Proteins PblA and PblB of Streptococcus mitis, which promote binding to human platelets, are encoded within a lysogenic bacteriophage.

The binding of platelets by bacteria is a proposed central mechanism in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis strain SF100 (an endocarditis isolate) was recently shown to be mediated in part by the surface proteins PblA and PblB. The genes encoding PblA and PblB are clustered with genes nearly identical to those of streptococcal phages r1t, 01205, and Dp-1, suggesting that pblA and pblB might reside within a prophage. To address this possibility, cultures of SF100 were exposed to either mitomycin C or UV light, both of which are known to induce the lytic cycle of many temperate phages. Both treatments caused a significant increase in the transcription of pblA. Treatment with mitomycin C or UV light also caused a substantial increase in the expression of PblA and PblB, as detected by Western blot analysis of proteins in the SF100 cell wall. By electron microscopy, phage particles were readily visible in the supernatants from induced cultures of SF100. The phage, designated SM1, had a double-stranded DNA genome of approximately 35 kb. Southern blot analysis of phage DNA indicated that pblA and pblB were contained within the SM1 genome. Furthermore, Western blot analysis of phage proteins revealed that both PblA and PblB were present in the phage particles. These findings indicate that PblA and PblB are encoded by a lysogenic bacteriophage, which could facilitate the dissemination of these potential virulence determinants to other bacterial pathogens.

Genetic loci of Streptococcus mitis that mediate binding to human platelets.

The direct binding of bacteria to platelets is a postulated major interaction in the pathogenesis of infective endocarditis. To identify bacterial components that mediate platelet binding by Streptococcus mitis, we screened a Tn916deltaE-derived mutant library of S. mitis strain SF100 for reduced binding to human platelets in vitro. Two distinct loci were found to affect platelet binding. The first contains a gene (pblT) encoding a highly hydrophobic, 43-kDa protein with 12 potential membrane-spanning segments. This protein resembles members of the major facilitator superfamily of small-molecule transporters. The second platelet binding locus consists of an apparent polycistronic operon. This region includes genes that are highly similar to those of Lactococcus lactis phage r1t and Streptococcus thermophilus phage 01205. Two genes (pblA and pblB) encoding large surface proteins are also present. The former encodes a 107-kDa protein containing tryptophan-rich repeats, which may serve to anchor the protein within the cell wall. The latter encodes a 121-kDa protein most similar to a tail fiber protein from phage 01205. Functional mapping by insertion-duplication mutagenesis and gene complementation indicates that PblB may be a platelet adhesin and that expression of PblB may be linked to that of PblA. The combined data indicate that at least two genomic regions contribute to platelet binding by S. mitis. One encodes a probable transmembrane transporter, while the second encodes two large surface proteins resembling structural components of lysogenic phages.

Pheromone cCF10 and plasmid pCF10-encoded regulatory molecules act post-transcriptionally to activate expression of downstream conjugation functions.

Expression of aggregation protein Asc10 from the prgB gene of conjugative plasmid pCF10 in Enterococcus faecalis is induced by the peptide pheromone cCF10. Genes required for Asc10 production, prgQ and prgS, lie 3-5 kb upstream, but can function at much greater distances. The prgQ transcripts encode a pheromone inhibitor peptide (iCF10) at the extreme 5' end. Neither production of this peptide nor translation of the 5' end of prgQ transcripts was found to be necessary for prgB expression. Pheromone cCF10 is required to activate prgB expression, even in the absence of iCF10 production, and does not affect initiation of transcription. The prgS gene encodes a 10.5 kDa protein that appears to be required for translation of prgB, and a non-coding RNA at the 3' end of prgS may be required for readthrough of transcription to prgB from the prgQ promoter. Although the entire positive control region is transcribed constitutively from the prgQ promoter, translation of PrgS and transcriptional readthrough to prgB occur only after induction with pheromone. The combined data are consistent with a model in which the positive regulatory molecules and pheromone cCF10 activate prgB expression post-transcriptionally.

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

Transfer of the conjugative plasmid pCF10 from Enterococcus faecalis donor strains is induced by a peptide pheromone (cCF10) secreted by recipient cells. High-efficiency transfer requires expression of an aggregation protein (Asc10) encoded by the prgB gene and positively regulated by genes in a region 3-5 kb upstream, containing prgQ-R-S. Transcriptional fusion data reported here support the results of recent molecular analysis of the 5' ends of prgB transcripts which indicated that prgB transcription occurs by readthrough from the prgQ promoter. A 530-nucleotide prgQ-encoded RNA molecule (Q(L)) with rRNA-like domains is required for Asc10 production. Q(L) and cCF10 were found to interact with the L6 and S5 ribosomal proteins, respectively. Mutational analysis of Q(L) indicates that this RNA may also directly interact with 16S RNA. Q(L) is present in ribosomes translating the prgB message, and pheromone cCF10 may affect the association of this RNA with translation complexes. Results suggest that the positive regulatory molecules act post-transcriptionally on the polycistronic message and modify a ribosome population to enhance pheromone-induced translation of prgB.

Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis.

A method was developed to detect 5' ends of bacterial RNAs expressed at low levels and to differentiate newly initiated transcripts from processed transcripts produced in vivo. The procedure involves use of RNA ligase to link a specific oligoribonucleotide to the 5' ends of cellular RNAs, followed by production of cDNA and amplification of the gene of interest by PCR. The method was used to identify the precise sites of transcription initiation within a 10-kb region of the pheromone-inducible conjugative plasmid pCF10 of Enterococcus faecalis. Results confirmed the 5' end of a very abundant, constitutively produced transcript (from prgQ) that had been mapped previously by primer extension and defined the initiation point of a less abundant, divergently transcribed message (from prgX). The method also showed that the 5' end of a pheromone-inducible transcript (prgB) that had been mapped by primer extension was generated by processing rather than new initiation. In addition, the results provided evidence for two promoters, 3 and 5 kb upstream of prgB, and indicated that only the transcripts originating 5 kb upstream may be capable of extending to prgB.

Genetic analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions.

The prgB gene encodes the surface protein Asc10, which mediates cell aggregation resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline resistance plasmid pCF10 in Enterococcus faecalis. Previous Tn5 insertional mutagenesis and sequencing analysis of a 12-kb fragment of pCF10 indicated that a region containing prgX, -Q, -R, -S, and -T, located 3 to 6 kb upstream of prgB, is required to activate the expression of prgB. Complementation studies showed that the positive regulatory region functions in cis in an orientation-dependent manner (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). In order to determine the involvement of each gene in the activation of prgB, Tn5 insertional mutagenesis and exonuclease III deletion analyses of the regulatory region were carried out. The results indicate that prgQ and -S are required for the expression of prgB, while prgX, -R, and -T are not required. Western blot (immunoblot) analysis of these mutants shows that prgQ is also essential for the expression of prgA (encoding the surface exclusion protein Sec10), which is located between prgB and the positive-control region. Complementation analysis demonstrates that a cis-acting regulatory element is located in the prgQ region and that pCF10 sequences in an untranslated region 3' from prgQ are an essential component of the positive-control system. Analyses of various Tn5 insertions in pCF10 genes suggest that transcription reading into this transposon is terminated in E. faecalis but that outward-reading transcripts may initiate from within the ends of Tn5 or from the junction sequences.

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

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.