A Mating Procedure for Genetic Transfer of Integrative and Conjugative Elements (ICEs) of Streptococci and Enterococci

DNA sequencing of whole bacterial genomes has revealed that the entire set of mobile genes (mobilome) represents as much as 25% of the bacterial genome. Despite the huge availability of sequence data, the functional analysis of the mobile genetic elements (MGEs) is rarely reported. Therefore, established laboratory protocols are needed to investigate the biology of this important part of the bacterial genome. Conjugation is a mechanism of horizontal gene transfer which allows the exchange of MGEs among strains of the same or different bacterial species. In streptococci and enterococci, integrative and conjugative elements (ICEs) represent a large part of the mobilome. Here, we describe an efficient and easy-to-perform plate mating protocol for in vitro conjugative transfer of ICEs in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis. Conjugative transfer is carried out on solid media and selection of transconjugants is performed with a multilayer plating. This protocol allows the transfer of large genetic elements with a size up to 81 kb, and a transfer frequency up to 6.7 × 10−3 transconjugants/donor cells.


Introduction
The three major mechanisms of horizontal gene transfer in bacteria are conjugation, transformation, and transduction. Mobile genetic elements (MGEs), including conjugative and integrative elements (ICEs) and prophages, shape the bacterial genome and are responsible for genome evolution [1]. Conjugation enables the genetic exchange of MGEs, which provide a major contribution to the spread of antimicrobial resistance and virulence, by recruiting new resistance and virulence genes and facilitating their dissemination [2]. Genome-wide DNA sequencing disclosed the presence of a large number of uncharacterized MGEs, whose open reading frames are often automatically annotated as conserved genes of unknown function [3]. In fact, the nature of the mobile elements and their transfer mechanisms have been clarified only in few cases [4][5][6][7]. ICEs account for the majority of streptococcal and enterococcal MGEs [8]. To elucidate transfer mechanisms and their regulation it is essential to develop an established protocol for efficient conjugal transfer of ICEs also from encapsulated clinical bacterial strains. In this work, we developed a successful plate mating protocol for in vitro transfer of large ICEs with a size up to 81 kb in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis.

Methods
A schematic diagram of the plate mating protocol is reported in Figure 1.

2.
Solid medium: Add 1.5% agar to TSB liquid medium and autoclave at 121 • C for 15 min. Equilibrate TSA (TSB and agar) at 48 • C for 20 min. Add 5% defibrinated horse blood and the appropriate antibiotics at the concentrations reported in step 1 when required. Mix and pour 25 mL in each Petri dish. Leave to solidify for 20 min, dry at 42 • C for 30 min and store at 4 • C.

3.
Dilute frozen cultures 100 fold in TSB containing antibiotics and incubate at 37 • C.

4.
Grow donor and recipient cells separately until late exponential phase.

5.
Record the OD 590 on a semi-log paper. 6.
Draw the growth curve and determine the duplication time.

3.
Centrifuge the mixed cells at room temperature for 15 min at 3000 × g.

4.
Discard supernatant and resuspend the pelleted cells in 0.1 mL of TSB.

5.
Plate mixture on a blood-agar plate and incubate at 37 • C for 4 hours in a 5% CO 2 enriched atmosphere. 6.
Harvest cells by scraping from the plate with a cotton swab and dissolve in a 1 mL of TSB/Glycerol 10%. 7.

1.
Prepare TSA medium and equilibrate at 48 • C for 20 min.

2.
Pour a 17 mL base layer of TSA in Petri dishes and let the medium solidify.

3.
Dispense 2 mL of TSB supplemented with 10% horse blood in 5 mL slip-cap tubes.

4.
Put 13 mL slip-cap tubes into a heat block at 48 • C and distribute 6 ml of TSA per tube.
Combine 6 ml of TSA with the 2 mL of TSB blood cells, shake, and pour onto plate. 7.
Add an 8 ml third layer of TSA containing the appropriate antibiotics (see Note 8).

1.
Fit blood-agar plates on grids of the transilluminator device (see Note 10).

2.
Pick single colony isolates from conjugation plates by using sterile toothpicks and transfer to the plates placed on the illuminated grid. 3.
Incubate at 37 • C overnight in a 5% CO 2 enriched atmosphere.
Isolate transconjugants from the genetic analysis plates on new plates containing the appropriate antibiotic. 6.
Grow single colony isolates in TSB containing the appropriate antibiotic. 8.

1.
Other media, such as brain heart infusion (BHI) (Oxoid, UK) for streptococci and enterococci can be used. For Bacillus subtilis the Luria-Bertani (LB) medium (BD, Difco) was used.

2.
Due to light sensitivity, wrap the microtubes containing novobiocin, tetracycline, and rifampicin antibiotics in aluminum foil. Antibiotics may be stored at −20 • C for extended periods. When preparing antibiotic solutions wear protective clothing, gloves, and eye/face protection.

3.
Disposable frozen mating starter cells can be stored at −70 • C for extended periods. The use of donor and recipient cells at a concentration of approximately 5 × 10 8 CFU mL −1 is mandatory 4.
Alternatively, fresh donor and recipient cell cultures may be used.

5.
Donor cells (100 µL) and recipient cells (900 µL) are also processed separately with the same procedure and included as controls for the conjugation experiment. The 1/10 donor/recipient cell ratio is mandatory. 6.
Mating reactions can be frozen at -70 • C and plated later. Comparable numbers of transconjugants can be obtained from fresh or frozen mating reactions. 7.
We constructed new Streptococcus pneumoniae strains, FP10 and FP11, to be used as standard conjugation recipients to transfer MGEs from the original encapsulated clinical isolates. These strains: (i) lack the capsule, (ii) contain a deletion in the comC gene for competence stimulating peptide (CSP) and are impaired in natural competence for genetic transformation, and (iii) harbor the str-41 (FP10) and the nov-1 (FP11) point mutations conferring resistance to streptomycin and novobiocin, respectively. The absence of the polysaccharide capsule on the bacterial surface increases the efficiency of ICEs conjugal transfer [9]. The impairment in natural competence for genetic transformation allows us to rule out the contribution of transformation to the genetic exchange during conjugation (F. Iannelli and G. Pozzi, unpublished). The availability of two strains with different resistance markers allows transconjugants selection and to transfer the genetic elements from transconjugants again. Plating of the mating reactions includes: (i) plates containing both antibiotics for the resistance marker of the donor genetic element and for the chromosomal resistance marker of recipient strain; (ii) plates containing antibiotic for the resistance marker of the genetic element of the donor strain; and (iii) plates containing antibiotic for the chromosomal resistance marker of recipient strain. Appropriate antibiotics are added to this 8 ml TSA layer at the following concentrations: 5 µg mL-1 chloramphenicol, 1 µg mL −1 erythromycin, 1000 µg mL −1 kanamycin, 10 µg mL −1 novobiocin, 400 µg mL −1 spectinomycin, 1000 µg mL −1 streptomycin, 5 µg mL −1 tetracycline, 25 µg mL −1 fusidic acid, 25 µg mL −1 rifampicin. The multilayer plating allows: (i) a slow diffusion of the antibiotics in the agar layer containing bacteria, (ii) the visualization of the colony's three-dimensional structure, (iii) a better count of the colonies since the plate is transparent and colonies are generally larger than when spread on a plate, and (iv) the prevention of contact with ambient air favoring the growth of fastidious bacteria. 9.
Incubation can be extended to 48 h if required. 10. At this stage, carefully check the phenotype of the colonies in order to exclude isolation of spontaneous mutants or colonies which might grow even in the absence of any genotype conferring resistance. We built a transilluminating box apparatus equipped with an inner white light illuminating an upper plexiglass cover. An overhead transparency plotted with petri dish-size grids overlays the plexiglass. Blood-agar plates can be adjusted over the grids so that each plate is divided into a total of 100 sectors [10]. Plates used for the genetic analysis of transconjugants include: (i) a plate containing both antibiotics for the resistance marker of the donor genetic element and for the chromosomal resistance marker of the recipient strain; (ii) a plate containing the antibiotic for the resistance marker of the donor genetic element; (iii) a plate containing the antibiotic for the chromosomal resistance marker of recipient strain; and (iv) a plate containing no antibiotics. In the absence of a transilluminator device, naked eye observation is possible, constructing a grid using a Petri dish lid. 11. Confirm the phenotypes of transconjugants by PCR genotyping using primers for the amplification of the ICE-chromosome junctions and for ICE internal region such as the resistance genes.

Results
In this work, we report an established plate mating protocol for the conjugal transfer of large ICEs up to 81 kb in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis (Tables 1 and 2). With this procedure, the transfer of the following genetic elements in the new S. pneumoniae recipients was obtained: (i) S. pneumoniae ICE Tn5253 (size 65 kb) carrying the cat and tet(M) resistance genes [5,11,12], (ii) S. pneumoniae ICE Tn5251 (size 18 kb) carrying the tet(M) [11], (iii) ICESp23FST81like of S. pneumoniae type 23F genome strain ATCC 700669 (size 81 kb) (conjugation frequency 2.3 × 10 −6 transconjugants per donor), and (iv) ICE Tn5253-like (size 81 kb) of S. pneumoniae type 6 genome strain 670-6B (conjugation frequency 2.7 × 10 −7 transconjugants per donor). ICE Tn5253 was successfully transferred from the representative transconjugant FR58 to S. pneumoniae strains with different capsular types, S. pyogenes, S. gordonii, S. agalactiae, and transferred back from representative transconjugants of each bacterial species to S. pneumoniae (conjugation frequency varying from 4.4 × 10 −7 to 6.7 × 10 −3 to transconjugants per donor). ICE Tn5251 is part of the composite S. pneumoniae ICE Tn5253 and uses its highly efficient conjugation machinery to spread among bacterial strains. This conjugation protocol also allows the detection of rare events such as the autonomous transfer of Tn5251, as an independent ICE, from the S. pneumoniae host to E. faecalis [11]. Finally, we applied this protocol to transfer S. pyogenes φ1207.3 phage (size 53 kb, carrying the mef (A)-msr (D) macrolide resistance genes [13]), which moves through a mechanism resembling conjugation [14][15][16]. Conjugal transfer from the original 2812A clinical strain to the FP10 recipient occurred at a frequency of 3.8 × 10 −5 transconjugants per donor and from the resulting transconjugant FR119 to S. pyogenes SF370 occurred with a frequency of 4.3 × 10 −6 transconjugants per donor. S. pneumoniae HB394 (A66 derivative, type 3 clinical strain) S. pneumoniae FR55 (SP18-BS74 derivative, type 6 clinical strain) S. agalactiae FR67 (H36B transconjugant derivative) S. gordonii FR43 (GP204 transconjugant derivative)

Concluding Remarks
In conclusion, the present conjugation protocol, based on plate mating, represents an efficient, low cost and easy-to-perform procedure to transfer large ICEs with a size up to 81 kb among streptococci and enterococci. This protocol allows compliance with much higher transfer frequencies and the transfer of elements that could not be moved using a classical filter mating protocol. Specifically, we obtained: (i) the autonomous transfer of ICE Tn5251 element from S. pneumoniae to E. faecalis (<1.8 × 10 −8 transconjugants per donor with the filter mating protocol [17]), and from here among different gram-positive species; (ii) the transfer of the 81 kb ICESp23FST81 element from the original clinical S. pneumoniae type 23 to pneumococcal conjugation recipients (<3.8 × 10 −8 transconjugants per donor with the filter mating protocol); and (iii) S. pyogenes φ1207.3 phage lysogenic transfer to the S. pneumoniae FP10 recipient with a 5.4 fold increase compared to that obtained with the filter mating protocol.