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Distributive Conjugal Transfer: New Insights into Horizontal Gene Transfer and Genetic Exchange in Mycobacteria

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  • Authors: Keith M. Derbyshire1, Todd A. Gray2
  • Editors: Graham F. Hatfull3, William R. Jacobs Jr.4
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    Affiliations: 1: Division of Genetics, Wadsworth Center, New York State Department of Health, and Department of Biomedical Sciences, University at Albany, Albany, NY 12201; 2: Division of Genetics, Wadsworth Center, New York State Department of Health, and Department of Biomedical Sciences, University at Albany, Albany, NY 12201; 3: University of Pittsburgh, Pittsburgh, PA; 4: Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, NY
    • Source: microbiolspec January 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.MGM2-0022-2013
  • Received 22 August 2013 Accepted 03 September 2013 Published 17 January 2014
  • Correspondence: Keith M. Derbyshire, [email protected]
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  • Abstract:

    The past decade has seen an explosion in the application of genomic tools across all biological disciplines. This is also true for mycobacteria, where whole-genome sequences are now available for pathogens and nonpathogens alike. Genomes within the complex (MTBC) bear the hallmarks of horizontal gene transfer (HGT). Conjugation is the form of HGT with the highest potential capacity and evolutionary influence. Donor and recipient strains of actively conjugate upon coculturing in biofilms and on solid media. Whole-genome sequencing of the transconjugant progeny demonstrated the incredible scale and range of genomic variation that conjugation generates. Transconjugant genomes are complex mosaics of the parental strains. Some transconjugant genomes are up to one-quarter donor-derived, distributed over 30 segments. Transferred segments range from ∼50 bp to ∼225,000 bp in length and are exchanged with their recipient orthologs all around the genome. This unpredictable genome-wide infusion of DNA sequences is called distributive conjugal transfer (DCT), to distinguish it from traditional -based conjugation. The mosaicism generated in a single transfer event resembles that seen from meiotic recombination in sexually reproducing organisms and contrasts with traditional models of HGT. This similarity allowed the application of a genome-wide association study approach to map the donor genes that confer a donor mating identity phenotype. The mating identity genes map to the locus, expanding the central role of ESX-1 function in conjugation. The potential for DCT to instantaneously blend genomes will affect how we view mycobacterial evolution and provide new tools for the facile manipulation of mycobacterial genomes.

  • Citation: Derbyshire K, Gray T. 2014. Distributive Conjugal Transfer: New Insights into Horizontal Gene Transfer and Genetic Exchange in Mycobacteria. Microbiol Spectrum 2(1):MGM2-0022-2013. doi:10.1128/microbiolspec.MGM2-0022-2013.

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2014-01-17
2019-09-17

Abstract:

The past decade has seen an explosion in the application of genomic tools across all biological disciplines. This is also true for mycobacteria, where whole-genome sequences are now available for pathogens and nonpathogens alike. Genomes within the complex (MTBC) bear the hallmarks of horizontal gene transfer (HGT). Conjugation is the form of HGT with the highest potential capacity and evolutionary influence. Donor and recipient strains of actively conjugate upon coculturing in biofilms and on solid media. Whole-genome sequencing of the transconjugant progeny demonstrated the incredible scale and range of genomic variation that conjugation generates. Transconjugant genomes are complex mosaics of the parental strains. Some transconjugant genomes are up to one-quarter donor-derived, distributed over 30 segments. Transferred segments range from ∼50 bp to ∼225,000 bp in length and are exchanged with their recipient orthologs all around the genome. This unpredictable genome-wide infusion of DNA sequences is called distributive conjugal transfer (DCT), to distinguish it from traditional -based conjugation. The mosaicism generated in a single transfer event resembles that seen from meiotic recombination in sexually reproducing organisms and contrasts with traditional models of HGT. This similarity allowed the application of a genome-wide association study approach to map the donor genes that confer a donor mating identity phenotype. The mating identity genes map to the locus, expanding the central role of ESX-1 function in conjugation. The potential for DCT to instantaneously blend genomes will affect how we view mycobacterial evolution and provide new tools for the facile manipulation of mycobacterial genomes.

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Distributive Conjugal Transfer: New Insights into Horizontal Gene Transfer and Genetic Exchange in Mycobacteria
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Citation: Derbyshire K, Gray T. 2014. Distributive conjugal transfer: new insights into horizontal gene transfer and genetic exchange in mycobacteria. microbiolspec 2(1): doi:10.1128/microbiolspec.MGM2-0022-2013
/content/microbiolspec/10.1128/microbiolspec.MGM2-0022-2013.fig1
FIGURE 1
directs both plasmid and chromosomal transfer in traditional conjugation systems. F episome plasmid transfer. The is nicked and a single strand is guided into the engaged recipient by a plasmid-encoded relaxase. Upon transfer, relaxase catalyzes recircularization at , and host polymerases synthesize a complementary strand. The recipient chromosome is unaltered, but the cell now exhibits a donor phenotype (blue). Hfr strains have a plasmid integrated into the chromosome, shown as a single strand for simplicity. The integrated functions as it would in the plasmid scenario above, except that transfer of the chromosome is usually incomplete. The linear chromosomal fragment must be incorporated into the recipient chromosome by homologous recombination for stable inheritance. Homologous recombination excludes the , and, while the transconjugant chromosome now has some donor sequences, it retains a recipient phenotype (yellow). For an Hfr transconjugant to become a donor, the entire donor chromosome must be transferred to regenerate .
microbiolspec/2/1/MGM2-0022-2013-fig1_thmb.gif
microbiolspec/2/1/MGM2-0022-2013-fig1.gif
Image of FIGURE 1

Click to view

FIGURE 1

directs both plasmid and chromosomal transfer in traditional conjugation systems. F episome plasmid transfer. The is nicked and a single strand is guided into the engaged recipient by a plasmid-encoded relaxase. Upon transfer, relaxase catalyzes recircularization at , and host polymerases synthesize a complementary strand. The recipient chromosome is unaltered, but the cell now exhibits a donor phenotype (blue). Hfr strains have a plasmid integrated into the chromosome, shown as a single strand for simplicity. The integrated functions as it would in the plasmid scenario above, except that transfer of the chromosome is usually incomplete. The linear chromosomal fragment must be incorporated into the recipient chromosome by homologous recombination for stable inheritance. Homologous recombination excludes the , and, while the transconjugant chromosome now has some donor sequences, it retains a recipient phenotype (yellow). For an Hfr transconjugant to become a donor, the entire donor chromosome must be transferred to regenerate .

Source: microbiolspec January 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.MGM2-0022-2013
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/content/microbiolspec/10.1128/microbiolspec.MGM2-0022-2013.fig2
FIGURE 2
An outline of the mycobacterial mating procedure. Selectable markers for donor and recipient are chromosomally encoded. Efficient transfer requires prolonged incubation (overnight) on solid medium. Transfer does not take place in liquid cultures, probably reflecting the need to force cell-cell contact on the solid medium. The transfer frequency is 1 event per 10 donors. However, this only reflects successful transfer of and therefore is an underestimate of the total number of events.
microbiolspec/2/1/MGM2-0022-2013-fig2_thmb.gif
microbiolspec/2/1/MGM2-0022-2013-fig2.gif
Image of FIGURE 2

Click to view

FIGURE 2

An outline of the mycobacterial mating procedure. Selectable markers for donor and recipient are chromosomally encoded. Efficient transfer requires prolonged incubation (overnight) on solid medium. Transfer does not take place in liquid cultures, probably reflecting the need to force cell-cell contact on the solid medium. The transfer frequency is 1 event per 10 donors. However, this only reflects successful transfer of and therefore is an underestimate of the total number of events.

Source: microbiolspec January 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.MGM2-0022-2013
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FIGURE 3
Donor chromosomal sites mobilize plasmids and mediate recircularization by gap repair in the recipient. Episomal plasmids are not subject to conjugal transfer unless they carry chromosomal DNA segments functionally defined as (basis of mobility) sites. Recovery of the transferred plasmids, and sequencing of the sites, revealed the presence of embedded recipient SNPs, suggesting a gap-repair mechanism. In this model, transfer would be initiated in the donor via a break in . Following transfer of the linear plasmid, the homologous region of the recipient chromosome would act as the template for gap repair to seal the break and recircularize the plasmid. As a consequence, recipient SNPs would be incorporated into the plasmid DNA. This model was confirmed by the use of two adjacent sites, separated by a nonhomologous sequence. The region of nonidentity was replaced by the intervening recipient chromosomal sequence upon transfer. In this two- model a break could occur at both s to create a gap spanning hypothetical gene . Alternatively, a break could occur at just one site (as shown) and then the nonhomologous region resected by exonucleases to generate ends suitable for gap repair, capture of gene and plasmid circularization.
microbiolspec/2/1/MGM2-0022-2013-fig3_thmb.gif
microbiolspec/2/1/MGM2-0022-2013-fig3.gif
Image of FIGURE 3

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FIGURE 3

Donor chromosomal sites mobilize plasmids and mediate recircularization by gap repair in the recipient. Episomal plasmids are not subject to conjugal transfer unless they carry chromosomal DNA segments functionally defined as (basis of mobility) sites. Recovery of the transferred plasmids, and sequencing of the sites, revealed the presence of embedded recipient SNPs, suggesting a gap-repair mechanism. In this model, transfer would be initiated in the donor via a break in . Following transfer of the linear plasmid, the homologous region of the recipient chromosome would act as the template for gap repair to seal the break and recircularize the plasmid. As a consequence, recipient SNPs would be incorporated into the plasmid DNA. This model was confirmed by the use of two adjacent sites, separated by a nonhomologous sequence. The region of nonidentity was replaced by the intervening recipient chromosomal sequence upon transfer. In this two- model a break could occur at both s to create a gap spanning hypothetical gene . Alternatively, a break could occur at just one site (as shown) and then the nonhomologous region resected by exonucleases to generate ends suitable for gap repair, capture of gene and plasmid circularization.

Source: microbiolspec January 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.MGM2-0022-2013
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FIGURE 4
Transconjugant chromosomes are mosaic blends of the parental strains. Whole-genome sequencing of transconjugants and alignment with the parental sequences used the presence or absence of parental SNPs to define tracts of transferred donor DNA. A Circos projection of the 7-Mb chromosome is shown as concentric circles, with the recipient genome on the outside (yellow), the donor on the inside (blue), and four transconjugants in between. The segment containing the gene used for postmating selection is indicated (green); the recipient antibiotic marker is episomally encoded. The outer three transconjugants have a donor mating identity, and all have inherited the locus from the donor strain (indicated at 0.1 Mb on the chromosome). The innermost transconjugant lacks the locus and is a recipient strain. These data and additional Circos plots can be seen in reference 15 .
microbiolspec/2/1/MGM2-0022-2013-fig4_thmb.gif
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FIGURE 4

Transconjugant chromosomes are mosaic blends of the parental strains. Whole-genome sequencing of transconjugants and alignment with the parental sequences used the presence or absence of parental SNPs to define tracts of transferred donor DNA. A Circos projection of the 7-Mb chromosome is shown as concentric circles, with the recipient genome on the outside (yellow), the donor on the inside (blue), and four transconjugants in between. The segment containing the gene used for postmating selection is indicated (green); the recipient antibiotic marker is episomally encoded. The outer three transconjugants have a donor mating identity, and all have inherited the locus from the donor strain (indicated at 0.1 Mb on the chromosome). The innermost transconjugant lacks the locus and is a recipient strain. These data and additional Circos plots can be seen in reference 15 .

Source: microbiolspec January 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.MGM2-0022-2013
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FIGURE 5
Models for chromosome fragmentation in DCT. DCT could follow one of two pathways, depending on whether fragmentation of the transferred chromosome occurs in the donor cell before transfer (left) or following transfer in the recipient (right and indicated by arrows). The left-hand model posits that multiple chromosome segments are cotransferred into the recipient, where they are recombined into the recipient chromosome to generate a mosaic pattern. Large chunk transfer (right) would be predicted to result in fewer large integrated segments rather than many widely distributed donor segments. Current results, as seen in Fig. 4 , are more consistent with the fragmentation-before-transfer model.
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FIGURE 5

Models for chromosome fragmentation in DCT. DCT could follow one of two pathways, depending on whether fragmentation of the transferred chromosome occurs in the donor cell before transfer (left) or following transfer in the recipient (right and indicated by arrows). The left-hand model posits that multiple chromosome segments are cotransferred into the recipient, where they are recombined into the recipient chromosome to generate a mosaic pattern. Large chunk transfer (right) would be predicted to result in fewer large integrated segments rather than many widely distributed donor segments. Current results, as seen in Fig. 4 , are more consistent with the fragmentation-before-transfer model.

Source: microbiolspec January 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.MGM2-0022-2013
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FIGURE 6
DCT brings large and small changes to transconjugant genomes. Transferred donor segments can be contiguous blocks hundreds of kilobases in length, spanning hundreds of genes. These large blocks can exchange recipient for donor orthologs (left). The large segments may also contain additional genes not present in the original recipient sequence (insertion) or may lack some genes that had been present (deletion). At the opposite end of the size spectrum, donor segments of <100 bp are often found in clusters to generate microcomplexity, with the potential to fine-tune genes or functional elements (right). Comparison of M. canettii genomes has identified similar mosaicism, in which sequences of entire genes are identical between some isolates, while other regions contain short regions of exchanged SNPs consistent with the recombinant patterns observed with DCT ( 14 ).
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FIGURE 6

DCT brings large and small changes to transconjugant genomes. Transferred donor segments can be contiguous blocks hundreds of kilobases in length, spanning hundreds of genes. These large blocks can exchange recipient for donor orthologs (left). The large segments may also contain additional genes not present in the original recipient sequence (insertion) or may lack some genes that had been present (deletion). At the opposite end of the size spectrum, donor segments of <100 bp are often found in clusters to generate microcomplexity, with the potential to fine-tune genes or functional elements (right). Comparison of M. canettii genomes has identified similar mosaicism, in which sequences of entire genes are identical between some isolates, while other regions contain short regions of exchanged SNPs consistent with the recombinant patterns observed with DCT ( 14 ).

Source: microbiolspec January 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.MGM2-0022-2013
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FIGURE 7
DCT generates instant diversity on a genome-wide scale. Progeny from a bacterial cross are shown for -mediated chromosomal transfer (left) and for DCT (right). In a single Hfr cross, transconjugants can acquire segments of DNA proximal to Depending on the length of transfer, all progeny will have overlapping regions of the donor chromosome extending from . By contrast, all DCT progeny are different because transfer initiates from sites all around the chromosome. In addition, DCT recombination results in both large segment exchanges and smaller regions of micro-heterogeneity, creating further diversity. The extreme diversity in the transconjugant population allows for rapid expansion under changing selective pressures.
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FIGURE 7

DCT generates instant diversity on a genome-wide scale. Progeny from a bacterial cross are shown for -mediated chromosomal transfer (left) and for DCT (right). In a single Hfr cross, transconjugants can acquire segments of DNA proximal to Depending on the length of transfer, all progeny will have overlapping regions of the donor chromosome extending from . By contrast, all DCT progeny are different because transfer initiates from sites all around the chromosome. In addition, DCT recombination results in both large segment exchanges and smaller regions of micro-heterogeneity, creating further diversity. The extreme diversity in the transconjugant population allows for rapid expansion under changing selective pressures.

Source: microbiolspec January 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.MGM2-0022-2013
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FIGURE 8
Mating identity is determined by genes within The . locus spans 26 homologous genes in the donor (blue) and recipient (orange) parental strains (top). Genes and define the ends of the locus. and encode the primary secreted substrates EsxA and EsxB. Whole-genome sequencing and genome-wide association mapping of F1 transconjugants that exhibited a donor phenotype revealed that all donor-proficient transconjugants had an locus of donor origin ( Fig. 4 ). Mapping of the ating entity () locus—the donor genes associated with the donor conjugal phenotype—to was confirmed by successively backcrossing donor-proficient transconjugants with the recipient, while maintaining the donor phenotype (transconjugant donor). Backcrossed transconjugants that showed a recipient conjugal phenotype (transconjugant recipient) all shared a region of their locus that was composed of recipient-derived genes, further refining the locus to six genes ( and ) near the 3′ end of . White-filled gene symbols represent repetitive elements.
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FIGURE 8

Mating identity is determined by genes within The . locus spans 26 homologous genes in the donor (blue) and recipient (orange) parental strains (top). Genes and define the ends of the locus. and encode the primary secreted substrates EsxA and EsxB. Whole-genome sequencing and genome-wide association mapping of F1 transconjugants that exhibited a donor phenotype revealed that all donor-proficient transconjugants had an locus of donor origin ( Fig. 4 ). Mapping of the ating entity () locus—the donor genes associated with the donor conjugal phenotype—to was confirmed by successively backcrossing donor-proficient transconjugants with the recipient, while maintaining the donor phenotype (transconjugant donor). Backcrossed transconjugants that showed a recipient conjugal phenotype (transconjugant recipient) all shared a region of their locus that was composed of recipient-derived genes, further refining the locus to six genes ( and ) near the 3′ end of . White-filled gene symbols represent repetitive elements.

Source: microbiolspec January 2014 vol. 2 no. 1 doi:10.1128/microbiolspec.MGM2-0022-2013
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