Plasmid-mediated drug resistance in mycobacteria: the tip of the iceberg?

Macrolides, such as clarithromycin, are crucial in the treatment of nontuberculous mycobacteria (NTM). NTM are notoriously innately drug resistant, which has made the dependence on macrolides for their treatment even more important. Not surprisingly, resistance to macrolides has been documented in some NTM, including Mycobacterium avium and Mycobacterium abscessus, which are the two NTM species most often identified in clinical isolates. Resistance is mediated by point mutations in the 23S ribosomal RNA or by methylation of the rRNA by a methylase (encoded by an erm gene). Chromosomally encoded erm genes have been identified in many of the macrolide-resistant isolates, but not in Mycobacterium chelonae. Now, Brown-Elliott et al. (J Clin Microbiol 61:e00428-23, 2023, https://doi.org/10.1128/JCM.00428-23) describe the identification of a new erm variant, erm(55), which was found either on the chromosome or on a plasmid in highly macrolide-resistant clinical isolates of M. chelonae. The chromosomal erm(55) gene appears to be associated with mobile elements; one gene is within a putative transposon and the second is in a large (37 kb) insertion/deletion. The plasmid carrying erm(55) also encodes type IV and type VII secretion systems, which are often linked on large mycobacterial plasmids and are hypothesized to mediate plasmid transfer. While the conjugative transfer of the erm(55)-containing plasmid between NTM has yet to be demonstrated, the inferences are clear, as evidenced by the dissemination of plasmid-mediated drug resistance in other medically important bacteria. Here, we discuss the findings of Brown-Elliott et al., and the potential ramifications on treatment of NTM infections.

The Many Hosts of Mycobacteria 9 (MHM9): A conference report.

The Many Hosts of Mycobacteria (MHM) meeting series brings together basic scientists, clinicians and veterinarians to promote robust discussion and dissemination of recent advances in our knowledge of numerous mycobacterial diseases, including human and bovine tuberculosis (TB), nontuberculous mycobacteria (NTM) infection, Hansen's disease (leprosy), Buruli ulcer and Johne's disease. The 9th MHM conference (MHM9) was held in July 2022 at The Ohio State University (OSU) and centered around the theme of "Confounders of Mycobacterial Disease." Confounders can and often do drive the transmission of mycobacterial diseases, as well as impact surveillance and treatment outcomes. Various confounders were presented and discussed at MHM9 including those that originate from the host (comorbidities and coinfections) as well as those arising from the environment (e.g., zoonotic exposures), economic inequality (e.g. healthcare disparities), stigma (a confounder of leprosy and TB for millennia), and historical neglect (a confounder in Native American Nations). This conference report summarizes select talks given at MHM9 highlighting recent research advances, as well as talks regarding the historic and ongoing impact of TB and other infectious diseases on Native American Nations, including those in Southwestern Alaska where the regional TB incidence rate is among the highest in the Western hemisphere.

The small mycobacterial ribosomal protein, bS22, modulates aminoglycoside accessibility to its 16S rRNA helix-44 binding site.

Treatment of tuberculosis continues to be challenging due to the widespread latent form of the disease and the emergence of antibiotic-resistant strains of the pathogen, Mycobacterium tuberculosis. Bacterial ribosomes are a common and effective target for antibiotics. Several second line anti-tuberculosis drugs, e.g. kanamycin, amikacin, and capreomycin, target ribosomal RNA to inhibit protein synthesis. However, M. tuberculosis can acquire resistance to these drugs, emphasizing the need to identify new drug targets. Previous cryo-EM structures of the M. tuberculosis and M. smegmatis ribosomes identified two novel ribosomal proteins, bS22 and bL37, in the vicinity of two crucial drug-binding sites: the mRNA-decoding center on the small (30S), and the peptidyl-transferase center on the large (50S) ribosomal subunits, respectively. The functional significance of these two small proteins is unknown. In this study, we observe that an M. smegmatis strain lacking the bs22 gene shows enhanced susceptibility to kanamycin compared to the wild-type strain. Cryo-EM structures of the ribosomes lacking bS22 in the presence and absence of kanamycin suggest a direct role of bS22 in modulating the 16S rRNA kanamycin-binding site. Our structures suggest that amino-acid residue Lys-16 of bS22 interacts directly with the phosphate backbone of helix 44 of 16S rRNA to influence the micro-configuration of the kanamycin-binding pocket. Our analysis shows that similar interactions occur between eukaryotic homologues of bS22, and their corresponding rRNAs, pointing to a common mechanism of aminoglycoside resistance in higher organisms.

Mycobacterium smegmatis: The Vanguard of Mycobacterial Research.

The genus Mycobacterium contains several slow-growing human pathogens, including Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium. Mycobacterium smegmatis is a nonpathogenic and fast growing species within this genus. In 1990, a mutant of M. smegmatis, designated mc2155, that could be transformed with episomal plasmids was isolated, elevating M. smegmatis to model status as the ideal surrogate for mycobacterial research. Classical bacterial models, such as Escherichia coli, were inadequate for mycobacteria research because they have low genetic conservation, different physiology, and lack the novel envelope structure that distinguishes the Mycobacterium genus. By contrast, M. smegmatis encodes thousands of conserved mycobacterial gene orthologs and has the same cell architecture and physiology. Dissection and characterization of conserved genes, structures, and processes in genetically tractable M. smegmatis mc2155 have since provided previously unattainable insights on these same features in its slow-growing relatives. Notably, tuberculosis (TB) drugs, including the first-line drugs isoniazid and ethambutol, are active against M. smegmatis, but not against E. coli, allowing the identification of their physiological targets. Furthermore, Bedaquiline, the first new TB drug in 40 years, was discovered through an M. smegmatis screen. M. smegmatis has become a model bacterium, not only for M. tuberculosis, but for all other Mycobacterium species and related genera. With a repertoire of bioinformatic and physical resources, including the recently established Mycobacterial Systems Resource, M. smegmatis will continue to accelerate mycobacterial research and advance the field of microbiology.

Pervasive translation in Mycobacterium tuberculosis.

Most bacterial ORFs are identified by automated prediction algorithms. However, these algorithms often fail to identify ORFs lacking canonical features such as a length of >50 codons or the presence of an upstream Shine-Dalgarno sequence. Here, we use ribosome profiling approaches to identify actively translated ORFs in Mycobacterium tuberculosis. Most of the ORFs we identify have not been previously described, indicating that the M. tuberculosis transcriptome is pervasively translated. The newly described ORFs are predominantly short, with many encoding proteins of ≤50 amino acids. Codon usage of the newly discovered ORFs suggests that most have not been subject to purifying selection, and hence are unlikely to contribute to cell fitness. Nevertheless, we identify 90 new ORFs (median length of 52 codons) that bear the hallmarks of purifying selection. Thus, our data suggest that pervasive translation of short ORFs in Mycobacterium tuberculosis serves as a rich source for the evolution of new functional proteins.

How can you predict which proteins an organism can make? To answer this question, scientists often use computer programs that can scan the genetic information of a species for open reading frames ­ a type of DNA sequence that codes for a protein. However, very short genes and overlapping genes are often missed through these searches. Mycobacteria are a group of bacteria that includes the species Mycobacterium tuberculosis, which causes tuberculosis. Previous work has predicted several thousand open reading frames for M. tuberculosis, but Smith et al. decided to use a different approach to determine whether there could be more. They focused on ribosomes, the cellular structures that assemble a specific protein by reading the instructions provided by the corresponding gene. Examining the sections of genetic code that ribosomes were processing in M. tuberculosis uncovered hundreds of new open reading frames, most of which carried the instructions to make very short proteins. A closer look suggested that only 90 of these proteins were likely to have a useful role in the life of the bacteria, which could open new doors in tuberculosis research. The rest of the sequences showed no evidence of having evolved a useful job, yet they were still manufactured by the mycobacteria. This pervasive production could play a role in helping the bacteria adapt to quickly changing environments by evolving new, functional proteins.

Spatiotemporal localization of proteins in mycobacteria.

Although prokaryotic organisms lack traditional organelles, they must still organize cellular structures in space and time, challenges that different species solve differently. To systematically define the subcellular architecture of mycobacteria, we perform high-throughput imaging of a library of fluorescently tagged proteins expressed in Mycobacterium smegmatis and develop a customized computational pipeline, MOMIA and GEMATRIA, to analyze these data. Our results establish a spatial organization network of over 700 conserved mycobacterial proteins and reveal a coherent localization pattern for many proteins of known function, including those in translation, energy metabolism, cell growth and division, as well as proteins of unknown function. Furthermore, our pipeline exploits morphologic proxies to enable a pseudo-temporal approximation of protein localization and identifies previously uncharacterized cell-cycle-dependent dynamics of essential mycobacterial proteins. Collectively, these data provide a systems perspective on the subcellular organization of mycobacteria and provide tools for the analysis of bacteria with non-standard growth characteristics.

Fluorescence Imaging-Based Discovery of Membrane Domain-Associated Proteins in Mycobacterium smegmatis.

Mycobacteria spatially organize their plasma membrane, and many enzymes involved in envelope biosynthesis associate with a membrane compartment termed the intracellular membrane domain (IMD). The IMD is concentrated in the polar regions of growing cells and becomes less polarized under nongrowing conditions. Because mycobacteria elongate from the poles, the observed polar localization of the IMD during growth likely supports the localized biosynthesis of envelope components. While we have identified more than 300 IMD-associated proteins by proteomic analyses, only a few of these have been verified by independent experimental methods. Furthermore, some IMD-associated proteins may have escaped proteomic identification and remain to be identified. Here, we visually screened an arrayed library of 523 Mycobacterium smegmatis strains, each producing a Dendra2-FLAG-tagged recombinant protein. We identified 29 fusion proteins that showed polar fluorescence patterns characteristic of IMD proteins. Twenty of these had previously been suggested to localize to the IMD based on proteomic data. Of the nine remaining IMD candidate proteins, three were confirmed by biochemical methods to be associated with the IMD. Taken together, this new colocalization strategy is effective in verifying the IMD association of proteins found by proteomic analyses while facilitating the discovery of additional IMD-associated proteins. IMPORTANCE The intracellular membrane domain (IMD) is a membrane subcompartment found in Mycobacterium smegmatis cells. Proteomic analysis of purified IMD identified more than 300 proteins, including enzymes involved in cell envelope biosynthesis. However, proteomics on its own is unlikely to detect every IMD-associated protein because of technical and biological limitations. Here, we describe fluorescent protein colocalization as an alternative, independent approach. Using a combination of fluorescence microscopy, proteomics, and subcellular fractionation, we identified three new proteins associated with the IMD. Such a robust method to rigorously define IMD proteins will benefit future investigations to decipher the synthesis, maintenance, and functions of this membrane domain and help delineate a more general mechanism of subcellular protein localization in mycobacteria.

Polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression in mycobacteria.

Genome-wide transcriptomic analyses have revealed abundant expressed short open reading frames (ORFs) in bacteria. Whether these short ORFs, or the small proteins they encode, are functional remains an open question. One quarter of mycobacterial mRNAs are leaderless, beginning with a 5'-AUG or GUG initiation codon. Leaderless mRNAs often encode unannotated short ORFs as the first gene of a polycistronic transcript. Here, we show that polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression. Detailed mutational analysis shows that one polycysteine short ORF controls expression of the downstream genes. Our data indicate that ribosomes stalled in the polycysteine tract block mRNA structures that otherwise sequester the ribosome-binding site of the 3'gene. We assessed endogenous proteomic responses to cysteine limitation in Mycobacterium smegmatis using mass spectrometry. Six cysteine metabolic loci having unannotated polycysteine-encoding leaderless short ORF architectures responded to cysteine limitation, revealing widespread cysteine-responsive attenuation in mycobacteria. Individual leaderless short ORFs confer independent operon-level control, while their shared dependence on cysteine ensures a collective response mediated by ribosome pausing. We propose the term ribulon to classify ribosome-directed regulons. Regulon-level coordination by ribosomes on sensory short ORFs illustrates one utility of the many unannotated short ORFs expressed in bacterial genomes.

Quantifying and Characterizing Distributive Conjugal Transfer in Mycobacterium smegmatis.

Horizontal gene transfer (HGT) in prokaryotes disseminates genetic information throughout a population and can facilitate adaptation and evolution of the species. Mycobacteria utilize an atypical method of conjugation called distributive conjugal transfer (DCT), which results in mosaic genomes and the potential for accelerated evolution beyond that enabled by the more classical oriT-mediated conjugation. The following is a description of the basic DCT protocol, some possible variations of the assay, and examples of downstream applications to better understand mycobacterial functions.

Zinc depletion induces ribosome hibernation in mycobacteria.

Bacteria respond to zinc starvation by replacing ribosomal proteins that have the zinc-binding CXXC motif (C+) with their zinc-free (C-) paralogues. Consequences of this process beyond zinc homeostasis are unknown. Here, we show that the C- ribosome in Mycobacterium smegmatis is the exclusive target of a bacterial protein Y homolog, referred to as mycobacterial-specific protein Y (MPY), which binds to the decoding region of the 30S subunit, thereby inactivating the ribosome. MPY binding is dependent on another mycobacterial protein, MPY recruitment factor (MRF), which is induced on zinc depletion, and interacts with C- ribosomes. MPY binding confers structural stability to C- ribosomes, promoting survival of growth-arrested cells under zinc-limiting conditions. Binding of MPY also has direct influence on the dynamics of aminoglycoside-binding pockets of the C- ribosome to inhibit binding of these antibiotics. Together, our data suggest that zinc limitation leads to ribosome hibernation and aminoglycoside resistance in mycobacteria. Furthermore, our observation of the expression of the proteins of C- ribosomes in Mycobacterium tuberculosis in a mouse model of infection suggests that ribosome hibernation could be relevant in our understanding of persistence and drug tolerance of the pathogen encountered during chemotherapy of TB.

Direct cell-cell contact activates SigM to express the ESX-4 secretion system in Mycobacterium smegmatis.

Conjugal cell-cell contact between strains of Mycobacterium smegmatis induces the esxUT transcript, which encodes the putative primary substrates of the ESAT-6 secretion system 4 (ESX-4) secretion system. This recipient response was required for conjugal transfer of chromosomal DNA from the donor strain. Here we show that the extracytoplasmic σ factor, SigM, is a cell contact-dependent activator of ESX-4 expression and is required for conjugal transfer of DNA in the recipient strain. The SigM regulon includes genes outside the seven-gene core esx4 locus that we show are also required for conjugation, and we show that some of these SigM-induced proteins likely function through ESX-4. A fluorescent reporter revealed that SigM is specifically activated in recipient cells in direct contact with donor cells. Coculture RNA-seq experiments indicated that SigM regulon induction occurred early and before transconjugants are detected. This work supports a model wherein donor contact with the recipient cell surface inactivates the transmembrane anti-SigM, thereby releasing SigM. Free SigM induces an extended ESX-4 secretion system, resulting in changes that facilitate chromosomal transfer. The contact-dependent inactivation of an extracytoplasmic σ-factor that tightly controls ESX-4 activity suggests a mechanism dedicated to detect, and appropriately respond to, external stimuli from mycobacteria.

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

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.

Application of Distributive Conjugal DNA Transfer in Mycobacterium smegmatis To Establish a Genome-Wide Synthetic Genetic Array.

Genetic redundancy can obscure phenotypic effects of single-gene mutations. Two individual mutations may be viable separately but are lethal when combined, thus synthetically linking the two gene products in an essential process. Synthetic genetic arrays (SGAs), in which defined mutations are combined, provide a powerful approach to identify novel genetic interactions and redundant pathways. A genome-scale SGA can offer an initial assignment of function to hypothetical genes by uncovering interactions with known genes or pathways. Here, we take advantage of the chromosomal conjugation system of Mycobacterium smegmatis to combine individual donor and recipient mutations on a genome-wide scale. We demonstrated the feasibility of a high-throughput mycobacterial SGA (mSGA) screen by using mutants of esx3, fxbA, and recA as query genes, which were combined with an arrayed library of transposon mutants by conjugation. The mSGA identified interacting genes that we had predicted and, most importantly, identified novel interacting genes-encoding both proteins and a noncoding RNA (ncRNA). In combination with other molecular genetic approaches, the mSGA has great potential to both reduce the high number of conserved hypothetical protein annotations in mycobacterial genomes and further define mycobacterial pathways and gene interactions.IMPORTANCE Mycobacterium smegmatis is the model organism of choice for the study of mycobacterial pathogens, because it is a fast-growing nonpathogenic species harboring many genes that are conserved throughout mycobacteria. In this work, we describe a synthetic genetic array (mSGA) approach for M. smegmatis, which combines mutations on a genome-wide scale with high efficiency. Analysis of the double mutant strains enables the identification of interacting genes and pathways that are normally hidden by redundant biological pathways. The mSGA is a powerful genetic tool that enables functions to be assigned to the many conserved hypothetical genes found in all mycobacterial species.

Defining a temporal order of genetic requirements for development of mycobacterial biofilms.

Most mycobacterial species spontaneously form biofilms, inducing unique growth physiologies and reducing drug sensitivity. Biofilm growth progresses through three genetically programmed stages: substratum attachment, intercellular aggregation and architecture maturation. Growth of Mycobacterium smegmatis biofilms requires multiple factors including a chaperonin (GroEL1) and a nucleoid-associated protein (Lsr2), although how their activities are linked remains unclear. Here it is shown that Lsr2 participates in intercellular aggregation, but substratum attachment of Lsr2 mutants is unaffected, thereby genetically distinguishing these developmental stages. Further, a suppressor mutation in a glycopeptidolipid synthesis gene (mps) that results in hyperaggregation of cells and fully restores the form and functions of Δlsr2 mutant biofilms was identified. Suppression by the mps mutation is specific to Δlsr2; it does not rescue the maturation-deficient biofilms of a ΔgroEL1 mutant, thereby differentiating the process of aggregation from maturation. Gene expression analysis supports a stepwise process of maturation, highlighted by temporally separated, transient inductions of iron and nitrogen import genes. Furthermore, GroEL1 activity is required for induction of nitrogen, but not iron, import genes. Together, the findings begin to define molecular checkpoints during development of mycobacterial biofilms.

Intercellular communication and conjugation are mediated by ESX secretion systems in mycobacteria.

Communal bacterial processes require intercellular communication mediated by secretion systems to coordinate appropriate molecular responses. Intercellular communication has not been described previously in mycobacteria. Here we show that the ESX secretion-system family member ESX-4 is essential for conjugal recipient activity in Mycobacterium smegmatis Transcription of esx4 genes in the recipient requires coculture with a donor strain and a functional ESX-1 apparatus in the recipient. Conversely, mutation of the donor ESX-1 apparatus amplifies the esx4 transcriptional response in the recipient. The effect of ESX-1 on esx4 transcription correlates with conjugal DNA transfer efficiencies. Our data show that intercellular communication via ESX-1 controls the expression of its evolutionary progenitor, ESX-4, to promote conjugation between mycobacteria.

Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape.

RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5' untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5' end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5' ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5' UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.

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

The last 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 non-pathogens alike. Genomes within the Mycobacterium tuberculosis 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 Mycobacterium smegmatis actively conjugate upon co-culturing 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 oriT-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 GWAS-like approach to map the donor genes that confer a donor mating identity phenotype. The mating identity genes map to the esx1 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.

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

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 Mycobacterium tuberculosis 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 Mycobacterium smegmatis 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 oriT-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 esx1 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.

Distributive conjugal transfer in mycobacteria generates progeny with meiotic-like genome-wide mosaicism, allowing mapping of a mating identity locus.

Horizontal gene transfer (HGT) in bacteria generates variation and drives evolution, and conjugation is considered a major contributor as it can mediate transfer of large segments of DNA between strains and species. We previously described a novel form of chromosomal conjugation in mycobacteria that does not conform to classic oriT-based conjugation models, and whose potential evolutionary significance has not been evaluated. Here, we determined the genome sequences of 22 F1-generation transconjugants, providing the first genome-wide view of conjugal HGT in bacteria at the nucleotide level. Remarkably, mycobacterial recipients acquired multiple, large, unlinked segments of donor DNA, far exceeding expectations for any bacterial HGT event. Consequently, conjugal DNA transfer created extensive genome-wide mosaicism within individual transconjugants, which generated large-scale sibling diversity approaching that seen in meiotic recombination. We exploited these attributes to perform genome-wide mapping and introgression analyses to map a locus that determines conjugal mating identity in M. smegmatis. Distributive conjugal transfer offers a plausible mechanism for the predicted HGT events that created the genome mosaicism observed among extant Mycobacterium tuberculosis and Mycobacterium canettii species. Mycobacterial distributive conjugal transfer permits innovative genetic approaches to map phenotypic traits and confers the evolutionary benefits of sexual reproduction in an asexual organism.