LpqM, a Mycobacterial Lipoprotein-Metalloproteinase, Is Required for Conjugal DNA Transfer inMycobacterium smegmatis

Abstract

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...

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…LpqM probably does not contribute directly to DNA transfer but instead might generate extracellular signal molecules through the proteolysis of proteins released by the Esx-1 or Tad systems (200). These peptide signals could in turn induce the expression of the DNA processing and transfer genes, reminiscent of the pheromone-inducible plasmid transfer systems of gram-positive bacteria.…”

Enhanced Priming of Adaptive Immunity by Mycobacterium smegmatis Mutants with High-Level Protein Secretion

“…Six independent transposon insertions in lpqM were responsible for higher levels of secretion of transgene products (PhoA and LpqH). LpqM, a putative lipoprotein-metalloproteinase, has been shown to be essential for efficient DNA transfer by conjugation (36). LpqM is a membraneassociated protein and requires an intact signal and lipobox for membrane localization.…”

Mycobacterial Biofilms Facilitate Horizontal DNA Transfer between Strains ofMycobacterium smegmatis

“…A mutant transposon insertion library was constructed in mc 2 155⌬esx-1 (29) by using a temperature-sensitive phage to deliver a mariner transposon encoding kanamycin resistance (Km r ) (3,39). Individual donor insertion mutants were screened for defects in conjugation by using a microtiter mating assay, and insertions were mapped as described previously (29). Briefly, chromosomal BamHI fragments encoding resistance to Km r were cloned, and the site of insertion was determined by DNA sequence analysis using primers that annealed to the Km r gene.…”

“…We have recently described a donor-defective transfer mutation in a gene encoding a membrane-localized lipoprotein called LpqM (29). Transfer from lpqM mutants is typically reduced Ͼ500-fold in plate matings.…”

“…We have exploited the hyperconjugative phenotype of esx-1 mutants so as to increase the sensitivity of a genetic screen for transfer-defective mutants (29). This strategy resulted in the identification of lsr2 as being important for DNA transfer in the donor and led us to investigate the dependence of conjugation on biofilm formation.…”