Blending genomes: distributive conjugal transfer in mycobacteria, a sexier form of HGT

Abstract

This review discusses a novel form of horizontal gene transfer (HGT) found in mycobacteria called Distributive Conjugal Transfer (DCT). While satisfying the criteria for conjugation, DCT occurs by a mechanism so distinct from oriT-mediated conjugation that it could be considered a fourth category of HGT. DCT involves the transfer of chromosomal DNA between mycobacteria and, most significantly, generates transconjugants with mosaic genomes of the parental strains. Multiple segments of donor chromosomal DNA can be co-transferred regardless of their location or the genetic selection and, as a result, the transconjugant genome contains many donor-derived segments; hence the name DCT. This distinguishing feature of DCT separates it from the other known mechanisms of HGT, which generally result in the introduction of a single, defined segment of DNA into the recipient chromosome (Fig. ). Moreover, these mosaic progeny are generated from a single conjugal event, which provides enormous capacity for rapid adaptation and evolution, again distinguishing it from the three classical modes of HGT. Unsurprisingly, the unusual mosaic products of DCT are generated by a conjugal mechanism that is also unusual. Here, we will describe the unique features of DCT and contrast those to other mechanisms of HGT, both from a mechanistic and an evolutionary perspective. Our focus will be on transfer of chromosomal DNA, as opposed to plasmid mobilization, because DCT mediates transfer of chromosomal DNA and is a chromosomally encoded process.

Fig. 1

Overview of the major mechanisms and products of bacterial HGT. To facilitate comparison between classes, a schematic representation of each is shown above a short list of attributes for that class. Donor cells have a green outline and contain dark blue DNA; recipient cells with a green outline contain pale blue DNA recipient, and varying amounts of transferred donor DNA; the dark blue arrow represents the origin of transfer, oriT, from which transfer is initiated in a 5′ to 3′ direction. *In transformation most acquired DNA segments are small, however, larger fragments (up to 40 kb) can be inherited if their gene content is selected (Blokesch, 2017).

Fig. 2

The experimental outline for DCT and the mosaic genomes it generates. Donor and recipient strains of mycobacteria with different antibiotic markers are expanded under selection to late mid-log densities (1), and equivalent amounts are combined, pelleted, resuspended and spotted on agar for 18–24 hr (2). The coculture polycolony is resuspended and plated on double selection plates to identify transconjugants, and on single selection plates to recover parents for calculating DNA transfer efficiencies (3). Transconjugants arising from independent matings from donors marked at varying chromosomal positions are subjected to whole genome sequencing (4). The many SNPs (>72,000 between the parental reference genomes) facilitate the identification of the parental origin of DNA comprising the transconjugant genomes with great precision. Circos plots of the recipient reference chromosome (outer ring, yellow), four independent transconjugants, and the donor reference chromosome (inner ring, blue). The transconjugants contain both recipient (yellow) and non-selected donor (blue) DNA. Green segments are those containing the selected kanamycin-resistance gene, which were located in different positions in the chromosome of each of the four donor strain derivatives. The three outermost transconjugants were also shown to have acquired the ability to be donors. Consistent with this phenotype, they each contain (blue) donor-derived DNA spanning esx1 and the mid locus (at 0.1 Mb), in contrast to the innermost transconjugant, which still has a recipient phenotype. Transconjugants show the random, complex, mosaicism that can be produced by a single DCT event (5). Modified from (Derbyshire & Gray, 2014)

Fig. 3

Localized recombinant architecture suggests different mechanisms that generate two potential levels of diversity. A single contiguous tract of donor DNA that has recombined into the transconjugant genome exemplifies large chunk exchange, which usually replaces the orthologous tract of recipient DNA and can also result in acquisition of novel genes (red arrow, left panel). Microcomplexity is likely generated by repair mechanisms that alternate between available donor and recipient templates, incorporating SNPs from each in quick succession. Collectively, these disparate recombination architectures can bring in new operons—and the pathways they encode—or make minor adjustments in regulatory elements or individual proteins. Modified from (Derbyshire & Gray, 2014).

Fig. 4

esx4 loci share common genes necessary for ESX function. The seven-gene core locus of esx4 is conserved in gene content and order in most mycobacteria, including M. tuberculosis and M. smegmatis donor and recipient strains (top panel). The esx1 locus is more complex and is more heterogeneous in the composition of genes near its 3′ boundary, particularly where the mating identity (mid) genes are located in M. smegmatis (bottom panel). The mid genes are expanded at the bottom to show the level of amino acid identity between donor and recipient strains (IS represents insertion sequence elements). The genes are colour coded to indicate orthologous genes in each ESX system. The M. smegmatis (Ms) and M. tuberculosis (Rv) gene numbers are given to indicate the location of each locus in its respective genome. The gene names follow the classification according to (Bitter et al., 2009). ecc genes are conserved components of all esx loci and are thought to encode core machinery. esp genes are specific to a locus, in this case esx1, and are thought to mediate locus-specific functions. EspB, PE35 and PPE68 are known to be secreted by ESX-1 (and figure 5). EsxB and EsxA form the heterodimer secreted by the ESX-1 system. The equivalent proteins in esx4, EsxU and EsxT, have yet to be shown to be secreted.

Fig. 5

General schematic of an ESX secretion apparatus based on the ESX-5 structure (Ates et al., 2016a, Beckham et al., 2017). The core components (Ecc) and secretion substrates (EsxBA, EspB, PE/PPE) encoded by each paralogous locus are dedicated to that specific apparatus, creating a series of operationally similar, but functionally non-redundant, secretion systems (Bitter et al., 2009, Houben et al., 2014). In ESX-1, EccA is thought to act as a chaperone delivering the secretion substrates to EccC, the membrane-bound ATPase, which then delivers the proteins to the inner membrane channel made by EccD. MycP is a protease, required for processing of some secreted substrates (eg. EspB; Ohol et al., 2010). EspG is a second chaperone dedicated to secretion of PE and PPE proteins (Abdallah et al., 2006, Abdallah et al., 2009). The channel protein and mechanism through which the ESX substrates traverse the outer mycomembrane is unknown. The protein cartoons are not drawn to scale.

Fig. 6

DCT can be conceptualized as a progression of sequential events. DCT begins with contact-dependent signaling between the donor and recipient that includes the polar-localized ESX-1 secretion apparatus from each strain (indicated by curved arrows). We hypothesize that proteins secreted decorate both donor and recipient cell surfaces (cups and circles). Interactions between the secreted proteins (joined cup and circle) trigger donor- and recipient-specific responses. At least one part of the recipient response is to signal induction of esx4, which is required for DCT. The dashed arrow in the recipient reflects the multiple steps between ESX-1 and esx4 induction. Following cell-cell contact, and the expression of genes required to transfer and receive DNA in donor and recipient, respectively, the donor chromosome is fragmented by an unidentified nuclease. (Alternatively, following transfer of the donor chromosome its fragmentation could occur by a nuclease in the recipient). Large, multiple, donor fragments are taken up by the recipient cell, but the DNA import machinery and the fate of the donor are not yet known. The imported donor fragments are integrated into the transconjugant genomes by homologous recombination or template repair mechanisms to create a transconjugant with a mosaic genome. The successful establishment of the transconjugant progeny within a growing population will be determined by the relative fitness conferred by the new combination of genetic variants in that transconjugant relative to the fitness of the parental strains.