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

Abstract

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.

Predicted transmembrane proteins with homology to Mef(A) are not responsible for complementing mef(A) deletion in the mef(A)–msr(D) macrolide efflux system in Streptococcus pneumoniae

“…Bacterial strains were grow grown in Tryptic Soy Broth (TSB) or Tryptic Soy Agar (TSA) supplemented with 3% defibrinated horse blood [ 29 ]. Transfer of Φ1207.3 or Φ1207.3Δ mef (A) from strains FR183 and FP40 to the deletion mutants was obtained through a mating protocol as already reported [ 30 ]. Briefly, donor and recipient cells were grown separately in TSB in the presence of the appropriate antibiotics.…”

“…Plates were incubated at 37 °C in the presence of 5% CO 2 for 4 h and cells were recovered with a cotton swab and resuspended in TSB. To select for recombinants, cell suspension was plated following a multilayer plating procedure [ 30 ].…”

Immune Memory After Respiratory Infection With Streptococcus pneumoniae Is Revealed by in vitro Stimulation of Murine Splenocytes With Inactivated Pneumococcal Whole Cells: Evidence of Early Recall Responses by Transcriptomic Analysis

“…The bacterial culture in mid-exponential phase (≈OD 590 = 0.6), was centrifuged at 2,000 x g for 10 minutes and resuspended in an appropriate volume of saline. Before the centrifugation, the culture was Gram-stained and bacterial vital counts were performed using the multilayer plating method ( Iannelli et al., 2021 ). Each mouse was anesthetized by intraperitoneal administration of 15 mg/kg tiletamine hydrochloride/zolazepam and 4 mg/kg xylazine and intranasally infected by instillation of 10^7 CFU of TIGR4, prepared as described above, in the volume of 25 μl/nostril in TSB.…”

“…Bacterial colony forming units (CFUs) were determined by plating appropriate dilutions of frozen lungs using a multilayer plating procedure ( Iannelli et al., 2021 ). The lower limit of detection was 10 CFU/ml.…”

Chromosomal integration of Tn5253 occurs downstream of a conserved 11-bp sequence of the rbgA gene in Streptococcus pneumoniae and in all the other known hosts of this integrative conjugative element (ICE)

“…Both streptococcal and enterococcal strains were grown in tryptic soy broth or tryptic soy agar (Difco) supplemented, where appropriate, with antibiotics. Plate mating conjugation experiments were performed as previously described [ 38 ]. Briefly, donor and recipient cells were grown until the end of exponential phase and mixed at a 1:10 ratio, then were collected by centrifugation, plated and incubated for 4 h. Cells were harvested by scraping the plates and recombinant strains were selected by a multilayer plating procedure in presence of the appropriate antibiotics.…”