Keith M. Derbyshire

The outs and ins of transposition: from Mu to Kangaroo

Transposons are ubiquitous in prokaryotic and eukaryotic organisms and are major determinants of genome structure. Transposition — the movement of discrete segments of DNA without a requirement for homology — occurs by a handful of mechanisms that are used over and over again in different combinations. Understanding these mechanisms provides an important key to unlocking the secrets of genome organization and evolution.

Systematic Genetic Nomenclature for Type VII Secretion Systems

CITATION: Bitter, W., et al. 2009. Systematic genetic nomenclature for type VII secretion systems. PLoS Pathogens, 5(10): 1-6, doi: 10.1371/journal.ppat.1000507.

The Specialized secretory apparatus ESX-1 is essential for DNA transfer in Mycobacterium smegmatis

Conjugal DNA transfer in Mycobacterium smegmatis occurs by a mechanism distinct from plasmid‐mediated DNA transfer. Previously, we had shown that the secretory apparatus, ESX‐1, negatively regulated DNA transfer from the donor strain; ESX‐1 donor mutants are hyper‐conjugative. Here, we describe a genome‐wide transposon mutagenesis screen to isolate recipient mutants. Surprisingly, we find that a majority of insertions map within the esx‐1 locus, which encodes the secretory apparatus. Thus, in contrast to its role in donor function, ESX‐1 is essential for recipient function; recipient ESX‐1 mutants are hypo‐conjugative. In addition to esx‐1 genes, our screen identifies novel non‐esx‐1 loci in the M. smegmatis genome that are required for both DNA transfer and ESX‐1 activity. DNA transfer therefore provides a simple molecular genetic assay to characterize ESX‐1, which, in Mycobacterium tuberculosis, is necessary for full virulence. These findings reinforce the functional intertwining of DNA transfer and ESX‐1 secretion, first described in the M. smegmatis donor. Moreover, our observation that ESX‐1 has such diametrically opposed effects on transfer in the donor and recipient, forces us to consider how proteins secreted by the ESX‐1 apparatus can function so as to modulate two seemingly disparate processes, M. smegmatis DNA transfer and M. tuberculosis virulence.

Transposition is modulated by a diverse set of host factors in Escherichia coli and is stimulated by nutritional stress

The role of host factors in regulating bacterial transposition has never been comprehensively addressed, despite the potential consequences of transposition. Here, we describe a screen for host factors that influence transposition of IS903, and the effect of these mutations on two additional transposons, Tn10 and Tn552. Over 20 000 independent insertion mutants were screened in two strains of Escherichia coli; from these we isolated over 100 mutants that altered IS903 transposition. These included mutations that increased or decreased the extent of transposition and also altered the timing of transposition during colony growth. The large number of gene products affecting transposition, and their diverse functions, indicate that the overall process of transposition is modulated at many different steps and by a range of processes. Previous work has suggested that transposition is triggered by cellular stress. We describe two independent mutations that are in a gene required for fermentative metabolism during anaerobic growth, and that cause transposition to occur earlier than normal during colony development. The ability to suppress this phenotype by the addition of fumarate therefore provides direct evidence that transposition occurs in response to nutritional stress. Other mutations that altered transposition disrupted genes normally associated with DNA metabolism, intermediary metabolism, transport, cellular redox, protein folding and proteolysis and together these define a network of host proteins that could potentially allow readout of the cells environmental and nutritional status. In summary, this work identifies a collection of proteins that allow the host to modulate transposition in response to cell stress.

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

We find that genome-wide DNA transfer by conjugation in mycobacteria affords bacteria that reproduce by binary fission the same advantages of sexual reproduction, and may explain the genomic evolution of Mycobacterium tuberculosis.

IS6110, a Mycobacterium tuberculosis Complex-Specific Insertion Sequence, Is Also Present in the Genome of Mycobacterium smegmatis, Suggestive of Lateral Gene Transfer among Mycobacterial Species

IS6110 is an insertion element found exclusively within the members of the Mycobacterium tuberculosis complex (MTBC), and because of this exclusivity, it has become an important diagnostic tool in the identification of MTBC species. The restriction of IS6110 to the MTBC is hypothesized to arise from the inability of these bacteria to exchange DNA. We have identified an IS6110-related element in a strain of Mycobacterium smegmatis. The presence of IS6110 indicates that lateral gene transfer has occurred among mycobacterial species, suggesting that the mycobacterial gene pool is larger than previously suspected.

Mycobacterial Biofilms Facilitate Horizontal DNA Transfer between Strains of Mycobacterium smegmatis

Conjugal transfer of chromosomal DNA between strains of Mycobacterium smegmatis occurs by a novel mechanism. In a transposon mutagenesis screen, three transfer-defective insertions were mapped to the lsr2 gene of the donor strain mc(2)155. Because lsr2 encodes a nonspecific DNA-binding protein, mutations of lsr2 give rise to a variety of phenotypes, including an inability to form biofilms. In this study, we show that efficient DNA transfer between strains of M. smegmatis occurs in a mixed biofilm and that the process requires expression of lsr2 in the donor but not in the recipient strain. Testing cells from different strata of standing cultures showed that transfer occurred predominantly at the biofilm air-liquid interface, as other strata containing higher cell densities produced very few transconjugants. These data suggest that the biofilm plays a role beyond mere facilitation of cell-cell contact. Surprisingly, we found that under standard assay conditions the recipient strain does not form a biofilm. Taking these results together, we conclude that for transfer to occur, the recipient strain is actively recruited into the biofilm. In support of this idea, we show that donor and recipient cells are present in almost equal numbers in biofilms that produce transconjugants. Our demonstration of genetic exchange between mycobacteria in a mixed biofilm suggests that conjugation occurs in the environment. Since biofilms are considered to be the predominant natural microhabitat for bacteria, our finding emphasizes the importance of studying biological and physical processes that occur between cells in mixed biofilms.

Unconventional conjugal DNA transfer in mycobacteria

Bacterial conjugation is an active process that results in unidirectional transfer of DNA from a donor to a recipient cell. Most transfer systems are plasmid-encoded and require proteins to act at a unique cis-acting site to initiate and complete DNA transfer. By contrast, the Mycobacterium smegmatis DNA transfer system is chromosomally encoded. Here we show that multiple cis-acting sequences present on the chromosome can mediate transfer of a non-mobilizable test plasmid. Moreover, unlike conventional plasmid transfer, recipient recombination functions are required to allow this plasmid, and derivatives of it, to re-circularize through a process similar to gap repair. Extended DNA homology with the recipient chromosome is required to facilitate repair, resulting in acquisition of recipient chromosomal DNA by the plasmid. Together, these results show that DNA transfer in M. smegmatis occurs by a mechanism different from that of prototypical plasmid transfer systems.

Chromosomal DNA transfer in Mycobacterium smegmatis is mechanistically different from classical Hfr chromosomal DNA transfer

Classical conjugal DNA transfer of chromosomal DNA in bacteria requires the presence of a cis‐acting site, oriT, in the chromosome. Acquisition of an oriT occurs if a conjugative plasmid integrates into the chromosome to form an Hfr donor strain, which can transfer extensive regions of chromosomal DNA. Because oriT sequences are unique, and because transfer occurs in a 5′ to 3′ direction, the frequency with which a particular gene is inherited by the recipient depends on the genes location: those closest to the 3′ side of oriT are transferred most efficiently. In addition, as the entire chromosome must be transferred to regenerate oriT, Hfr transconjugants never become donors. Here we describe novel aspects of a chromosomal DNA transfer system in Mycobacterium smegmatis. We demonstrate that there are multiple transfer initiations from a donor chromosome and, as a result, the inheritance of any gene is location‐independent. Transfer is not contiguous; instead, multiple non‐linked segments of DNA can be inherited in a recipient. However, we show that, with appropriate selection, segments of DNA at least 266 kb in length can be transferred. In further contrast to Hfr transfer, transconjugants can become donors, suggesting that the recipient chromosome contains multiple cis‐acting sequences required for transfer, but lacks the trans‐acting transfer functions. We exploit these observations to map a donor‐determining locus in the M. smegmatis chromosome using genetic linkage analysis. Together, these studies further underline the unique nature of the M. smegmatis chromosomal transfer system.