Genetic characterization of the conjugative DNA processing system of enterococcal plasmid pCF10

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 poorly-transformed bacteria.     

The peptide pheromone-inducible conjugation system of Enterococcus faecalis plasmid pCF10: cell-cell signalling, gene transfer, complexity and evolution

Expression of a large set of gene products required for conjugative transfer of the antibiotic resistance plasmid pCF10 is controlled by cell-cell communication between plasmid-free recipient cells and plasmid-carrying donor cells using a peptide mating pheromone cCF10. Most of the recent experimental analysis of this system has focused on the molecular events involved in initiation of the pheromone response in the donor cells, and on the mechanisms by which the donor cells control self-induction by endogenously produced pheromone. Recently, studies of the molecular machinery of conjugation encoded by the pheromone-inducible genes have been initiated. In addition, the system may serve as a useful bacterial model for addressing the evolution of biological complexity.     

Staphylococcus aureus and Staphylococcus epidermidis peptide pheromones produced by the accessory gene regulator agr system

The accessory gene regulator (agr) system of staphylococci regulates the expression of virulence factors in response to cell density. The extracellular signaling molecule encoded by this system is a thiolactone-containing pheromone peptide whose primary sequence varies among staphylococcal strains. A post-translational modification of the peptide is believed to be carried out by an enzyme with a novel function, AgrB. Staphylococcal pheromones show cross-inhibiting properties: Pheromones of self and pheromones of non-self induce and suppress the agr response, respectively, and have therefore been proposed as novel anti-staphylococcal drugs. As inhibition of agr leads to diminished expression of toxins, but to increased expression of colonization factors and biofilm formation, their therapeutic potential remains yet to be evaluated in depth.     

IL-10: the master regulator of immunity to infection

IL-10 is an anti-inflammatory cytokine. During infection it inhibits the activity of Th1 cells, NK cells, and macrophages, all of which are required for optimal pathogen clearance but also contribute to tissue damage. In consequence, IL-10 can both impede pathogen clearance and ameliorate immunopathology. Many different types of cells can produce IL-10, with the major source of IL-10 varying in different tissues or during acute or chronic stages of the same infection. The priming of these various IL-10-producing populations during infections is not well understood and it is not clear whether the cellular source of IL-10 during infection dictates its cellular target and thus its outcome. In this article we review the biology of IL-10, its cellular sources, and its role in viral, bacterial, and protozoal infections.     

Specific control of endogenous cCF10 pheromone by a conserved domain of the pCF10-encoded regulatory protein PrgY in Enterococcus faecalis

Conjugative transfer of Enterococcus faecalis plasmid pCF10 is induced by the heptapeptide pheromone cCF10. cCF10 produced by plasmid-free recipient cells is detected by pCF10-containing donor cells, which respond by induction of plasmid-encoded transfer functions. The pCF10-encoded membrane protein PrgY is essential to prevent donor cells from responding to endogenously produced pheromone while maintaining the ability to respond to pheromone from an exogenous source; this function has not been identified in any nonenterococcal prokaryotic signaling system. PrgY specifically inhibited endogenous cCF10 and cPD1 (a pheromone that induces transfer of closely related plasmid pPD1) but not cAD1 (which is specific for less-related plasmid pAD1). Ectopic expression of PrgY in plasmid-free recipient cells reduced pheromone activity in culture supernatants and reduced the ability of these cells to acquire pCF10 by conjugation but did not have any effect on the interaction of these cells with exogenously supplied cCF10. The cloned prgY gene could complement a pCF10 prgY null mutation, and complementation was used to identify point mutations impairing PrgY function. Such mutations also abolished the inhibitory effect of PrgY expression in recipients on pheromone production and on acquisition of pCF10. Most randomly generated point mutations identified in the genetic screen mapped to a predicted extracellular domain in the N terminus of PrgY that is conserved in a newly identified family of related proteins from disparate species including Borrelia burgdorferi, Archaeoglobus fulgidus, Arabidopsis thaliana, and Homo sapiens. The combined genetic and physiological data suggest that PrgY may sequester or inactivate cCF10 as it is released from the membrane.     

The prgQ gene of the Enterococcus faecalis tetracycline resistance plasmid pCF10 encodes a peptide inhibitor, iCF10.

Conjugative transfer of the Enterococcus faecalis tetracycline resistance plasmid pCF10 is stimulated by a peptide pheromone, cCF10. Once a recipient strain acquires pCF10 and thus becomes a pheromone-responsive donor, cCF10 activity is no longer detected in culture filtrates. Here we show that pCF10 encodes a peptide inhibitor, iCF10, secreted by donor cells; this inhibitor antagonizes the cCF10 activity in culture filtrates. In order to detect and quantitate iCF10, we developed a reverse-phase high-performance liquid chromatography assay in which the inhibitor peptide elutes separately from the pheromone; this type of assay enabled us to determine that lack of pheromone activity in donor culture filtrates was due to secretion of a mixture of iCF10 and cCF10, rather than abolition of cCF10 secretion. The gene encoding iCF10, prgQ, is located on the EcoRI-C fragment of pCF10. The open reading frame comprising the prgQ gene encodes a 23-amino-acid precursor that resembles a signal peptide. This precursor is cleaved to the mature heptapeptide iCF10 during the secretion process.     

Mammalian target of rapamycin: master regulator of cell growth in the nervous system

The mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine protein kinase that regulates a number of diverse biologic processes important for cell growth and proliferation, including ribosomal biogenesis and protein translation. In this regard, hyperactivation of the mTOR signaling pathway has been demonstrated in numerous human cancers, including a number of inherited cancer syndromes in which individuals have an increased risk of developing benign and malignant tumors. Three of these inherited cancer syndromes (Lhermitte-Duclos disease, neurofibromatosis type 1, and tuberous sclerosis complex) are characterized by significant central nervous system dysfunction and brain tumor formation. Each of these disorders is caused by a genetic mutation that disrupts the expression of proteins which negatively regulate mTOR signaling, indicating that the mTOR signaling pathway is critical for appropriate brain development and function. In this review, we discuss our current understanding of the mTOR signaling pathway and its role in promoting ribosome biogenesis and cell growth. We suggest that studies of this pathway may prove useful in identifying molecular targets for biologically-based therapies of brain tumors associated with these inherited cancer syndromes as well as sporadic central nervous system tumors.     

Structure of cCF10, a peptide sex pheromone which induces conjugative transfer of the Streptococcus faecalis tetracycline resistance plasmid, pCF10

The peptide pheromone, cCF10, which induces aggregation and high frequency plasmid transfer in Streptococcus faecalis cells carrying the tetracycline resistance plasmid, pCF10, was isolated and its structure determined. The molecular weight of cCF10 is 789, and its amino acid sequence is H-Leu-Val-Thr-Leu-Val-Phe-Val-OH. Pheromone activity, as determined by a clumping induction assay, was detectable at a concentration of 2.5 x 10(-11) M. A peptide of the same sequence as that of the cCF10 produced by S. faecalis cells was synthesized by the liquid-phase method. The synthetic pheromone showed biological activity and chromatographic behavior that was identical to that of the cCF10 of bacterial origin. When the response of S. faecalis cells to various concentrations of synthetic cCF10 was monitored by measuring both the frequency of plasmid transfer and the synthesis of pheromone-inducible antigens, an excellent correlation was observed between donor ability and the appearance of a 150-kilodalton protein that appears to be involved in formation of mating aggregates. The dose-response data in the range of concentrations where the amount of pheromone became limiting (10(-11)-10(-12) M) were consistent with the notion that as few as one or two molecules per donor cell may be sufficient to induce a mating response.     

Effects of early plant growth regulator treatments on flavonoid levels in grapefruit

Early foliar spray treatments containing gibberellic acid(GA₃) significantly lower the concentration ofthe bitter flavonoid naringin in fruit tissues. Sprayscontaining a surfactant and different levels ofGA₃ (5, 50, 100, and 500 ppm) or abscisic acid(ABA) (5, 25, and 50 ppm) were applied to young,developing fruit on mature grapefruit (Citrusparadisi) trees during the period from April to June,beginning just after fruit set. The fruit were allowedto mature and were harvested early the following year.Harvested fruit were evaluated for weight, juicecharacteristics, and flavonoid concentrations.GA₃ application resulted in larger mature fruit,which yielded juice with the same soluble solids valueas juice from control fruit, but with slightly loweracid percentages and lower concentrations of naringin.ABA treatment had little effect on juice solublesolids, acid content and naringin content except atthe highest concentration of 50 ppm, which lowerednaringin levels slightly in juice.     

Effects of abscisic acid on the levels of endogenous gibberellin-like substances in Solanum andigena

Summary Treatment of young, fully expanded leaves of Solanum andigena with synthetic abscisic acid resulted in marked increases in gibberellin-like substances. Abscisic acid treatment caused increases in the gibberellin content of leaves excised from both short day and long day grown plants.     

Effect of pheromone induction on transfer of the Enterococcus faecalis plasmid pCF10 in intestinal mucus ex vivo

The effect of synthetic sex pheromone on pheromone-inducible conjugation between the isogenic Enterococcus faecalis strains OG1RF and OG1SS was investigated in (i) Todd-Hewitt broth medium and (ii) intestinal mucus isolated from germ-free rats. In broth, the presence of synthetic pheromone cCF10 had no detectable effect on the transfer kinetics observed for the tetracycline resistance encoding plasmid pCF10. In mucus, presence of the same pheromone significantly increased the transfer efficiency observed during the first 2 h of conjugation, while the effect was less pronounced later in the experiment. We suggest that due to differences in diffusion rates and medium-binding of the pheromones, the effect of the synthetic cCF10 was immediately dominated by the effect of pheromones produced by the recipient E. faecalis strain in broth, while this happened later in mucus.     

Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis.

The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcus faecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J. Christie and G. M. Dunny, Plasmid 15:230-241, 1986). When S. faecalis cells were mixed with Bacillus subtilis in broth, tetracycline resistance was transferred from S. faecalis. The tetracycline-resistant B. subtilis cells contained a 16-kb region of pCF10 (distinct from TRA) that carried the tetracycline resistance determinant (Tetr). This Tetr element was found to transfer between S. faecalis and B. subtilis strains in the absence of plasmids. Genetic and molecular techniques were used to establish locations of the element at several different sites on the B. subtilis chromosome. The Tetr element could be transferred in filter matings from B. subtilis to S. faecalis strains and between recombination-proficient and -deficient S. faecalis strains in the absence of any plasmid DNA. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The Tetr element was shown to transpose from the S. faecalis chromosome to various locations within the hemolysin plasmid pAD1. Together, the data indicate that the Tetr element, termed transposon Tn925, is very similar to the conjugative transposon Tn916 in both structure and function. A derivative of Tn925, containing transposon Tn917 inserted into a site approximately 3 kb from one end, exhibited elevated transfer frequencies and may provide a useful means for delivering Tn917 by conjugation into various gram-positive species.     

Paralogous antirepressors acting on the master regulator for biofilm formation in Bacillus subtilis

Matrix production during biofilm formation by Bacillus subtilis is governed by a gene control circuit at the heart of which are three dedicated regulatory proteins, the antirepressor SinI, the repressor SinR and the downstream regulator SlrR. Matrix production is triggered by the synthesis of SinI, which binds to and inactivates SinR, thereby derepressing genes for matrix production as well as the gene for SlrR. Recently, two additional regulators of matrix genes were identified: SlrA, which was reported to be an activator of SlrR, and YwcC, a repressor of SlrA synthesis (Kobayashi, 2008). We present evidence indicating that SlrA, which is a paralogue of SinI, is like SinI, an antirepressor that binds to, and inactivates, SinR. We also show that SlrA does not activate SlrR for expression of matrix genes. Instead, SlrR binds to, and inhibits the activity of, SlrA. Thus, the YwcC-SlrA-SinR-SlrR pathway is a negative feedback loop in which SlrA indirectly stimulates the synthesis of SlrR, and SlrR, in turn, inhibits the activity of SlrA. Finally, we report that under standard laboratory conditions SlrA makes only a small contribution to the expression of genes for matrix production. We propose that in response to an unknown signal recognized by the YwcC repressor, SlrA transiently boosts matrix production.     

Processing and export of peptide pheromones and bacteriocins in Gram-negative bacteria

Cell-density-dependent gene expression is widespread in bacteria and is mediated by extracellular communication molecules. Gram-negative bacteria often use N-acyl homoserine lactones, whereas cell-cell signaling in Gram-positive bacteria is accomplished using post-translationally processed peptide pheromones. In many Gram-positive bacteria, export of these peptides requires the activity of a dedicated ATP-binding cassette (ABC) transporter, which cleaves off a typical leader peptide termed the double-glycine leader sequence concomitant with translocation across the membrane. Inspection of bacterial genome sequences has revealed the presence of similar ABC transporters, as well as genes encoding peptides with double-glycine-type leader sequences in Gram-negative bacteria, and it is suggested that the postulated transported peptides could perform a signaling function.