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...

Succinyl-CoA:3-ketoacid CoA transferase (SCOT; EC 2.8.3.5) activates the acetoacetate in ketone bodies by transferring the CoA group from succinyl-CoA to acetoacetate to produce acetoacetyl-CoA and succinate. In the reaction, a glutamate residue at the active site of the enzyme forms a thioester bond with CoA and in this form the enzyme is subject to autolytic fragmentation. The crystal structure of pig heart SCOT has been solved and refined to 1.7 A resolution in a new crystal form. The structure shows the active-site glutamate residue in a conformation poised for autolytic fragmentation, with its side chain accepting one hydrogen bond from Asn281 and another from its own amide N atom. However, the conformation of this glutamate side chain would have to change for the residues that are conserved in the CoA transferases (Gln99, Gly386 and Ala387) to participate in stabilizing the tetrahedral transition states of the catalytic mechanism. The structures of a deletion mutant in two different crystal forms were also solved.

Surprisingly little is known about the role of host factors in regulating transposition, despite the potentially deleterious rearrangements caused by the movement of transposons. An extensive mutant screen was therefore conducted to identify Escherichia coli host factors that regulate transposition. An E. coli mutant library was screened using a papillation assay that allows detection of IS903 transposition events by the formation of blue papillae on a colony. Several host mutants were identified that exhibited a unique papillation pattern: a predominant ring of papillae just inside the edge of the colony, implying that transposition was triggered within these cells based on their spatial location within the colony. These mutants were found to be in pur genes, whose products are involved in the purine biosynthetic pathway. The transposition ring phenotype was also observed with Tn552, but not Tn10, establishing that this was not unique to IS903 and that it was not an artifact of the assay. Further genetic analyses of purine biosynthetic mutants indicated that the ring of transposition was consistent with a GTP requirement for IS903 and Tn552 transposition. Together, our observations suggest that transposition occurs during late stages of colony growth and that transposition occurs inside the colony edge in response to both a gradient of exogenous purines across the colony and the developmental stage of the cells.Transposons are defined as distinct regions of DNA that have the ability to move from one genomic location to another, a process mediated by element-encoded transposases (8, 9). While the main focus of transposition research has been in determining the detailed mechanisms of transposition, relatively little attention has been paid to how transposition is regulated in vivo or the in vivo requirements for the process (35). Transposon movement can result in gene inactivation (by insertional inactivation or deletion) or activation (by creation or introduction of promoters upstream of genes). In addition, intramolecular transposition can result in extensive DNA rearrangements, including deletions, inversions, and duplications of chromosome segments; these events are thought to play an important role in facilitating genome evolution (7-9). As the consequences of transposition can be either dire or favorable for the host organism, it is intuitive that the host has the capability to regulate this process. There are multiple examples of how transposons regulate their own movement, but very little work has focused on the role played by the host in regulating transposition. To address this, we have generated an insertion mutant library of Escherichia coli, which we have used to screen for host genes involved in regulation of IS903 transposition (E. Twiss, A. Coros, N. Tavakoli, and K. M. Derbyshire, unpublished data).The first comprehensive genetic screen for host mutants affecting transposition was carried out in 1985; it revealed a role for dam methylation in decreasing the rate of IS10, IS903, and IS50 transposi...

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