Thursday, November 21, 2024
11/21/2024
Distributive Conjugal Transfer (DCT) is a unique conjugation process, first described in Mycobacterium smegmatis, which is so robust that it emulates the genome mixing capacity of sexual reproduction: transconjugant genomes are mosaic blends of each parent. Thus, DCT is a new paradigm of conjugation, evolution, and horizontal gene transfer (HGT)-mediated gene flow within a bacterial gene pool. Our long- term goals are to elucidate the mechanism of DCT and understand its role in driving mycobacterial evolution. We recently defined a three-gene mating identity locus (mid) as critical for donor-recipient compatibility. Remarkably, one gene within the locus, midA, confers mycobacterial self-identity (kin). If the identical midA gene is expressed in both donor and recipient, DCT is prevented as the cells sense kin. The focus of this proposal is to characterize this novel form of kin recognition in mycobacteria, and to determine how it triggers/blocks DCT. Sequence comparisons of the three mid proteins in other mycobacteria shows that they are highly polymorphic. We hypothesize that this sequence diversity is driven by the need to express self-identity. MidA is predicted to have a small N-terminal cytoplasmic domain and a large extracellular C-terminal domain. We propose a model in which direct cell contact between donor and recipient would allow the interaction of the surface exposed MidA proteins to sense kin. The N-terminal cytoplasmic domain would trigger a transcriptional response in the recipient that either activates (non- kin) or prevents (kin) progression to conjugation. The two Aims are designed to characterize the mycobacterial kin recognition protein(s), determine how mid discriminates between contacting mycobacteria, and elucidate the molecular genetic pathways triggered by mid. Aim1. Define and characterize the mid locus and the encoded kin functions. Aim 2. Identify and characterize the genes responsible for kin recognition in donor and recipient strains by transcriptional profiling wild-type and mutant strains in co-culture. These objectives will be achieved by employing a combination of molecular genetic, genomic, and biochemical approaches to dissect the mid locus, and transcriptional profiling of donor and recipient strains in coculture to determine the transcriptional responses to kin recognition and DCT. Together, these studies will elucidate the molecular basis of kin recognition in mycobacteria and provide further mechanistic insights on this novel mechanism of HGT.
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