Green Fluorescent Protein-Labeled Monitoring Tool To Quantify Conjugative Plasmid Transfer between Gram-Positive and Gram-Negative Bacteria

Abstract

On the basis of pIP501, a green fluorescent protein (GFP)-tagged monitoring tool was constructed for quantifying plasmid mobilization among Gram-positive bacteria and between Gram-positive Enterococcus faecalis and Gram-negative Escherichia coli. Furthermore, retromobilization of the GFP-tagged monitoring tool was shown from E. faecalis OG1X into the clinical isolate E. faecalis T9.

TEXT

The mechanisms of conjugative transfer in Gram-negative bacteria are fairly well understood (e.g., see references 2, 9, 18, 33, 36, and 41), whereas conjugation in Gram-positive bacteria has been studied in greater detail for only the last decade leading to the first model of a type IV secretion-like system in Gram-positive bacteria (1, 2, 16, 17, 41). The antibiotic resistance plasmid pIP501 from Streptococcus agalactiae has a very broad host range for conjugative plasmid transfer and mobilization. Its host range includes virtually all tested Gram-positive bacteria, including the multicellular filamentous streptomycetes and Gram-negative Escherichia coli (17, 24).

For Gram-positive systems, molecular tools for in situ detection of horizontal gene transfer by conjugation are still very limited in contrast to Gram-negative systems (4, 7, 8, 29, 37, 38). Nieto and Espinosa (30) have constructed a green fluorescent protein (GFP)-tagged derivative of plasmid pMV158 from Streptococcus pneumoniae, pMV158GFP, that was shown to be mobilizable to different low-GC Gram-positive bacteria like Enterococcus faecalis and Lactococcus lactis. Lorenzo-Díaz and Espinosa applied pMV158GFP to intra- and interspecies mobilization between different Gram-positive bacteria in large-scale filter mating assays (26).

Recently, Babic and coworkers (3) demonstrated conjugative transfer of the integrative and conjugative element ICEBs1 from Bacillus subtilis donor cells to B. subtilis recipient cells in real time using a lacO or LacI-GFP system for visualization of transfer events.

Here, we report the construction and mobilization of a GFP-tagged mobilizable plasmid based on the pIP501 tra region to monitor horizontal gene transfer between Gram-positive bacteria and between Gram-positive and Gram-negative bacteria by the formation of a green fluorescent phenotype in transconjugants. The mobilizable plasmid is based on a nisin-inducible expression system (NICE) and replicates in both Gram-positive and Gram-negative bacteria.

Plasmid construction.

The oriT region from the broad-host-range plasmid pIP501 (oriT_pIP50) was subcloned with primer pair oriT-HindIII-fw (fw stands for forward) and oriT-HindIII-re (re stands for reverse) (see Table S1 in supplemental material) via HindIII into plasmid pJP_relGFP encoding a gfp gene improved for expression in prokaryotes (28, 31). All bacterial strains and plasmids used in this work are described in Table 1. The gfp-oriT_pIP501 cassette was inserted into the E. coli shuttle plasmid pMSP3535VA (6) via XmaI/XbaI with primer pair P_rel-gfp-XmaI-fw and oriT-XbaI-re under the control of a nisin-inducible nisA promoter. Then, the λ phage t₀ terminator was cloned downstream of the oriT region via XbaI and XhoI sites with primer pair t₀-term-XbaI-fw and t₀-term-XbaI-re using the expression vector pQTEV (35) as the template, thus generating plasmid pVA-gfp-oriT (Fig. 1).

Table 1.

Bacterial strains and plasmids used in this study

Bacterial strain or plasmid	Relevant genotype or phenotypea	Source or reference
Bacterial strains
Escherichia coli XL10	Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F′ proAB lacIqZΔM15 Tn10(Tetr) Amy Cmr]	Stratagene
Enterococcus faecalis
JH2-2	Rifr Fusr	23
OG1RF	Rifr Fusr	10
OG1X	Smr	22
T9	Tetr	21
Bacillus subtilis subsp. natto DSM 4451	Smr	DSMZb
Plasmids
pIP501	Tra Cmr MLSr	12
pMSP3535VA	pVA380-1 and ColE1 replicons nisRK PnisA Kmr	6
pJPrelGFP	Prel gfp	31
pQTEV	Pt4lacIq His7 Ampr	35
pJPrelGFP-oriT	pJPrelGFP oriTpIP501	This work
pVA-gfp-oriT	pMSP3535VA gfp oriTpIP501 t0-termpQTEV	This work

^aMLS^r, macrolide-lincosamide-streptogramin B resistance; Hte, high transformation efficiency; Amy, amylase.

^bDSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures).

Fig 1.

The pIP501 oriT region was subcloned into plasmid pJP_relGFP via HindIII. The gfp-oriT cassette was then cloned into plasmid pMSP3535VA via XmaI and XbaI sites, followed by insertion of the t₀ termination sequence via XbaI and XhoI, generating pVA-gfp-oriT.

Assessment of green fluorescence by FACS.

The fluorescence of E. faecalis OG1X(pVA-gfp-oriT) was quantified by fluorescence-activated cell sorting (FACS) (FACScan flow cytometer; BD Biosciences, Heidelberg, Germany) after induction of the nisA promoter with 100 ng · ml⁻¹ nisin on brain heart infusion (BHI) (Condalab, Madrid, Spain) agar plates supplemented with streptomycin (1,000 μg · ml⁻¹) and kanamycin (2,000 μg · ml⁻¹). More than 95% of the analyzed cells were shifted to a green fluorescent phenotype, indicating efficient induction of the nisA promoter and expression of GFP with the plasmid construct (data not shown).

pIP501-mediated mobilization of the GFP-tagged plasmid to E. faecalis and B. subtilis.

pVA-gfp-oriT was tested for its ability to be mobilized by pIP501 in filter matings. The donor E. faecalis OG1X harboring pIP501 and pVA-gfp-oriT and the recipient E. faecalis JH2-2 were grown to an optical density at 600 nm (OD₆₀₀) of 0.5, mixed in a 1:10 ratio, and passed through a sterile nitrocellulose membrane filter (0.45 μm) (Millipore, Schwalbach, Germany). After overnight incubation on BHI agar at 37°C, the cells were recovered in 1 ml phosphate-buffered saline (PBS), and serial dilutions were plated on BHI agar with kanamycin (400 μg · ml⁻¹) and/or fusidic acid (50 μg · ml⁻¹) to enumerate transconjugants and recipients, respectively. The mean mobilization rate from three independent experiments was 1.42 · 10⁻⁵ ± 3.11 · 10⁻⁶ transconjugants per recipient cell. Mobilization rates were approximately three times lower than pIP501 transfer rates in experiments under the same conditions, suggesting a possible cis-acting preference of the Orf1 relaxase in agreement with observations made for the TraA relaxase of plasmid pRetCF2d and relaxase Orf28 of the conjugative transposon Tn1549 (32, 40).

Transconjugants were verified by green fluorescent phenotype after induction of the nisA promoter on BHI agar supplemented with kanamycin (400 μg · ml⁻¹), fusidic acid (50 μg · ml⁻¹), and nisin (100 ng · ml⁻¹) and amplification of the gfp gene by PCR with primer pair P_rel-gfp-XmaI-fw and oriT-XbaI-re (data not shown).

Since pIP501 can be transferred and stably maintained in various Gram-positive bacteria, we tried to mobilize pVA-gfp-oriT to B. subtilis in a triparental mating with the help of pIP501: E. faecalis JH2-2(pVA-gfp-oriT), E. faecalis OG1RF(pIP501), and B. subtilis subsp. natto DSM 4451 were grown to an OD₆₀₀ of 0.5 and mated overnight at 30°C in a ratio of 1:1:10. Serial dilutions were plated on Luria-Bertani agar supplemented with kanamycin (20 μg · ml⁻¹) and/or streptomycin (1,000 μg · ml⁻¹) to enumerate transconjugants and recipients, respectively. The mean mobilization rate obtained from three independent experiments was 4.10 · 10⁻⁶ ± 4.74 · 10⁻⁷ transconjugants per recipient. Thus, the mobilization rates are similar to those obtained for mobilization of pMV158 from E. faecalis OG1X to B. subtilis MB46 SL601 by pIP501 (24). Mobilization of pVA-gfp-oriT was verified by a green fluorescent phenotype for B. subtilis subsp. natto DSM 4451(pVA-gfp-oriT) (not shown) after induction of the nisA promoter on LB agar supplemented with kanamycin (20 μg · ml⁻¹), streptomycin (1,000 μg · ml⁻¹), and nisin (100 μg · ml⁻¹) and amplification of the gfp gene by PCR (data not shown).

Interestingly, even after induction of the nisA promoter with different nisin concentrations (10, 100, and 1,000 ng · ml⁻¹), only 5% of the Bacillus transconjugants expressed a green fluorescent phenotype. It has been previously demonstrated that the NICE system can be used for efficient inducible gene expression in Gram-positive bacteria like Lactococcus, Bacillus, and Enterococcus (6, 11, 27). However, Hirt and coworkers (20) demonstrated weak expression of the pCF10 encoded surface protein PrgB with plasmid pMSP3535 in B. subtilis. This finding together with our results leads to the hypothesis that the NICE vectors pMSP3535 and pMSP3535VA might not be suitable for efficient gene expression in Bacillus species. Thus, GFP expression levels in our case might not have reached a certain threshold in most B. subtilis cells that is necessary for developing a green fluorescent phenotype (15).

pIP501-mediated mobilization from the Gram-positive E. faecalis to the Gram-negative E. coli.

pVA-gfp-oriT was shown to be suited to monitor the transfer of plasmid from the Gram-positive E. faecalis to the Gram-negative E. coli by formation of a green fluorescent phenotype in an E. coli recipient. The donor E. faecalis OG1X(pVA-gfp-oriT), the helper E. faecalis OG1RF(pIP501), and the recipient E. coli XL10 were grown to an OD₆₀₀ of 0.5 and mated overnight at 37°C in a ratio of 1:1:10. Serial dilutions were plated on Luria-Bertani agar supplemented with kanamycin (50 μg · ml⁻¹) and/or tetracycline (10 μg · ml⁻¹) to enumerate transconjugants and recipients, respectively. pIP501-mediated mobilization of pVA-gfp-oriT from E. faecalis OG1X to E. coli XL10 occurred with a rather low frequency of 2.30 · 10⁻⁸ ± 2.07 · 10⁻⁸ transconjugants per recipient cell. However, the nisA promoter in E. coli was functional, leading to a green fluorescent phenotype in approximately 95% of the induced E. coli cells (data not shown). Trieu-Cuot and coworkers (39) showed mobilization of shuttle vectors containing the RK2 oriT region from Gram-negative E. coli to Gram-positive E. faecalis with mobilization frequencies in the same range.

Retromobilization of pVA-gfp-oriT into the clinical strain E. faecalis T9.

To investigate whether mobilization of pVA-gfp-oriT can also occur into pathogenic enterococcal strains, we tested the clinical E. faecalis T9 isolate (21). Molecular characterization of E. faecalis T9 revealed the presence of pSK41-like nes and traE, traG, and traK genes confirmed by dot blotting (Fig. 2; see Table S1 in the supplemental material for the primers used in dot blots), thus indicating the presence of a putative conjugative element in E. faecalis T9. However, plasmid isolation by the method of Woodford et al. (43) with the modifications of Werner et al. (42) and plasmid profiling by S1 nuclease macrorestriction (5, 13, 25) of E. faecalis T9 did not confirm the presence of plasmids (not shown).

Fig 2.

Dot blot hybridization of nes (A), traE (B), traG (C), and traK (D). PCR products were generated with digoxigenin-labeled pSK41-derived probes. Spot 1, negative control (no DNA template was applied to the PCR); spot 2, PCR product of S. aureus(pSK41) as a positive control, spot 3, PCR product of E. faecalis T9.

To prove the presence of a conjugative element in E. faecalis T9, a biparental retromobilization experiment was performed: E. faecalis T9 as a helper strain and recipient and E. faecalis OG1X(pVA-gfp-oriT) as a donor were grown to an OD₆₀₀ of 0.5 and mated overnight at 37°C in a ratio of 1:10 (donor/recipient [D/R]). Serial dilutions were plated on BHI agar supplemented with kanamycin (500 μg · ml⁻¹) and/or tetracycline (10 μg · ml⁻¹) to enumerate transconjugants and recipients, respectively. Retromobilization of pVA-gfp-oriT into E. faecalis T9 occurred with a frequency of 1.62 · 10⁻⁵ ± 1.04 · 10⁻⁵, indicating a putative pSK41-like conjugative element in E. faecalis T9. Transconjugants were verified by PCR amplification of the gfp gene, plasmid isolation, and fluorescence microscopy after induction of the nisA promoter on BHI agar supplemented with kanamycin (500 μg · ml⁻¹), tetracycline (10 μg · ml⁻¹), and nisin (100 ng · ml⁻¹) (data not shown). To the best of our knowledge, retromobilization into E. faecalis has been demonstrated for the first time.

To further investigate the conjugative element present in E. faecalis T9, E. faecalis T9 harboring pVA-gfp-oriT was used as a donor to mobilize the GPF-tagged plasmid in a biparental mating to B. subtilis subsp. natto DSM 4451. The strains were grown to an OD₆₀₀ of 0.5 and mated overnight at 30°C. Serial dilutions were plated on LB agar supplemented with kanamycin (20 μg · ml⁻¹) and/or streptomycin (1,000 μg · ml⁻¹) to enumerate transconjugants and recipients, respectively. Green fluorescent B. subtilis transconjugants were obtained with a mean mobilization rate of 3.10 · 10⁻⁷ ± 3.32 · 10⁻⁸ per recipient, indicating that mobilizable plasmids can be transferred to B. subtilis and E. faecalis by the conjugative element in E. faecalis T9. These results suggest that the pIP501 oriT region is recognized by the pSK41-like relaxase present in E. faecalis T9. In favor of this hypothesis, Garcillan-Barcia et al. reported that pIP501 and pSK41 relaxases belong to the same mobilization protein (MOB) family (14).

To date, we have no information about traits that can be transferred by the conjugative element in E. faecalis T9. Most widely spread integrative and conjugative elements (ICEs) in E. faecalis such as Tn916, Tn1545, and Tn1549 are genetically linked to genes conferring antibiotic resistance and have a broad host range (13). However, none of these transposons shows similarities to the pSK41 tra region originating from Staphylococcus aureus. Considering the clinical origin of E. faecalis T9, a pSK41-like plasmid might have been integrated into the chromosome of E. faecalis T9, thus allowing the capture of different traits from the clinical background.

It has been demonstrated that Inc18 plasmids like pIP501 might be involved in the spread of vancomycin resistance genes and the emergence of vancomycin-resistant S. aureus (VRSA) (44, 45). Recent studies indicate that the pIP501 replicon has a high prevalence in clinical Enterococcus faecium isolates and that it is often genetically linked with vanA resistance genes in enterococci (19, 34). Thus, the constructed mobilizable plasmid might be a powerful tool to screen for pIP501-like and pSK41-like conjugative elements in Enterococcus and Staphylococcus isolates.