Biomedical subjects

Gary M Dunny

Publications and source records attributed to Gary M Dunny.

At least 19 recordsLinked to original sources

PubMed2006

Analysis of the amino acid sequence specificity determinants of the enterococcal cCF10 sex pheromone in interactions with the pheromone-sensing machinery.

The level of expression of conjugation genes in Enterococcus faecalis strains carrying the pheromone-responsive transferable plasmid pCF10 is determined by the ratio in the culture medium of two types of signaling peptides, a pheromone (cCF10) and an inhibitor (iCF10). Recent data have demonstrated that both peptides target the cytoplasmic receptor protein PrgX. However, the relative importance of the interaction of these peptides with the pCF10 protein PrgZ (which enhances import of cCF10) versus PrgX is not fully understood, and there is relatively little information about specific amino acid sequence determinants affecting the functional interactions of cCF10 with these proteins in vivo. To address these issues, we used a pheromone-inducible reporter gene system where various combinations of PrgX and PrgZ could be expressed in an isogenic host background to examine the biological activities of cCF10, iCF10, and variants of cCF10 isolated in a genetic screen. The results suggest that most of the amino acid sequence determinants of cCF10 pheromone activity affect interactions between the peptide and PrgX, although some sequence variants that affected peptide/PrgZ interactions were also identified. The results provide functional data to complement ongoing structural studies of PrgX and increase our understanding of the functional interactions of cCF10 and iCF10 with the pheromone-sensing machinery of pCF10.

Amino Acid Sequence↗

PubMed2006

Molecular basis for control of conjugation by bacterial pheromone and inhibitor peptides.

In many bacteria expression of lateral gene transfer and of virulence factors is controlled by cell-cell signalling systems. Molecular interactions of microbial signal molecules with their cognate receptors are not well understood. For the Enterococcus faecalis conjugative plasmid pCF10, the PrgX protein serves as a molecular switch controlling expression of conjugation and virulence genes encoded by the plasmid. The induction state of a pCF10-carrying donor cell is determined by the ratio of two signalling peptides, cCF10 pheromone and iCF10 inhibitor. Recent analysis of PrgX/cCF10 interactions suggests a mechanism for conversion to the induced state. However, the means by which iCF10 peptide antagonizes cCF10 activity is unclear, and it has been suggested that inhibitor peptides block import of pheromone peptides. We now show that both of these peptides interact with the same binding pocket of PrgX, but they differentially alter the conformation of the protein and its oligomerization state, resulting in opposing biological activities.

Bacterial Proteins↗

PubMed2006

Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis.

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 delta eep10 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.

Conjugation, Genetic↗

PubMed2006

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.

Base Sequence↗

PubMed2006

Pheromone-inducible conjugation in Enterococcus faecalis: a model for the evolution of biological complexity?

Pheromone-inducible transfer of the plasmid pCF10 in Enterococcus faecalis is regulated using a complicated network of proteins and RNAs. The plasmid itself has been assembled from parts garnered from a variety of sources, and many aspects of the system resemble a biological kluge. Recently several new functions of various pCF10 gene products that participate in regulation of plasmid transfer have been identified. The results indicate that selective pressures controlling the evolution of the plasmid have produced a highly complex regulatory network with multiple biological functions that may serve well as a model for the evolution of biological complexity.

Bacterial Proteins↗

PubMed2006

Comparison of OG1RF and an isogenic fsrB deletion mutant by transcriptional analysis: the Fsr system of Enterococcus faecalis is more than the activator of gelatinase and serine protease.

The FsrABC system of Enterococcus faecalis controls the expression of gelatinase and a serine protease via a quorum-sensing mechanism, and recent studies suggest that the Fsr system may also regulate other genes important for virulence. To investigate the possibility that Fsr influences the expression of additional genes, we used transcriptional profiling, with microarrays based on the E. faecalis strain V583 sequence, to compare the E. faecalis strain OG1RF with its isogenic mutant, TX5266, an fsrB deletion mutant. We found that the presence of an intact fsrB influences expression of numerous genes throughout the growth phases tested, namely, late log to early stationary phase. In addition, the Fsr regulon is independent of the activity of the proteases, GelE and SprE, whose expression was confirmed to be activated at all three time points tested. While expression of some genes (i.e., ef1097 and ef0750 to -757, encoding hypothetical proteins) was activated in late log phase in OG1RF versus the fsrB deletion mutant, expression of ef1617 to -1634 (eut-pdu orthologues) was highly repressed by the presence of an intact Fsr at entry into stationary phase. This is the first time that Fsr has been characterized as a negative regulator. The newly recognized Fsr-regulated targets include other factors, besides gelatinase, described as important for biofilms (BopD), and genes predicted to encode the surface proteins EF0750 to -0757 and EF1097, along with proteins implicated in several metabolic pathways, indicating that the FsrABC system may be an important regulator in strain OG1RF, with both positive and negative effects.

Bacterial Proteins↗

PubMed2005

Structure of peptide sex pheromone receptor PrgX and PrgX/pheromone complexes and regulation of conjugation in Enterococcus faecalis.

Many bacterial activities, including expression of virulence factors, horizontal genetic transfer, and production of antibiotics, are controlled by intercellular signaling using small molecules. To date, understanding of the molecular mechanisms of peptide-mediated cell-cell signaling has been limited by a dearth of published information about the molecular structures of the signaling components. Here, we present the molecular structure of PrgX, a DNA- and peptide-binding protein that regulates expression of the conjugative transfer genes of the Enterococcus faecalis plasmid pCF10 in response to an intercellular peptide pheromone signal. Comparison of the structures of PrgX and the PrgX/pheromone complex suggests that pheromone binding destabilizes PrgX tetramers, opening a 70-bp pCF10 DNA loop required for conjugation repression.

Base Sequence↗

PubMed2005

A paracrine peptide sex pheromone also acts as an autocrine signal to induce plasmid transfer and virulence factor expression in vivo.

The peptide pheromone cCF10 of Enterococcus faecalis is an intercellular signal for induction of conjugative transfer of plasmid pCF10 from donor cells to recipient cells. When a donor cell is exposed to recipient-produced cCF10, expression of the pCF10-encoded aggregation substance of pCF10 (Asc10) and other conjugation gene products is activated. Asc10 also increases enterococcal virulence in several models, and when donor cells are grown in animals or in plasma, Asc10 expression is induced by means of the cCF10-sensing machinery. Plasmid pCF10 carries two genes that function to prevent self-induction by endogenous cCF10 in donor cells. The membrane protein PrgY reduces endogenous pheromone activity in donor cells, and the inhibitor peptide iCF10 neutralizes the residual endogenous cCF10 that escapes PrgY. In the current study, we found that E. faecalis strains with allelic replacements abolishing active cCF10 production showed reduced ability to acquire pCF10 by conjugation; prgY-null mutations had no phenotype in the cCF10-negative strains. We observed that expression of the mRNA for iCF10 was reduced in this background and that these mutations also blocked plasma induction of Asc10 expression. These findings support a model in which plasma induction in wild-type donors results from iCF10 inactivation by a plasma component, causing disruption of a precisely maintained balance of iCF10 to cCF10 activity and allowing subsequent induction by endogenous cCF10. Although cCF10 has traditionally been viewed as an intercellular signal, these results show that pCF10 has also adapted cCF10 as an autocrine signal that activates expression of virulence and conjugation functions.

Amino Acid Sequence↗

PubMed2005

Development of a method for markerless genetic exchange in Enterococcus faecalis and its use in construction of a srtA mutant.

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, genetic analysis of these organisms has thus far been limited in scope due to the lack of advanced genetic tools. To broaden the repertoire of genetic tools available for manipulation of E.faecalis, we investigated the use of phosphoribosyl transferases as elements of a counterselection strategy. We report here the development of a counterselectable markerless genetic exchange system based on the upp-encoded uracil phosphoribosyl transferase of E. faecalis. Whereas wild-type E. faecalis is sensitive to growth inhibition by the toxic base analog 5-fluorouracil (5-FU), a mutant bearing an in-frame deletion of upp is resistant to 5-FU. When a cloned version of upp was ectopically introduced into the deletion mutant, sensitivity to 5-FU growth inhibition was restored, thereby providing the basis for a two-step integration and excision strategy for the transfer of mutant alleles to the enterococcal chromosome by recombination. This method was validated by the construction of a DeltasrtA mutant of E. faecalis and by the exchange of the surface protein Asc10, encoded on the pheromone-responsive conjugative plasmid pCF10, with a previously isolated mutant allele. Analysis of the DeltasrtA mutant indicated that SrtA anchors Asc10 to the enterococcal cell wall, facilitating the pheromone-induced aggregation of E. faecalis cells required for high-frequency conjugative plasmid transfer in liquid matings. The system of markerless exchange reported here will facilitate detailed genetic analysis of these important pathogens.

Aminoacyltransferases↗

PubMed2005

Quantitative analysis of group II intron expression and splicing in Lactococcus lactis.

The group II intron Ll.ltrB is found within the ltrB relaxase gene of the conjugative element pRS01 in Lactococcus lactis. Precise splicing of the intron is essential for pRS01 transfer. The transcription regulation and in vivo splicing activity of Ll.ltrB have not been investigated thoroughly in L. lactis in the natural pRS01 context. We developed absolute quantitative real-time reverse transcription-PCR assays to quantify RNA levels of the 5' exon (ltrBE1) and the spliced relaxase (ltrB) and intron-encoded protein (ltrA) genes, as well as Ll.ltrB splicing activity under different physiological conditions. The mRNA levels for the ATP-binding protein OppD were assayed for comparison to the ltrB transcripts. The oppD mRNA ranged from 10- to 10,000-fold higher than ltrB region genes. ltrBE1 expression was growth-phase dependent. The mRNA level of ltrA was almost constant during all growth phases and in all media tested. Ll.ltrB in vivo splicing activity ranged from (6.5 +/- 2.1)% to (22.1 +/- 8.0)%. Acid challenge significantly decreased both ltrB region mRNA levels and intron splicing activity. The presence of recipient cells, different mating environments, and temperature stress had no significant effects on expression and splicing. Western blotting showed that the level of LtrB protein expressed from an intronless ltrB gene was much higher (about 20-fold) than the level of protein expressed from an intron-containing construct. Interestingly, LtrB protein showed a tendency to function in cis on its oriT target. The low level of ltrB transcript and relatively inefficient splicing of the intron may limit Ll.ltrB mobility and dissemination in nature.

Bacterial Proteins↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

Characterization of the pheromone response of the Enterococcus faecalis conjugative plasmid pCF10: complete sequence and comparative analysis of the transcriptional and phenotypic responses of pCF10-containing cells to pheromone induction.

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.

Chromosome Mapping↗

PubMed2004

Enterococcal peptide sex pheromones: synthesis and control of biological activity.

The enterococcal pheromone-inducible plasmids such as pCF10 represent a unique class of mobile genetic elements whose transfer functions are induced by peptide sex pheromones. These pheromones are excreted by potential recipient cells and detected by plasmid-containing donor cells at the cell surface, where the pheromone is imported and signals induction of the plasmid transfer system. Pheromone is processed from a chromosomally encoded lipoprotein and excreted by both the donor and recipient cells, but a carefully controlled detection system prevents a response to self-pheromone while still allowing an extremely sensitive response to exogenous pheromone.

Amino Acid Sequence↗

PubMed2004

Characterization of cis-acting prgQ mutants: evidence for two distinct repression mechanisms by Qa RNA and PrgX protein in pheromone-inducible enterococcal plasmid pCF10.

The pCF10-encoded negative regulators PrgX and Qa (prgQ antisense) RNA inhibit pCF10 transfer by blocking prgQ transcription extension past a potential transcription terminator sequence IRS1. To identify potential target sites for negative regulation, we isolated and analysed 13 cis-acting mutations in the prgXQ region. Determination of the 3' end of Qa RNA showed that eight mutations mapped in the region encoding Qa RNA. Four mutations were in the Qa promoter region and one was in IRS1. Three mutations in Qa greatly reduced the intracellular level of this RNA but did not affect that of PrgX. However, both Qa RNA and PrgX protein were reduced in three Qa promoter region mutants and the expression of prgQ transcripts extending 3' from IRS1 became constitutive. Qa RNA could mediate its negative regulatory activity in the absence of PrgX, and this activity was not abolished by cCF10, the peptide pheromone that induces pCF10 transfer. RNA analysis showed that Qa RNA abolished transcription readthrough. Based on the experimental data as well as computer analysis of predicted secondary structures of prgQ mRNA in the presence or absence of Qa, we concluded that Qa RNA is a pheromone-insensitive effector of prgQ mRNA termination or degradation at IRS1. In cells lacking a Qa target sequence, expression of PrgX repressed transcription from the prgQ promoter, and this repression was relieved by addition of exogenous cCF10. Thus, even though the synthesis of these negative regulators is coupled, they each act independently on separate targets to regulate expression of conjugation functions.

Bacterial Proteins↗

PubMed2004

Enterococcus faecalis pheromone-responsive protein PrgX: genetic separation of positive autoregulatory functions from those involved in negative regulation of conjugative plasmid transfer.

The pCF10 plasmid in Enterococcus faecalis transfers from donor cells to recipients upon induction via peptide pheromone. Two plasmid-encoded negative regulators produced from the same transcript, PrgX protein and Qa RNA, repress conjugation genes in uninduced donor cells. PrgX positively autoregulates production of both itself and mature Qa RNA, and is believed to repress the prgQ promoter in a pheromone-sensitive fashion. Previous analysis of PrgX was complicated because mutations in prgX affecting regulation of conjugation also disrupted PrgX autoregulation, suggesting the two functions might be inseparable. In this study, we isolated 14 single amino acid substitutions in PrgX that reduced or eliminated repression of prgQ, without affecting autoregulation or DNA binding. PrgX was shown to bind to its cognate pheromone, cCF10, and most of the mutations lowered the affinity of PrgX for cCF10. Dimerization was affected by five of the mutations and the data indicate that it is required, but insufficient for pheromone induction. We propose a new model for the mechanism used by PrgX for regulation of the prgQ promoter, PrgX autoregulation, and Qa RNA processing.

Bacterial Proteins↗

PubMed2004

Esp-independent biofilm formation by Enterococcus faecalis.

Enterococcus faecalis is a gram-positive opportunistic pathogen known to form biofilms in vitro. In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices. However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown. Recent work has suggested that a specific cell surface protein (Esp) of E. faecalis is critical for biofilm formation by this organism. However, in the same study, esp-deficient strains of E. faecalis were found to be capable of biofilm formation. To test the hypothesis that Esp is dispensable for biofilm formation by E. faecalis, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains. Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of esp, but also in the absence of the entire pathogenicity island that harbors the esp coding sequence. Using scanning electron microscopy to evaluate biofilms of E. faecalis OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels. Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions. Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by E. faecalis. In summary, E. faecalis forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein.

Bacterial Proteins↗

PubMed2004

A conjugation-based system for genetic analysis of group II intron splicing in Lactococcus lactis.

The conjugative element pRS01 from Lactococcus lactis encodes the putative relaxase protein LtrB. The ltrB gene is interrupted by the functional group II intron Ll.ltrB. Accurate splicing of the two ltrB exons is required for synthesis of the mRNA encoding the LtrB conjugative relaxase and subsequent plasmid transfer. A conjugation-based genetic assay was developed to identify Ll.ltrB mutations that affect splicing. In this assay a nonsplicing, transfer-defective pRS01 derivative (pM1014) and a shuttle vector carrying the ltrB region, including the Ll.ltrB intron (pCOM9), are used. pCOM9 provides splicing-dependent complementation of the transfer defect of pM1014. Site-directed mutations within Ll.ltrB, either in the catalytic RNA or in the intron-encoded protein gene ltrA, were generated in the context of pCOM9. When these mutants were tested in the conjugation-based assay, significantly reduced mating was observed. Quantitative molecular analysis of in vivo splicing activity confirmed that the observed mating defects resulted from reduced splicing. Once the system was validated for the engineered mutants, random mutagenesis of the intron followed by genetic and molecular screening for splicing defects resulted in identification of point mutations that affect splicing.

Bacterial Proteins↗

PubMed2004

Conserved target for group II intron insertion in relaxase genes of conjugative elements of gram-positive bacteria.

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.

Amino Acid Sequence↗