The sex pheromone plasmids in Enterococcus faecalis are one of the most efficient conjugative plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed 10 ؊1 transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of plasmid pCF10 and the analysis of the transcription profile of plasmid genes, relative to conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by the prgB gene) was present on the cell surface for a long period of time after pheromone-induced transcription of prgB and plasmid transfer ability had ceased. This observation could have relevance for the virulence of E. faecalis.The advent of microarray technology allows for a comprehensive analysis of gene expression patterns associated with various biological processes, providing insights into complex regulatory networks. One of the most complex processes is the transfer of large portions of genetic material from a donor cell into a recipient cell by means of conjugation. The plasmids of the sex pheromone family in Enterococcus faecalis are among the most efficient bacterial conjugation systems known (16). The family consists of over 20 plasmids and shows extensive sequence homologies (28). E. faecalis strains can host several of these plasmids. This is exemplified by strain V583, the first vancomycin-resistant isolate in the United States (45), chosen for genome sequencing by The Institute for Genomic Research (TIGR; www.tigr.org). V583 contains two sex pheromone plasmids with homology to the well-characterized pAD1 (pTEF1) and pCF10 (pTEF2) plasmids, respectively. The complete sequences for the pheromone plasmids pAD1 and pAM373 became available recently (14,19). Analysis of the sequences of this group of plasmids allows comparisons and insights into the evolution of these elements.Although the sex pheromone plasmids can be disseminated among enterococcal populations very efficiently, plasmid transfer is highly regulated and only induced by recipient cells in close proximity to plasmid donors. The recipient cells secret 7-to 8-amino-acid-long hydrophobic sex pheromones that are bound by a plasmid-encoded binding protein (44, 51). The pheromone is then taken up into the cell (32) and releases a transcriptional block of the PrgX/TraA family of repressors (5). One of the early transcripts after induction encodes for the surface protein aggregation substance (AS) (9). Expression of AS results in tight physical contact between donor and recipient, allows for plasmid transfer rates of up to 10 Ϫ1 transconjugants/donor (16), and is nece...

SummaryThe DNA-processing region of the Enterococcus faecalis pheromone-responsive plasmid pCF10 is highly similar to that of the otherwise unrelated plasmid pRS01 from Lactococcus lactis. A transferproficient pRS01 derivative was unable to mobilize plasmids containing the pCF10 origin of transfer, oriT. In contrast, pRS01 oriT-containing plasmids could be mobilized by pCF10 at a low frequency. Relaxases PcfG and LtrB were both capable of binding to single-stranded oriT DNAs; LtrB was highly specific for its cognate oriT, whereas PcfG could recognize both pCF10 and pRS01 oriT. However, pcfG was unable to complement an ltrB insertion mutation. Genetic analysis showed that pcfF of pCF10 and ltrF of pRS01 are also essential for plasmid transfer. Purified PcfF and LtrF possess double-stranded DNA binding activities for the inverted repeat within either oriT sequence. PcfG and LtrB were recruited into their cognate F-oriT DNA complex through direct interactions with their cognate accessory protein. PcfG also could interact with LtrF when pCF10 oriT was present. In vivo cross-complementation analysis showed that ltrF partially restored the pCF10DpcfF mutant transfer ability when provided in trans, whereas pcfF failed to complement an ltrF mutation. Specificity of conjugative DNA processing in these plasmids involves both DNA-protein and protein-protein interactions.

The lactococcal group II intron Ll.ltrB interrupts the ltrB relaxase gene within a region that encodes a conserved functional domain. Nucleotides essential for the homing of Ll.ltrB into an intronless version of ltrB are found exclusively at positions required to encode amino acids broadly conserved in a family of relaxase proteins of gram-positive bacteria. Two of these relaxase genes, pcfG from the enterococcal plasmid pCF10 and the ORF4 gene in the streptococcal conjugative transposon Tn5252, were shown to support Ll.ltrB insertion into the conserved motif at precisely the site predicted by sequence homology with ltrB. Insertion occurred through a mechanism indistinguishable from retrohoming. Splicing and retention of conjugative function was demonstrated for pCF10 derivatives containing intron insertions. Ll.ltrB targeting of a conserved motif of a conjugative element suggests a mechanism for group II intron dispersal among bacteria. Additional support for this mechanism comes from sequence analysis of the insertion sites of the E.c.I4 family of bacterial group II introns.Group II introns are self-splicing and mobile RNAs (14) encoded within the genomes of bacteria (27) and in the organelle DNAs of plants, fungi, and protists (3). These RNAs have a conserved secondary structure with six sets of basepairing regions, domains I through VI, arranged radially around a central circle (29). Much of domain IV is looped out of the secondary structure and determines the synthesis of an intron-encoded protein (IEP) with maturase, endonuclease, and reverse transcriptase (RT) activities (28, 33). Maturase assists in splicing, probably by enhancing the formation or stabilization of a catalytic ribozyme structure which mediates splicing (28) via a mechanism very similar to that of eukaryotic spliceosomal introns (38).Group II introns home at high frequency into intronless alleles of the same exon gene and transpose at low frequency into ectopic sites with sequence similarity to the natural site (26). Since group II intron mobility occurs through an RNA intermediate, the terms retrohoming and retrotransposition are used to designate these events (9, 21). During retrohoming, the IEP, which is associated with the spliced-out intron RNA, recognizes nucleotides in the distal 5Ј portion of the DNA target and EBS/␦ ribonucleotides in the intron base pair with IBS/␦Ј nucleotides in the DNA target site. This process is followed by reverse splicing of the intron RNA into target DNA (20,28,34,40). The IEP then interacts with the 3Ј portion of the DNA target, and the IEP endonuclease nicks the antisense strand of recipient DNA a few base pairs downstream of the insertion site (20,28). Using the 3Ј end of the nicked target DNA as a primer, IEP RT reverse transcribes the intron RNA via a mechanism reminiscent of non-long terminal repeat retrotransposon target DNA-primed reverse transcription (14, 28). In retrohoming by yeast mitochondrial group II introns aI1 and aI2, completion of the insertion event generally occurs by double-strand...

Conjugation is a major contributor to lateral gene transfer in bacteria, and pheromone-inducible conjugation systems in Enterococcus faecalis play an important role in the dissemination of antibiotic resistance and virulence in enterococci and related bacteria. We have genetically dissected the determinants of DNA processing of the enterococcal conjugative plasmid pCF10. Insertional inactivation of a predicted relaxase gene pcfG, via insertion of a splicing-deficient group II intron, severely reduced pCF10 transfer. Restoration of intron splicing ability by genetic complementation restored conjugation. The pCF10 origin of transfer (oriT) was localized to a 40-nucleotide sequence within a non-coding region with sequence similarity to origins of transfer of several other plasmids in gram positive bacteria. Deletion of the oriT reduced pCF10 transfer by more than five orders of magnitude without affecting pCF10-dependent mobilization of co-resident oriT-containing plasmids. Although the host range for pCF10 replication is limited to enterococci, we found that the pCF10 conjugation system promotes mobilization of oriT-containing plasmids to multiple bacterial genera. Therefore, this transfer system may have applications for gene delivery to a variety of poorlytransformed bacteria.

Objective HIV pathogenesis is characterized by destructive imbalances between virus-mediated immune damage, anti-viral immune responses, and immune activation. We characterized the effects of successful antiretroviral therapy (ART) to identify the breadth and patterns of HIV-associated gene expression. Methods In a prospective observational, longitudinal cohort study of 10 ART-naive Ugandans with AIDS (median 30 CD4+/μL), we measured mRNA gene profiles in peripheral blood using Affymetrix U133_Plus2.0 microarrays at 0, 2, 4, 8, and 24 weeks after ART initiation. Results We identified 160 mRNA transcripts that were consistently down-regulated and 48 that were up-regulated after ART at each point over 24 weeks based on linear regression modeling (adjusted-P<.05), Of these 208 transcripts, approximately half represent heretofore unrecognized ART-responsive genes and one-third have no known function. The down-regulated genes with known function encoded mediators of innate anti-viral responses, including antiviral restriction factors, pattern recognition receptors, and interferon response proteins, as well as mediators of immune activation, cellular proliferation, and apoptosis. Conclusions By using ART to block the viral stimulus, we identified transcripts involved in innate antiviral immunity, including antiviral restriction factors and pattern recognition receptors, that were not previously known to be induced by HIV infection.