Cloning and molecular analysis of genes affecting expression of binding substance, the recipient-encoded receptor(s) mediating mating aggregate formation in Enterococcus faecalis

Abstract

Transfer of the conjugative plasmid pCF10 in Enterococcus faecalis strains involves production of a plasmid-encoded aggregation substance on the surface of donor cells in response to stimulation by a pheromone secreted by recipient cells. Aggregation substance then facilitates attachment to recipient cells via a chromosomally encoded receptor, termed binding substance (BS). A BS mutant, strain INY3000, generated by random Tn916 insertions, was previously found to carry copies of the transposon at four unique sites (K. M. Trotter and G. M. Dunny, Plasmid 24:57-67, 1990). In the present study, DNA flanking the Tn916 insertions was used to complement the BS mutation of INY3000 following Tn916 excision from cloned chromosomal fragments. Complementation results showed that three of the four regions mutated in INY3000 play some role in BS expression. Tn5 mutagenesis and DNA sequence analysis of the complementing fragment from one of these regions indicated the presence of three genes (ebsA, ebsB, and ebsC) that affect BS expression. The ebsA and ebsB genes encode peptides likely to function in cell wall metabolism, whereas ebsC may encode a product that suppresses the function or expression of EbsB.

Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori

“…Since that time, however, the characterization of DHQases from several sources has thrown new light on the distribution of these two classes of enzyme, especially in prokaryotes. Type I DHQases have been identified in the shikimate pathway of both the Gramnegative organism Salmonella typi [9] and the Gram-positive organism Enterococcus faecalis [10], as well as in the inducible quinate pathway of the Gram-negative Acinetobacter calcoaceticus [49]. An even greater number of type II enzymes have been identified, primarily in the shikimate pathway of microorganisms.…”

“…Escherichia coli DHQase is monofunctional (encoded by the aroD gene) and has been cloned, sequenced and overexpressed [7]. Salmonella typhi [8,9], Enterococcus faecalis [10] and Bacillus subtilis [11] all possess monofunctional DHQases. In plants such as Physcomitrella patens [12], Nicotiana tabacum [13] and Pisum sati um [14], DHQase is part of a bifunctional polypeptide that also carries shikimate dehydrogenase activity, the next enzyme in the pathway.…”

Pheromone‐inducible expression of an aggregation protein in Enterococcus faecalis requires interaction of a plasmid‐encoded RNA with components of the ribosome

“…E. faecalis strain OG1RF (Dunny et al, 1981) was used as the host strain for all plasmids, and was transformed by electroporation as described (Bensing and Dunny, 1993).…”

Peptides and ATP binding cassette peptide transporters

“…Asc10 contributes to the formation of a stable mating complex between donor and recipient cells and establishes an efficient transfer of the conjugative plasmid [6].…”