids following yeast assembly, colonies were pooled rather than individually screened. Transformation to E. coli. Total DNA was isolated as previously described [24]. Isolated DNA (0.52 μL) was added to E. coli Epi300 electro-competent cells (40 μL) and electroporated using the Gene Pulser Xcell Electroporation System (2.5 kV voltage, 25 μF capacitance, and 200 Ω resistance). Following a recovery in 1 mL of SOC medium for 1 hour at 37°C (225 RPM), a 100250 μL aliquot of the transformants was plated on LB medium supplemented with gentamicin (40 μg mL-1). The cells transformed with Golden Gate-compatible plasmids (pSC5GGv1/v2, pSC5-toxic1, pSC5-toxic2, and pSC5-toxic3) were instead plated on LB plates containing gentamicin (40 μg mL-1) and arabinose (100 μg mL-1). Screening in E. coli. For Golden Gate-compatible plasmids, white colonies were then screened by multiplex PCR for insertion of the cassette of interest. For all other assembled plasmids, the transformed E. coli was pooled and conjugated to S. cerevisiae, and DNA was re-isolated and transformed back to E. coli to focus screening on functional conjugative plasmids. Once in E. coli, all plasmids were genotypically screened using multiplex PCR and restriction enzyme digest analysis. Sequencing. The plasmids, pTA-Mob 2.0 Tp/To, underwent Sanger DNA sequencing (London Regional Genomics Centre at Robarts Research Institute) to ensure the introduction of the correct mutations using the primers listed in Supplemental Table S2. Selected plasmids were sequenced at CCIB DNA Core at Massachusetts General Hospital or at the Western University sequencing facility.

2.5. Conjugation

Both donor (E. coli) and recipient (S. cerevisiae) strains were prepared and frozen prior to conjugation experiments. For E. coli strains, saturated overnight cultures inoculated with a single colony were diluted to OD600 of 0.1 in 50 mL of LB medium supplemented with appropriate antibiotics (Table 1) and grown until an OD600 of 1.0 was reached. The cells were pelleted (3,000 × RCF, 15 minutes) in a 50 mL Falcon tube and resuspended in 500 μL ice-cold 10% glycerol. Then, 100 μL aliquots in Eppendorf tubes were frozen in a -80°C ethanol bath and stored at -80°C. For S. cerevisiae recipient strain preparation, a culture was started from a single colony and grown in 5 mL of 2 × YPDA medium supplemented with ampicillin (100 μg mL-1) for 7 hours. After, this culture was diluted in 50 mL of 2 x YPDA medium supplemented with ampicillin (100 μg mL-1) and grown until an OD600 of 3.0 was reached (~17 hours). The cells were pelleted (3,000 × RCF, 5 minutes) in a 50 mL Falcon tube and resuspended in 1 mL of ice-cold 10% glycerol. Then, 250 μL aliquots in Eppendorf tubes were frozen in a -80°C ethanol bath and stored at -80°C.


PlasmidPlasmid size (kb)E. coli markerYeast markerCitation

pTA-Mob 2.057aacC1HIS3/URA3Soltysiak et al. (2019)
pAGE1.018catHIS3Brumwell et al. (2019)
pAGE2.0.T19catTRP1This study
pAGE2.0-i20catHIS3This study
pAGE2.0-iTraJ20catHIS3This study
pRS3211blaShBleShapiro et al. (unpublished)
M1C1: pTA-Mob 2.0 ΔTrbO-FiwA54aacC1HIS3/URA3This study
M1C2: pTA-Mob 2.0 ΔTrbO-FiwA54aacC1HIS3/URA3This study
M2C5: pTA-Mob 2.0 ΔKlaC-KleA53aacC1HIS3/URA3This study
M3C1: pTA-Mob 2.0 ΔIstA-TraB52aacC1HIS3This study
M3C2: pTA-Mob 2.0 ΔIstA-TraB52aacC1HIS3This study
M4C16: pTA-Mob 2.0 ΔIstA-TraE46aacC1HIS3This study
M4C19: pTA-Mob 2.0 ΔIstA-TraE46aacC1HIS3This study
M5C1: pTA-Mob 2.0 ΔTrbN-TraE40aacC1HIS3This study
M5C3: pTA-Mob 2.0 ΔTrbN-TraE40aacC1HIS3This study
M6C2: pTA-Mob 2.0 ΔTrbN-TraE, ΔKlaC-KleA35aacC1HIS3This study
M6C4: pTA-Mob 2.0 ΔTrbN-TraE, ΔKlaC-KleA35aacC1HIS3This study
M7C2: pTA-Mob 2.0 ΔTrbN-TraE, ΔTrfA-TraJ37aacC1HIS3This study
M8C8: pTA-Mob 2.0 ΔTrbM-TraE, ΔKlaC-KleA, ΔTrfA-TraJ31aacC1HIS3This study
M8C10: pTA-Mob 2.0 ΔTrbM-TraE, ΔKlaC-KleA, ΔTrfA-TraJ31aacC1HIS3This study
M3C1_F1: pTA-Mob 2.0 ΔIstA-TraB52aacC1HIS3This study
M3C1_F2: pTA-Mob 2.0 ΔIstA-TraB52aacC1HIS3This study
M3C1_F3: pTA-Mob 2.0 ΔIstA-TraB52aacC1HIS3This study
M3C1_F4: pTA-Mob 2.0 ΔIstA-TraB52aacC1HIS3This study
M3C1_F5: pTA-Mob 2.0 ΔIstA-TraB52aacC1HIS3This study
pTA-Mob 2.0 Tp56aacC1HIS3/URA3This study
pTA-Mob 2.0 To56aacC1HIS3/URA3This study
pSC5: pTA-Mob 2.0 ΔIstA-TraB56aacC1HIS3/yNATThis study
pTA-Mob 2.1: pTA-Mob 2.0 ΔTraJ56aacC1HIS3/URA3This study
pSC5.1: pTA-Mob 2.0 ΔIstA-TraB, ΔTraJ55aacC1HIS3/yNATThis study
pSC5GGv1: pTA-Mob 2.0 ΔIstA-TraB, +mRFP57aacC1HIS3/yNATThis study
pSC5GGv2: pTA-Mob 2.0 ΔIstA-TraB, +mRFP57aacC1HIS3/yNATThis study
pSC5GGv1_ShBle: pTA-Mob 2.0 ΔIstA-TraB57aacC1HIS3/yNAT/ShBleThis study
pSC5-toxic1: pTA-Mob 2.0 ΔIstA-TraB, +ACL011759aacC1HIS3/yNATThis study
pSC5-toxic2: pTA-Mob 2.0 ΔIstA-TraB, +HindII57aacC1HIS3/yNATThis study
pSC5-toxic3: pTA-Mob 2.0 ΔIstA-TraB, +HindII57aacC1HIS3/yNATThis study

On the day of conjugation, conjugation plates (20 mL, 1.8% agar, 10% LB medium, complete minimal glucose broth lacking histidine) were dried for 30 minutes. Aliquots of the donor (E. coli) and recipient (S. cerevisiae) strains were removed from the freezer and thawed on ice for approximately 20 minutes. Next, 50 μL of S. cerevisiae was added to the 100 μL of E. coli and mixed by gentle pipetting before being transferred to the plate and spread evenly. Alternatively, when the yeast toxic plasmids were being tested, 10 μL of the recipient S. cerevisiae strain was used. Once dried, the plates were incubated at 30°C for 3 hours, or 12 hours when wild yeast strains were used as the recipient. The plates were scraped with 2 mL of sterile double-distilled water (sddH2O), mixed by vortexing for 5 seconds, and 100 μL plated on respective selection media (25 mL, 2% agar supplemented with ampicillin 100 μg mL-1) listed in Table 1. In the case of wild yeast strains, they were plated on 1×YPDA medium supplemented with nourseothricin (100 μg mL-1) and two technical replicates of each dilution (100–10-1) were plated on selective plates. For experiments evaluating conjugation of pSC5 in cis and trans, dilution series of 100–10-2 were generated and plated on selective media while dilution series of 10-4–10-7 were generated and plated on non-selective medium (1 × YPDA supplemented with ampicillin 100 μg mL-1); and two technical replicates were plated for each dilution.

2.6. RNA Isolation and Quantitative Reverse Transcriptase-Polymerase Chain Reaction

For RNA isolation, the E. coli strains carrying conjugative plasmids pTA-Mob 2.1 and pSC5.1 were grown in LB medium supplemented with gentamicin (40 μg mL-1) overnight at 37°C shaking at 225 RPM. In the morning, RNA was isolated as previously described [43]. Following DNase treatment with TURBO DNA-free™ Kit (Invitrogen Cat #: AM1907, USA), the RNA concentration and the integrity was verified [43].

cDNA was prepared from 500 ng of RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat #: 4368814, USA). qRT-PCR was performed using six biological and three technical replicates, on a ViiA7 system of QuantStudio Real-Time PCR System (Applied Biosystems, USA) using the SYBR™ Select Master Mix (Applied Biosystems, Cat #: 472908, USA) under the following conditions: 50°C for 2 minutes, 95°C for 2 minutes followed by 40 cycles of: 95°C for 1 second, 60°C for 30 seconds. Expression levels were normalized against two reference genes (rrsA and cysG) as previously described [29]. Primer sequences used for the qRT-PCR expression analyses are listed in Supplemental Table S2.

2.7. Statistical Analysis

The pairwise comparisons between groups were made using Student’s -test with either equal or unequal variance based on the result of an -test. Data were expressed as either ±95% confidence interval (CI) or as mean ± standard error of the mean (SEM) or as mean ± standard deviation of at least three biological replicates. The tests were considered statistically significant when (), (), or ().

3. Results

3.1. Development of Streamlined Conjugation Plasmids

As a first step (Figure 1) toward creating an optimized and minimized conjugative plasmid for yeast, 55 single genes or small genetic regions were individually deleted from our previously established trans-kingdom conjugation plasmid, pTA-Mob 2.0 [28] (Supplemental Figure S1, Supplemental Table S1). To validate these plasmid variants, up to two clones of each were tested for conjugation from E. coli to S. cerevisiae, and the genes/regions deleted were classified as essential (no conjugation), semi-essential (decreased conjugation), or non-essential (near wild type conjugation) for bacteria-to-yeast conjugation (Supplemental Table S3). Based on this data, four streamlined plasmids were created where clusters of non-essential genes were simultaneously removed (plasmids M1–M4, Table 1, Supplemental Table S2). Plasmids M1–M4 were then conjugated from E. coli to yeast, and we observed a significant increase in successful conjugation efficiency for plasmid M3 clone 1 (M3C1), monitored by yeast colony formation on selective media (Figure 2). Sequencing both M3 clones, M3C1 and M3C2, revealed multiple mutations in each clone, which are likely responsible for the increase in conjugation efficiency for M3C1 (Supplemental Table S4).

To identify which mutations in M3C1 were responsible for the increased conjugation efficiency, we performed a fragment swapping experiment between M3C1 and M3C2 to produce five hybrid plasmids M3C1_F1–F5 (Figure 3). Each hybrid plasmid was created from four fragments that were amplified from M3C2 and one fragment from M3C1. Hybrid plasmid M3C1_F4 (fragment 4 originated from M3C1) had the closest conjugation efficiency compared to M3C1 (Figure 3). There were two mutated regions in fragment 4 of M3C1: a cluster of mutations in the promoter of traJ, and a single mutation in the open reading frame (ORF) of traJ (Supplemental Table S4). To validate which mutation(s) contributed to the increased conjugative phenotype, the promoter or ORF traJ mutations were introduced into pTA-Mob 2.0 and tested for conjugation efficiency. Only the mutations in the promoter region of traJ improved conjugation efficiency (Figure 3). Additionally, we continued to minimize M3C1 by creating new plasmids with additional non-essential genes removed to obtain M5–M8 plasmids (Figure 2). All M5–M8 minimized plasmids still produced more colonies when conjugated to yeast as compared to the original pTA-Mob 2.0 (Figure 2).

3.2. Creation of Superior Conjugative Plasmid pSuperCon5

Based on the identified traJ promoter mutations, we created the pSuperCon5 (pSC5) plasmid with additional elements to enable the delivery of our improved conjugative plasmids to diverse yeast and diatoms. The pSC5 plasmid was built based on M3C1 and contains two copies of the nourseothricin resistance gene (yNAT and dNAT): one optimized for selection in diverse yeast and one for diatoms (Figure 4(a)). Conjugation frequency for pSC5 from E. coli to S. cerevisiae was increased approximately 10- or 23-fold compared to pTA-Mob 2.0 when tested in cis (mobilizing itself) or trans (mobilizing another plasmid), respectively (Figure 4(b), Supplemental Table S5 & S6). No significant difference in conjugation frequency was observed when plasmids were transferred between E. coli strains (Figure 4(c), Supplemental Table S5 and S6).

In order to more precisely evaluate the frequency of bacteria-to-yeast conjugation, we performed additional experiments to monitor the effect of conjugation plasmid-containing E. coli on yeast viability. Cells from E. coli-to-yeast conjugation experiments were plated on non-selective yeast medium supplemented with ampicillin to inhibit E. coli growth. More yeast colonies grew when pSC5 was used versus pTA-Mob 2.0 (Supplemental Table S7), indicating that E. coli carrying pSC5 has fewer adverse effects on yeast when they are co-cultured. To determine if the same effect could be observed when different donor cells were used, we performed conjugation with Sinorhizobium meliloti as a donor, as it was previously shown to conjugate to yeast [25]. Similarly, a higher number of yeast colonies grew on non-selective plates when S. meliloti harboring pSC5 was used when compared to pTA-Mob 2.0 (Supplemental Table S8). In addition, a higher number of colonies on selective plates were observed following conjugation of pSC5 from S. meliloti to S. cerevisiae compared to pTA-Mob 2.0 (Supplemental Figure S2, Supplemental Table S9).

Additional experiments will need to be performed to determine if there is a link between the increased number of yeast colonies on non-selective/selective plates and the lower expression of traJ (Supplemental Figure S3) in plasmids carrying the promoter mutation.

3.3. Conjugation to Diverse Yeast Species

The significantly improved frequency of conjugation with the pSC5 plasmid suggests it may be effective for conjugation beyond a standard laboratory strain of S. cerevisiae and may have utility in transferring DNA from bacteria to diverse yeast species. To test the ability of pSC5 to transfer DNA to diverse yeast strains, we selected four Metschnikowia and six Candida species as conjugative recipients. Previously, we have demonstrated that small DNA fragments (yNAT selection marker) can be delivered to most of these species by electroporation [46]. Conjugation to these diverse yeasts was performed with the same protocol used for S. cerevisiae, modified to allow for selection on complete yeast media containing antibiotics. Transconjugant colonies were obtained for all species (Figure 5(a), Supplemental Figure S4, S5), and 1–8 colonies for each species were genotyped by PCR for the presence of the yNAT marker. Of the 10 species tested, seven tested positive by PCR for the presence of the yNAT marker, suggesting successful conjugation had occurred (Supplemental Figure S6). A plasmid rescue experiment, where total yeast genomic DNA is electroporated into E. coli, was performed for selected colonies for each of the seven species, as well as S. cerevisiae. pSC5 plasmids from the seven yeast species were successfully recovered in E. coli; however, all except those recovered from S. cerevisiae showed rearrangements when diagnostic restriction enzyme digestion was performed (Figures 5(b) and 5(c)). Furthermore, only the plasmids recovered from S. cerevisiae were still able to conjugate (Supplemental Figure S7).

3.4. Domestication of pSC5 for Golden Gate Assembly

Next, we sought to modify the pSC5 plasmid to make it readily amenable to cloning for easy incorporation of any desired DNA fragment, to facilitate downstream applications of bacterial-to-yeast conjugation. To this end, we eliminated existing BsaI restriction sites from pSC5 and created a single BsaI-based Golden Gate cloning-compatible site to enable efficient plasmid manipulation [48, 49]. In addition, we incorporated a landing pad with mRFP driven by an arabinose-inducible promoter (Figure 6(a)). In this modified plasmid, Golden Gate assembly can readily be used to replace the mRFP gene with any gene of interest, allowing for an easy visual screen for correct gene insertion events (white versus red bacterial colonies).

To validate this system, we inserted a second antibiotic marker (ShBle) for yeast into pSC5GGv1 (Figure 6(a)) to create pSC5GGv1_ShBle, which provides resistance to zeocin. White bacterial colonies were selected (Figure 6(b)) and genotyped with diagnostic multiplex PCR and restriction digest (data not shown). These validated colonies were conjugated to S. cerevisiae and tested for survival on single or double antibiotic selection (Figure 6(c)). Successful exconjugants which received pSC5GGv1_ShBle were able to grow on media supplemented with zeocin, nourseothricin, or both (Figure 6(c)).

3.5. Proof of Concept for Conjugation-Mediated Delivery as an Antifungal

To demonstrate that pSC5-based conjugating plasmids could be used as an antifungal, we developed a system where each donor E. coli strain carried two plasmids: a control plasmid (pAGE2.0.T) that can be selected on media lacking tryptophan and either pSC5 or pSC5-toxic gene plasmid that can be selected on media lacking histidine (Figure 7(a)). We used Golden Gate assembly to create three pSC5-toxic plasmids, each carrying a gene that should be partially or fully toxic to yeast. To prevent toxicity in E. coli, we inserted a yeast ACT1 intron [50] into each toxic gene. Next, we cloned the A. laidlawii toxic gene [47] into pSC5GGv1 to generate pSC5-toxic1, or an Haemophilus influenzae HindII restriction gene into pSC5GGv1 and pSC5GGv2 to generate pSC5-toxic2 and pSC5-toxic3, respectively. The pSC5 and pSC5-toxic gene plasmids can act in cis, mobilizing themselves, as well as in trans, mobilizing the control plasmid pAGE2.0.T. Using a control E. coli strain carrying plasmids pSC5 and pAGE2.0.T, we observed a similar colony number on both selection plates. For conjugation with pSC5-toxic gene plasmids, substantially fewer colonies grew on minimal media lacking histidine. The most substantial difference in yeast colony formation was with donor E. coli carrying pSC5-toxic3 (Figure 7(b), Supplemental Table S10). This provides a proof of concept that bacteria-to-yeast conjugation can be used to effectively deliver plasmid-based antifungals.

4. Discussion

Conjugation-based techniques, such as the one described here, provide a unique and functional method to deliver plasmids between microbial species in vitro and in vivo. While there are innumerable possible applications for these systems, many have focused on the use of plasmid-encoded CRISPR-based genetic manipulation systems to modify the genomes of the recipient microbes [19, 23, 51]. Indeed, CRISPR-based gene targeting and manipulation systems offer a breadth of applications that can be paired with conjugation (or other methods of DNA delivery, such as phage transduction) to achieve desired manipulation of a target microbial population. The majority of this work to date has focused on bacterial species. For instance, CRISPR-based systems have been used to induce lethal DNA damage in key bacterial pathogens, including E. coli, Staphylococcus aureus, and Clostridium difficile, to effectively eradicate unwanted bacterial populations, including drug-resistant bacteria [13, 14, 52], and specific pathogenic species or sub-populations [13, 1619, 53, 54]. In addition to directly killing bacterial populations, CRISPR systems can also be applied to modify virulence determinants to erode microbial pathogenicity [53, 55], or alter drug-resistance genes to restore antimicrobial susceptibilities [15, 23, 5660]. To enable the application of these CRISPR systems in vivo, many have relied on the use of bacterial conjugation or phage transduction as methods to deliver the relevant CRISPR components [13, 17, 2123, 61]. While this has been effective for delivery to bacterial strains, it has limited the applications in fungi, which lack well-established tools for conjugation or virus-based gene delivery [62, 63].

To address the bottleneck of improving DNA delivery to yeast, we performed experiments to optimize the conjugative plasmid pTA-Mob 2.0 [28]. We first evaluated whether plasmid derivatives with targeted deletions of the conjugative plasmid could improve DNA transfer to S. cerevisiae. After testing 57 single-gene and four cluster-gene deletion plasmids, one with superior conjugative properties (M3C1) was identified. Sequence analysis of M3C1 revealed that in addition to the designed deletions, M3C1 had unintended mutations that were likely introduced during PCR amplification or plasmid assembly. The mutations responsible for improved conjugation to S. cerevisiae were narrowed down to the promoter region of traJ (Tp) using a fragment swapping experiment. Following this discovery, five derivative plasmids of M3C1 were built containing the Tp mutation: four minimized versions (M5–M8) and pSuperCon5 (pSC5, containing selectable markers for diverse yeast species [46] and diatoms [24]). Each derivative plasmid of M3C1, including the smallest 31 kb plasmid M8, outperformed the original 57 kb pTA-Mob 2.0 plasmid when tested for DNA transfer to S. cerevisiae. Notably, using pSC5 compared to pTA-Mob 2.0, we observed an increase in conjugation to S. cerevisiae 10- or 23-fold in either a cis or trans setup, respectively. Yet, no increase in plasmid transfer was observed when pSC5 was conjugated between E. coli strains. This improved conjugation to S. cerevisiae could be partially explained by the increased S. cerevisiae viability during the co-culture conjugation step when plasmids harboring the Tp mutation are used. The same effect was also observed when S. meliloti was used as a conjugative donor, suggesting the mechanism may be independent of the bacterial host. We also demonstrated that the Tp mutation results in a lower expression of the traJ gene. TraJ has been demonstrated as an essential conjugative protein that negatively autoregulates the expression of the relaxase operon [64]. Therefore, decreased expression of traJ could have a significant effect on the expression of all the conjugative machinery proteins. Further investigation will focus on resolving the link between traJ downregulation and increased yeast viability or DNA transfer during the co-culture conjugation step.

The significantly improved pSC5 plasmid allowed for DNA transfer to seven Metschnikowia and Candida yeast species, though relatively few colonies were obtained for each of them. One explanation for the low conjugative transfer could be that the S. cerevisiae centromere along with the origin of replication was not functional in these yeasts. In such a case, survival of these yeasts would only be possible if the conjugative plasmid was integrated into the yeast genome. Using a plasmid rescue experiment, we showed that plasmids could be recovered in E. coli, although none of them had the correct size or ability to conjugate. Since E. coli can assemble linear fragments into plasmids [65], it is most likely that some of the linear yeast fragments with integrated conjugative plasmids were assembled into plasmids in E. coli. Despite not being able to replicate as an episome in diverse yeasts, the improved conjugative plasmids, especially pSC5GGv1_ShBle with two antibiotic resistance genes, provide a great initial resource for DNA delivery. For applications where replicative plasmids are necessary for the yeast species of interest, specific origins and centromeres will need to be identified and incorporated into the conjugative plasmid as was done for S. cerevisiae [66, 67].

Our improved conjugative plasmids hold promise as a novel antifungal. As a proof of concept, we cloned restriction nucleases onto pSC5GGv2 plasmid and demonstrated that >99% of yeast cells that receive the plasmid DNA can be eliminated. However, additional improvements in the conjugation frequency will need to be achieved before this technology can be used in antifungal treatments. In the future, our Golden Gate-compatible plasmids can be engineered with programmable systems such as CRISPR/Cas9 to target specific yeast strains. This coincides with the development and optimization of numerous CRISPR-based editing platforms optimized for a diversity of yeast species [6872], including Candida pathogens [73]. Recent work has demonstrated the utility of CRISPR systems for modifying fungal genes involved in virulence [7481] and antifungal drug resistance [74, 77, 82, 83] in diverse Candida pathogens, and combining these CRISPR systems with this trans-kingdom conjugation system could facilitate the delivery of CRISPR to fungi in different environmental contexts.

Data Availability

Raw data used to support the findings presented in this study are available from the corresponding author upon request.

Disclosure

The views, opinions, and/or findings expressed are those of the author and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government.

Conflicts of Interest

The authors declare no competing financial interests.

Authors’ Contributions

B.J.K. conceived the idea. R.R.C., A.S., M.M.S., R.S.S., and B.J.K. designed experiments. R.R.C., A.S., M.M.S., M.A-T., S.L.B., S.H., J.S.M., D.P.N., H.H.S., J.S., M.P.M.S., C.T., K.V-B., and E.J.L.W. performed the experiments. R.R.C., A.S., M.M.S., M.A-T., S.H., R.S.S., and B.J.K. analyzed the data. A.S. performed all statistical analysis. R.R.C., A.S., R.S.S., and B.J.K. wrote the article. R.R.C., A.S., S.H., S.L.B., R.S.S., M.A.L, G.B.G., D.R.E., and B.J.K. edited manuscript. All authors read the manuscript. R.R.C., A.S., and M.M.S are co-first authors.

Acknowledgments

This research was funded by Defense Advanced Research Projects Agency (DARPA), Agreement Number: D18AC00035 to B.J.K. Natural Sciences and Engineering Research Council of Canada (NSERC), grant number: RGPIN-2018-06172 to B.J.K.). CIHR Project Grant (PJT 162195) to R.S.S. Natural Sciences and Engineering Research Council of Canada (NSERC) grants to M.A.L. CIFAR Azrieli Global Scholar Award (Fungal Kingdom: Threats and Opportunities) to R.S.S. CIHR Project Grant (PJT 159708) to D.R.E., G.B.G, and B.J.K. In addition, the following trainees were sponsored by NSERC Scholarships: R.R.C, S.H., J.M., D.P.N., M.P.M.S, E.J. L. W.; EvoFunPath Fellowship (NSERC CREATE) – M.A-T.

Supplementary Materials

Supplemental Figure S1. Deletion plasmid assembly strategy. Supplemental Figure S2. Bacterial conjugation from S. meliloti to S. cerevisiae. Supplemental Figure S3. Quantitative real-time polymerase chain reaction (qRT-PCR) of traJ expression. Supplemental Figure S4. Bacterial conjugation from E. coli to diverse yeast species. Supplemental Figure S5. Conjugation frequency of pSC5 to Metschnikowia gruessi. Supplemental Figure S6. Genotyping transconjugants of diverse yeast species. Supplemental Figure S7. Phenotypic E. coli to E. coli conjugation screen of recovered transconjugant plasmids from diverse yeast species. Supplemental Table S1. Description of pTA-Mob 2.0 deletion plasmid library. Supplemental Table S2. List of primers used to amplify the assembly fragments and genotype the plasmids created in this study. Supplemental Table S3. Conjugation phenotype of pTA-Mob 2.0 deletion plasmid library. Supplemental Table S4. Whole plasmid sequencing of minimal conjugative plasmid 3 (M3C1 and M3C2). Supplemental Table S5. Cis- and trans- conjugation of super conjugative plasmid (pSC5). Supplemental Table S6. Recipient yeast cell concentrations used in conjugation experiments of Figure 4. Supplemental Table S7. S. cerevisiae cell viability following conjugation with different E. coli strains. Supplemental Table S8. S. cerevisiae cell viability following conjugation with different S. meliloti strains Supplemental Table S9. S. cerevisiae transconjugant colony count following conjugation with S. meliloti. Supplemental Table S10. Yeast transconjugant colony counts for the conjugation-based antifungal experiment (Figure 7). (Supplementary Materials)

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