SummaryConjugal DNA transfer in Mycobacterium smegmatis occurs by a mechanism distinct from plasmidmediated 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.

SummaryThe 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 IS 903 , and the effect of these mutations on two additional transposons, Tn 10 and Tn 552 . Over 20 000 independent insertion mutants were screened in two strains of Escherichia coli ; from these we isolated over 100 mutants that altered IS 903 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 cell's 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.

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.Genetic exchange is thought to be a driving force behind the ability of bacterial species to evolve and adjust to environmental challenges. Lateral gene transfer (LGT) in bacteria is mediated by one of three processes, namely, conjugation, transformation, or transduction; examples of these processes have been described for almost all bacterial species (9,19). By contrast, the Mycobacterium tuberculosis complex (MTBC) species, comprising M. tuberculosis, M. africanum, M. bovis, M. cannetti, M. caprae, M. microti, and M. pinnipedi (10, 15), are clonal populations evolved from a single progenitor species that has diversified by the acquisition of spontaneous mutations rather than by LGT. Genome comparisons between these seven species show that they have almost identical 16S rRNA sequences and highly similar genome sequences, and there is no strong evidence for genetic exchange (Ͼ99% identity [2,3,16]). The lack of clearly documented LGT among members of the MTBC is thought to be a consequence of the organisms' solitary lifestyles within their hosts, preventing their contact with other mycobacterial species, or perhaps even other bacteria. Thus, it has become generally accepted within the scientific community that the MTBC species do not undergo genetic exchange (10,15,16).IS6110 is an insertion element that is found exclusively within the MTBC; the assumption has been that this restriction is a result of the lack of genetic exchange with other mycobacterial species. A benefit of this exclusivity is that IS6110 has become an important diagnostic tool in the differentiation of MTBC species from other mycobacteria. Moreover, the element's presence in multiple copies, and at differing locations in the genome, has provided an excellent method by which strains can be genotyped; because of these characteristics, IS6110 has been used extensively for epidemiological studies (12,18,20).Our studies have focused on DNA transfer between strains of M. smegmatis. This work has shown that DNA transfer occurs by a process most similar to conjugation: distinct donor and recipient strains exist and transconjugants are detected only after prolonged cell-cell contact (14,21,22). The transfer process is chromosomally encoded and can occur only from a donor to a recipient. The donor and recipient strains are independent isolates of M. smegmatis with distinct colony morphologies (13). The genetic basis for donor and recipient abilit...