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.Biofilms are dynamic communities of microorganisms that form on surfaces or at air-liquid interfaces (17,20,41). They arise following the attachment of bacteria to a surface; the bacteria then grow, differentiate, and multiply. The colonizing bacteria produce extracellular polymers, which encapsulate the cells and trap particulate matter, nutrients, and other bacteria that in turn contribute to the further development of the biofilm. Thus, as the biofilm develops it becomes increasingly heterogeneous. Microbial life is thought to exist predominantly in a biofilm, and biofilms can have either beneficial or harmful impacts on their environments (23). From a medical standpoint, biofilms can create serious problems. Bacteria within a biofilm are inherently more resistant to antibiotics, which makes their eradication difficult and is particularly problematic for patients with surgical implants resulting in chronic infections (19,33).Mycobacteria are known to form biofilms; however, relatively little is known about the mechanism of biofilm formation and development or its role in the biology of Mycobacterium species. For practical reasons, most biofilm studies have focused on the more rapidly growing and less pathogenic species, namely, Mycobacterium fortuitum, M. marinum, and M. smegmatis (16, 18, 36). In particular, genetic studies of M. smegmatis have provided insight into some of the key factors required f...
We have previously described a novel conjugal DNA transfer process that occurs in Mycobacterium smegmatis. To identify donor genes required for transfer, we have performed a transposon mutagenesis screen; we report here that LpqM, a putative lipoprotein-metalloproteinase, is essential for efficient DNA transfer. Bioinformatic analyses predict that LpqM contains a signal peptide necessary for the protein's targeting to the cell envelope and a metal ion binding motif, the likely catalytic site for protease activity. Using targeted mutagenesis, we demonstrate that each of these motifs is necessary for DNA transfer and that LpqM is located in the cell envelope. The requirement for transfer is specific to the donor strain; an lpqM knockout mutant in the recipient is still proficient in transfer assays. The activity of LpqM is conserved among mycobacteria; homologues from both Mycobacterium tuberculosis and Mycobacterium avium can complement lpqM donor mutants, suggesting that the homologues recognize and process similar proteins. Lipoproteins constitute a significant proportion of the mycobacterial cell wall, but despite their abundance, very few have been assigned an activity. We discuss the potential role of LpqM in DNA transfer and the implications of the conservation of LpqM activity in M. tuberculosis.In previous work, we have described a novel conjugation system in Mycobacterium smegmatis (15). Although this process meets the criteria of conjugation (successful transfer requires prolonged cell-cell contact and is DNase resistant), the mechanism of transfer is unique (27)(28)(29). Transfer is chromosomally encoded, and despite exhaustive bioinformatics searches, we have yet to identify any genes encoding obvious transfer functions. A comprehensive transposon mutagenesis screen of the donor strain failed to identify transfer-defective mutants. Instead, the screen identified hyperconjugative mutants that mapped to a large, 30-kb locus, esx-1 (7). esx-1 encodes a secretory apparatus, ESX-1, and we hypothesized that the secretion of proteins by ESX-1 negatively regulates transfer, either because the secreted proteins physically block transfer or because they act as intercellular quorum sensors. In a more recent study of the M. smegmatis recipient strain, we showed that a functional ESX-1 apparatus is essential for recipient activity (4). Recipient activity required secretion of at least two esx-1-encoded proteins, EsxA and EsxB. We have therefore proposed that in the recipient, as in the donor, ESX-1 is secreting proteins that regulate, rather than mediate, DNA transfer. The ESX-1 apparatus is highly conserved, and esx-1-encoded mutants of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium marinum are attenuated (reviewed in references 1 and 6). Proteins known to be secreted by ESX-1 from M. tuberculosis include EsxA and EsxB (formerly known as ESAT-6 and CFP10, respectively), EspA, and Rv3881 (10,12,17,25). Further underscoring the functional involvement of ESX-1 in both conjugation and virulence, o...
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