Chapter 7 : Conjugation and Genetic Exchange in Enterococci

Expression of pCF10-encoded surface proteins observed by field emission scanning electron microscopy. The cells shown are from cultures of isogenic strains of E. faecalis carrying either the shuttle vector pWM402 (right panel) or pINY1801 (a chimeric plasmid containing the cloned prgA [surface exclusion protein, Sec10]) and prgB [aggregation substance, Asc10] genes of pCFlO cloned into pWM402). The latter plasmid confers constitutive expression of Asc10 and Sec10 on its host cell ( 29 ). The cells were preserved by a cryofixation procedure similar to that described by Erlandsen et al. ( 73 ). Note the smooth appearance of the bacterial cell surface (right panel) and surface display of the plasmid-encoded proteins as discrete entities with well-defined structural features. Micrographs were kindly supplied by Dr. Stanley Erlandsen, University of Minnesota.

Production, release, and sensing of enterococcal peptides at the cell surface. In the recipient cell shown at the left, the lipoprotein signal peptide is processed by Eep in the cell membrane and the mature pheromone is secreted to the exterior, where it can diffuse into the extracellular medium or remain associated with the cell wall. The plasinid-containing donor cell depicted on the right can bind exogenous pheromone via the plasmid-encoded, peptide-specific binding protein (PrgZ/TraC) and use the chromosomal-encoded Opp system to import the peptide. This cell retains the ability to produce pheromone; however, the plasmid-encoded regulatory protein (PrgY/TraB) in the membrane reduces the endogenous pheromone, thus blocking an autocrine loop where endogenous pheromone could be immediately bound and reinternalized by the producing cell. In addition, the inhibitor peptide is secreted into the growth medium where it serves to neutralize any pheromone released by the same cell. The level of inhibitor secreted seems to be just enough to neutralize any endogenous peptide, while still allowing for exquisite sensitivity to exogenous pheromone.

Comparison of pADl, pCF10, pPDl, and pAM373 in regions that include determinants that relate to regulation of the pheromone response. ORFs indicated by similar colors are homologues in the different plasmids.

Model showing the control circuitry for the pADl and pCFlO pheromone response. P0 is a primary promoter that is active to some extent even in the uninduced state, giving rise to m3 (for pADl) and Qs (for pCF10), which are terminated at t2/IRS1 (noted as 1 in the figure). Induction results in up-regulation from P0, which gives rise to significant amounts of m3* (for pADl) and QL (for pCF10), which are terminated at t2/IRS2 (noted as 2 in the figure). Induction also results in extension through t2/IRS2 and into regions that include determinants that positively regulate conjugation genes. The positive regulators appear to differ significantly for the two plasmids. The short component of the speckled transcript represents mD (for pADl) and Qa (for pCF10), which are expressed under the Pa promoter and, at least in the case of pADl, enhance termination at t1. Pa is also believed to govern expression of TraA/PrgX, which negatively regulates expression from P0 and is able to bind to pheromone. Expression from Pa is down-regulated upon induction.

Map of the conjugative transposon ?η916 showing ORFs believed to be related to conjugation. The transposon is approximately 18 kb in length. Apparent relationships with genes/ORFs in the database are noted.