Recipient strains of Streptococcus faecalis produce a trypsin sensitive, heat resistant, nuclease resistant factor, designated clumping-inducing agent (CIA) which causes strains carrying certain conjugative plasmids to aggregate. RNA and protein synthesis but not DNA synthesis are required for aggregation to occur. Recipient filtrates that contain CIA activity also induce donors to mate at high frequencies. Introduction of a transferable plasmid into strains producing CIA dramatically reduces the amount of CIA activity produced by the strain but allows the strain to respond to exogenously added CIA. Our data suggest that CIA represents a bacterial sex hormone (pheromone).

In gram-positive bacteria, many important processes are controlled by cell-to-cell communication, which is mediated by extracellular signal molecules produced by the bacteria. Most of these signaling molecules are peptides or modified peptides. Signal processing, in most cases, involves either transduction across the cytoplasmic membrane or import of the signal and subsequent interaction with intracellular effectors. Concentrations of signal in the nanomolar range or below are frequently sufficient for biological activity. The microbial processes controlled by extracellular signaling include the expression of virulence factors, the expression of gene transfer functions, and the production of antibiotics.

In a previous report (1978, Proc. Nat. Acad. Sci. USA 75, 3479-3483), we showed that recipient strains of Streptococcusfaecalis excrete a heat-stable substance (sex pheromone) which induces donor cells carrying certain conjugative plasmids to become adherent, generating the cell-to-cell contact necessary for plasmid transfer. Since donors themselves could be induced to aggregate or "clump" by recipient filtrates, the substance was referred to as "clumping-inducing agent" (CIA). In this report, we present a simplified assay for CIA and determine the level of activity in filtrates prepared at various stages of growth. We also present evidence that recipient cells produce multiple pheromones, each specific for donors harboring a particular class of plasmids. Whereas a recipient that acquires a conjugative plasmid no longer produces the corresponding CIA, it still produces CIAs specific for donors with different conjugative plasmids. In addition, an analysis of 100 clinical isolates of S. faecalis showed that drug-resistant strains are significantly more likely to respond to and produce CIA activities than drug-sensitive strains. A model is discussed describing the relationships of sex pheromones to the mating process.

The Lactococcus lactis group II intron Ll.ltrB is similar to mobile yeast mtDNA group II introns, which encode reverse transcriptase, RNA maturase, and DNA endonuclease activities for site-specific DNA insertion. Here, we show that the Lactococcal intron can be expressed and spliced efficiently in Escherichia coli. The intron-encoded protein LtrA has reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II intron-encoded protein. As for the yeast mtDNA introns, the DNA endonuclease activity of the Lactococcal intron is associated with RNP particles containing both the intron-encoded protein and the excised intron RNA. Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cleaves the antisense strand. The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA in E. coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA. Furthermore, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component. Expression in E. coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.

Enterococcus faecalis is a Gram-positive commensal bacterium of the gastrointestinal tract. E. faecalis is also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite the clinical significance of the enterococci, genetic analysis has been restricted by limitations inherent in the available genetic tools. To facilitate genetic manipulation of E. faecalis, we developed a conjugative delivery system for high-frequency introduction of cloned DNA into target strains of E. faecalis and a host-genotype-independent counterselectable marker for use in markerless genetic exchange. We used these tools to construct a collection of E. faecalis mutant strains carrying defined mutations in several genes, including ccfA, eep, gelE, sprE, and an alternative sigma factor (sigH). Furthermore, we combined these mutations in various permutations to create double mutants, triple mutants, and a quadruple mutant of E. faecalis that enabled tests of epistasis to be conducted on the pheromone biosynthesis pathway. Analysis of cCF10 pheromone production by the mutants revealed that both the ccfA2 and Δeep10 mutations are epistatic to mutations in gelE/sprE. To our knowledge, this represents the first example of epistasis analysis applied to a chromosomally encoded biosynthetic pathway in enterococci. Thus, the advanced tools for genetic manipulation of E. faecalis reported here enable efficient and sophisticated genetic analysis of these important pathogens.