Limits...
Chromosomal transfers in mycoplasmas: when minimal genomes go mobile.

Dordet-Frisoni E, Sagné E, Baranowski E, Breton M, Nouvel LX, Blanchard A, Marenda MS, Tardy F, Sirand-Pugnet P, Citti C - MBio (2014)

Bottom Line: One main mechanism involves conjugation, a process that allows the simultaneous transfer of significant amounts of DNA upon cell-to-cell contact.In these organisms, HGT was long thought to be marginal.The transfer involved DNA blocks containing up to 80 genes that were incorporated into the host chromosome by homologous recombination.

View Article: PubMed Central - PubMed

Affiliation: University of Melbourne, Department of Veterinary Science, Werribee, Victoria, Australia.

Show MeSH

Related in: MedlinePlus

Schematic of the overall strategy used to analyze DNA transfer events in 5632GT transconjugants by high-throughput sequencing. (A) NGS1 corresponds to the analysis of the 5632GT transconjugants population generated in 10 parallel mating experiments using the 5632T-H3 clone (Tet marker at nt 529737) and individual PG2G clones, each having a gentamicin marker inserted at a different locus (Table 1). For each mating, 20 individual transconjugants were randomly picked, and all were identified as having the 5632 genomic backbone by molecular typing. Transconjugants were then subjected to two parallel protocols. In protocol 1, individual DNA extractions were conducted, and the 200 extracted DNAs were pooled before sequencing. In protocol 2, individual cultures of the transconjugants were pooled prior to DNA extraction and sequencing. NGS2 is the analysis of 5632GT transconjugants derived from mating 5632T-H3 with PG2G-10 (Gm gene, nt 104815) (Table 1), using protocol 2 for DNA extraction. (B) Analyses of the sequencing reads obtained for panel A. Sequences strictly identical between 5632 and PG2 were removed, while those specific to 5632 or PG2 were mapped on the corresponding genomes using Integrative Genomics Viewer (IGV) (42).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4251992&req=5

fig2: Schematic of the overall strategy used to analyze DNA transfer events in 5632GT transconjugants by high-throughput sequencing. (A) NGS1 corresponds to the analysis of the 5632GT transconjugants population generated in 10 parallel mating experiments using the 5632T-H3 clone (Tet marker at nt 529737) and individual PG2G clones, each having a gentamicin marker inserted at a different locus (Table 1). For each mating, 20 individual transconjugants were randomly picked, and all were identified as having the 5632 genomic backbone by molecular typing. Transconjugants were then subjected to two parallel protocols. In protocol 1, individual DNA extractions were conducted, and the 200 extracted DNAs were pooled before sequencing. In protocol 2, individual cultures of the transconjugants were pooled prior to DNA extraction and sequencing. NGS2 is the analysis of 5632GT transconjugants derived from mating 5632T-H3 with PG2G-10 (Gm gene, nt 104815) (Table 1), using protocol 2 for DNA extraction. (B) Analyses of the sequencing reads obtained for panel A. Sequences strictly identical between 5632 and PG2 were removed, while those specific to 5632 or PG2 were mapped on the corresponding genomes using Integrative Genomics Viewer (IGV) (42).

Mentions: To better define which portions of the M. agalactiae chromosome can be transferred, 10 individual PG2G clones having the selective marker inserted at different loci (Table 1) were chosen from a well-characterized PG2G minilibrary (23, 25). These potential donors were then individually mated in parallel experiments with one single 5632T clone, 5632T-H3, as the recipient strain (Fig. 2). Again, clone 5632T-H3 had the Tet selective marker inserted outside any known mobile element (nt 529737). Transconjugants were obtained in all mating attempts, indicating that the 10 individual PG2G clones are able to conjugate with 5632T-H3 regardless of the position of the selective marker (Table 1). From each mating, 20 individual transconjugants were then randomly picked, grown, and typed by PCR as described above. Results indicated that the 200 transconjugants all possessed the two selective markers and displayed the 5632 genomic backbone (see Materials and Methods; also, see Fig. S1 in the supplemental material), confirming this strain as the recipient.


Chromosomal transfers in mycoplasmas: when minimal genomes go mobile.

Dordet-Frisoni E, Sagné E, Baranowski E, Breton M, Nouvel LX, Blanchard A, Marenda MS, Tardy F, Sirand-Pugnet P, Citti C - MBio (2014)

Schematic of the overall strategy used to analyze DNA transfer events in 5632GT transconjugants by high-throughput sequencing. (A) NGS1 corresponds to the analysis of the 5632GT transconjugants population generated in 10 parallel mating experiments using the 5632T-H3 clone (Tet marker at nt 529737) and individual PG2G clones, each having a gentamicin marker inserted at a different locus (Table 1). For each mating, 20 individual transconjugants were randomly picked, and all were identified as having the 5632 genomic backbone by molecular typing. Transconjugants were then subjected to two parallel protocols. In protocol 1, individual DNA extractions were conducted, and the 200 extracted DNAs were pooled before sequencing. In protocol 2, individual cultures of the transconjugants were pooled prior to DNA extraction and sequencing. NGS2 is the analysis of 5632GT transconjugants derived from mating 5632T-H3 with PG2G-10 (Gm gene, nt 104815) (Table 1), using protocol 2 for DNA extraction. (B) Analyses of the sequencing reads obtained for panel A. Sequences strictly identical between 5632 and PG2 were removed, while those specific to 5632 or PG2 were mapped on the corresponding genomes using Integrative Genomics Viewer (IGV) (42).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4251992&req=5

fig2: Schematic of the overall strategy used to analyze DNA transfer events in 5632GT transconjugants by high-throughput sequencing. (A) NGS1 corresponds to the analysis of the 5632GT transconjugants population generated in 10 parallel mating experiments using the 5632T-H3 clone (Tet marker at nt 529737) and individual PG2G clones, each having a gentamicin marker inserted at a different locus (Table 1). For each mating, 20 individual transconjugants were randomly picked, and all were identified as having the 5632 genomic backbone by molecular typing. Transconjugants were then subjected to two parallel protocols. In protocol 1, individual DNA extractions were conducted, and the 200 extracted DNAs were pooled before sequencing. In protocol 2, individual cultures of the transconjugants were pooled prior to DNA extraction and sequencing. NGS2 is the analysis of 5632GT transconjugants derived from mating 5632T-H3 with PG2G-10 (Gm gene, nt 104815) (Table 1), using protocol 2 for DNA extraction. (B) Analyses of the sequencing reads obtained for panel A. Sequences strictly identical between 5632 and PG2 were removed, while those specific to 5632 or PG2 were mapped on the corresponding genomes using Integrative Genomics Viewer (IGV) (42).
Mentions: To better define which portions of the M. agalactiae chromosome can be transferred, 10 individual PG2G clones having the selective marker inserted at different loci (Table 1) were chosen from a well-characterized PG2G minilibrary (23, 25). These potential donors were then individually mated in parallel experiments with one single 5632T clone, 5632T-H3, as the recipient strain (Fig. 2). Again, clone 5632T-H3 had the Tet selective marker inserted outside any known mobile element (nt 529737). Transconjugants were obtained in all mating attempts, indicating that the 10 individual PG2G clones are able to conjugate with 5632T-H3 regardless of the position of the selective marker (Table 1). From each mating, 20 individual transconjugants were then randomly picked, grown, and typed by PCR as described above. Results indicated that the 200 transconjugants all possessed the two selective markers and displayed the 5632 genomic backbone (see Materials and Methods; also, see Fig. S1 in the supplemental material), confirming this strain as the recipient.

Bottom Line: One main mechanism involves conjugation, a process that allows the simultaneous transfer of significant amounts of DNA upon cell-to-cell contact.In these organisms, HGT was long thought to be marginal.The transfer involved DNA blocks containing up to 80 genes that were incorporated into the host chromosome by homologous recombination.

View Article: PubMed Central - PubMed

Affiliation: University of Melbourne, Department of Veterinary Science, Werribee, Victoria, Australia.

Show MeSH
Related in: MedlinePlus