Regulation of Mannitol Metabolism in Enterococcus faecalis and Association with parEF0409 Toxin-Antitoxin Locus Function.

The parEF0409 type I toxin-antitoxin locus is situated between genes for two paralogous mannitol family phosphoenolpyruvate phosphotransferase systems (PTSs). In order to address the possibility that parEF0409 function was associated with sugar metabolism, genetic and phenotypic analyses were performed on the flanking genes. It was found that the genes were transcribed as two operons: the downstream operon essential for mannitol transport and metabolism and the upstream operon performing a regulatory function. In addition to genes for the PTS components, the upstream operon harbors a gene similar to mtlR, the key regulator of mannitol metabolism in other Gram-positive bacteria. We confirmed that this gene is essential for the regulation of the downstream operon and identified putative phosphorylation sites required for carbon catabolite repression and mannitol-specific regulation. Genomic comparisons revealed that this dual-operon organization of mannitol utilization genes is uncommon in enterococci and that the association with a toxin-antitoxin system is unique to Enterococcus faecalis. Finally, we consider possible links between parEF0409 function and mannitol utilization. IMPORTANCE Enterococcus faecalis is both a common member of the human gut microbiota and an opportunistic pathogen. Its evolutionary success is partially due to its metabolic flexibility, in particular its ability to import and metabolize a wide variety of sugars. While a large number of phosphoenolpyruvate phosphotransferase sugar transport systems have been identified in the E. faecalis genome bioinformatically, the specificity and regulation of most of these systems remain undetermined. Here, we characterize a complex system of two operons flanking a type I toxin-antitoxin system required for the transport and metabolism of the common dietary sugar mannitol. We also determine the phylogenetic distribution of mannitol utilization genes in the enterococcal genus and discuss the significance of the association with toxin-antitoxin systems.

Charged Residues Flanking the Transmembrane Domain of Two Related Toxin-Antitoxin System Toxins Affect Host Response.

A majority of toxins produced by type I toxin-antitoxin (TA-1) systems are small membrane-localized proteins that were initially proposed to kill cells by forming non-specific pores in the cytoplasmic membrane. The examination of the effects of numerous TA-1 systems indicates that this is not the mechanism of action of many of these proteins. Enterococcus faecalis produces two toxins of the Fst/Ldr family, one encoded on pheromone-responsive conjugative plasmids (FstpAD1) and the other on the chromosome, FstEF0409. Previous results demonstrated that overexpression of the toxins produced a differential transcriptomic response in E. faecalis cells. In this report, we identify the specific amino acid differences between the two toxins responsible for the differential response of a gene highly induced by FstpAD1 but not FstEF0409. In addition, we demonstrate that a transporter protein that is genetically linked to the chromosomal version of the TA-1 system functions to limit the toxicity of the protein.

The Fst/Ldr Family of Type I TA System Toxins: Potential Roles in Stress Response, Metabolism and Pathogenesis.

The parpAD1 locus was the first type I toxin-antitoxin (TA) system described in Gram-positive bacteria and was later determined to be the founding member of a widely distributed family of plasmid- and chromosomally encoded TA systems. Indeed, homology searches revealed that the toxin component, FstpAD1, is a member of the Fst/Ldr superfamily of peptide toxins found in both Gram-positive and Gram-negative bacteria. Regulation of the Fst and Ldr toxins is distinct in their respective Gram-positive and Gram-negative hosts, but the effects of ectopic over-expression are similar. While, the plasmid versions of these systems appear to play the canonical role of post-segregational killing stability mechanisms, the function of the chromosomal systems remains largely obscure. At least one member of the family has been suggested to play a role in pathogenesis in Staphylococcus aureus, while the regulation of several others appear to be tightly integrated with genes involved in sugar metabolism. After a brief discussion of the regulation and function of the foundational parpAD1 locus, this review will focus on the current information available on potential roles of the chromosomal homologs.

Enterococcal Genetics.

The study of the genetics of enterococci has focused heavily on mobile genetic elements present in these organisms, the complex regulatory circuits used to control their mobility, and the antibiotic resistance genes they frequently carry. Recently, more focus has been placed on the regulation of genes involved in the virulence of the opportunistic pathogenic species Enterococcus faecalis and Enterococcus faecium. Little information is available concerning fundamental aspects of DNA replication, partition, and division; this article begins with a brief overview of what little is known about these issues, primarily by comparison with better-studied model organisms. A variety of transcriptional and posttranscriptional mechanisms of regulation of gene expression are then discussed, including a section on the genetics and regulation of vancomycin resistance in enterococci. The article then provides extensive coverage of the pheromone-responsive conjugation plasmids, including sections on regulation of the pheromone response, the conjugative apparatus, and replication and stable inheritance. The article then focuses on conjugative transposons, now referred to as integrated, conjugative elements, or ICEs, and concludes with several smaller sections covering emerging areas of interest concerning the enterococcal mobilome, including nonpheromone plasmids of particular interest, toxin-antitoxin systems, pathogenicity islands, bacteriophages, and genome defense.

Examination of Enterococcus faecalis Toxin-Antitoxin System Toxin Fst Function Utilizing a Pheromone-Inducible Expression Vector with Tight Repression and Broad Dynamic Range.

Tools for regulated gene expression in Enterococcus faecalis are extremely limited. In this report, we describe the construction of an expression vector for E. faecalis, designated pCIE, utilizing the PQ pheromone-responsive promoter of plasmid pCF10. We demonstrate that this promoter is tightly repressed, responds to nanogram quantities of the peptide pheromone, and has a large dynamic range. To demonstrate its utility, the promoter was used to control expression of the toxic peptides of two par family toxin-antitoxin (TA) loci present in E. faecalis, parpAD1 of the pAD1 plasmid and parEF0409 located on the E. faecalis chromosome. The results demonstrated differences in the modes of regulation of toxin expression and in the effects of toxins of these two related systems. We anticipate that this vector will be useful for further investigation of par TA system function as well as the regulated expression of other genes in E. faecalisIMPORTANCEE. faecalis is an important nosocomial pathogen and a model organism for examination of the genetics and physiology of Gram-positive cocci. While numerous genetic tools have been generated for the manipulation of this organism, vectors for the regulated expression of cloned genes remain limited by high background expression and the use of inducers with undesirable effects on the cell. Here we demonstrate that the PQ pheromone-responsive promoter is repressed tightly enough to allow cloning of TA system toxins and evaluate their effects at very low induction levels. This tool will allow us to more fully examine TA system function in E. faecalis and to further elucidate its potential roles in cell physiology.

The Type I toxin-antitoxin par locus from Enterococcus faecalis plasmid pAD1: RNA regulation by both cis- and trans-acting elements.

The pAD1 par determinant was the first Type I toxin-antitoxin system identified in Gram-positive bacteria and has recently been shown to be the prototype of a family of loci that is widespread in these organisms. All family members have (i) convergently transcribed toxin message and regulatory RNAs, (ii) three non-contiguous complementary regions for potential interaction, and (iii) intramolecular structures within the toxin message that modulate translation and transcript stability. Therefore, the detailed information available on the par locus provides a paradigm for studying the function and mechanism of regulation of the related loci.

Identification of a conserved branched RNA structure that functions as a factor-independent terminator.

Anti-Q is a small RNA encoded on pCF10, an antibiotic resistance plasmid of Enterococcus faecalis, which negatively regulates conjugation of the plasmid. In this study we sought to understand how Anti-Q is generated relative to larger transcripts of the same operon. We found that Anti-Q folds into a branched structure that functions as a factor-independent terminator. In vitro and in vivo, termination is dependent on the integrity of this structure as well as the presence of a 3' polyuridine tract, but is not dependent on other downstream sequences. In vitro, terminated transcripts are released from RNA polymerase after synthesis. In vivo, a mutant with reduced termination efficiency demonstrated loss of tight control of conjugation function. A search of bacterial genomes revealed the presence of sequences that encode Anti-Q-like RNA structures. In vitro and in vivo experiments demonstrated that one of these functions as a terminator. This work reveals a previously unappreciated flexibility in the structure of factor-independent terminators and identifies a mechanism for generation of functional small RNAs; it should also inform annotation of bacterial sequence features, such as terminators, functional sRNAs, and operons.

Antibiotic resistant enterococci-tales of a drug resistance gene trafficker.

Enterococci have been recognized as important hospital-acquired pathogens in recent years, and isolates of E. faecalis and E. faecium are the third- to fourth-most prevalent nosocomial pathogen worldwide. Acquired resistances, especially against penicilin/ampicillin, aminoglycosides (high-level) and glycopeptides are therapeutically important and reported in increasing numbers. On the other hand, isolates of E. faecalis and E. faecium are commensals of the intestines of humans, many vertebrate and invertebrate animals and may also constitute an active part of the plant flora. Certain enterococcal isolates are used as starter cultures or supplements in food fermentation and food preservation. Due to their preferred intestinal habitat, their wide occurrence, robustness and ease of cultivation, enterococci are used as indicators for fecal pollution assessing hygiene standards for fresh- and bathing water and they serve as important key indicator bacteria for various veterinary and human resistance surveillance systems. Enterococci are widely prevalent and genetically capable of acquiring, conserving and disseminating genetic traits including resistance determinants among enterococci and related Gram-positive bacteria. In the present review we aimed at summarizing recent advances in the current understanding of the population biology of enterococci, the role mobile genetic elements including plasmids play in shaping the population structure and spreading resistance. We explain how these elements could be classified and discuss mechanisms of plasmid transfer and regulation and the role and cross-talk of enterococcal isolates from food and food animals to humans.

Characterization of the effects of an rpoC mutation that confers resistance to the Fst peptide toxin-antitoxin system toxin.

Overexpression of the Fst toxin in Enterococcus faecalis strain OG1X leads to defects in chromosome segregation, cell division and, eventually, membrane integrity. The M7 mutant derivative of OG1X is resistant to most of these effects but shows a slight growth defect in the absence of Fst. Full-genome sequencing revealed two differences between M7 and its OG1X parent. First, OG1X contains a frameshift mutation that inactivates the etaR response regulator gene, while M7 is a wild-type revertant for etaR. Second, the M7 mutant contains a missense mutation in the rpoC gene, which encodes the ß' subunit of RNA polymerase. Mutagenesis experiments revealed that the rpoC mutation was primarily responsible for the resistance phenotype. Microarray analysis revealed that a number of transporters were induced in OG1X when Fst was overexpressed. These transporters were not induced in M7 in response to Fst, and further experiments indicated that this had a direct protective effect on the mutant cells. Therefore, exposure of cells to Fst appears to have a cascading effect, first causing membrane stress and then potentiation of these effects by overexpression of certain transporters.

The par toxin-antitoxin system from Enterococcus faecalis plasmid pAD1 and its chromosomal homologs.

The par post-segregational killing locus present on Enterococcus faecalis plasmid pAD1 was the first Type I toxin-antitoxin system described in Gram-positive bacteria. Translation of the 33 amino acid Fst toxin, encoded on RNA I, is suppressed by a 66 nucleotide regulatory RNA, RNA II. RNA I and RNA II are transcribed convergently and interact at dispersed regions of complementarity, establishing a stable complex that accumulates in plasmid-containing cells. RNA II is slowly removed from the complex, allowing translation of RNA I in plasmid-free segregants. Intramolecular structures are also important for regulating translation of RNA I. The Fst toxin contains a putative transmembrane domain and is believed to exert its function at the bacterial cytoplasmic membrane, although its precise target and mode of action have yet to be determined. Numerous chromosomal homologs of pAD1 par have been identified in Gram-positive bacteria suggesting that this locus may play important roles in cellular function.

Note: Thermal conductivity measurement of individual poly(ether ketone)/carbon nanotube fibers using a steady-state dc thermal bridge method.

Customized engineered fibers are currently being used extensively in the aerospace and automobile industries due to the ability to "design in" specific engineering characteristics. Understanding the thermal conductivity of these new fibers is critical for thermal management and design optimization. In the current investigation, a steady-state dc thermal bridge method (DCTBM) is developed to measure the thermal conductivity of individual poly(ether ketone) (PEK)/carbon nanotube (CNT) fibers. For non-conductive fibers, a thin platinum layer was deposited on the test articles to serve as the heater and temperature sensor. The effect of the platinum layer on the thermal conductivity is presented and discussed. DCTBM is first validated using gold and platinum wires (25 µm in diameter) over a temperature ranging from room temperature to 400 K with ±11% uncertainty, and then applied to PEK/CNT fibers with diverse CNT loadings. At a 28 wt. % CNT loading, the thermal conductivity of fibers at 390 K is over 27 Wm(-1)K(-1), which is comparable to some engineering alloys.

RNA-mediated reciprocal regulation between two bacterial operons is RNase III dependent.

UNLABELLED: In bacteria, RNAs regulate gene expression and function via several mechanisms. An RNA may pair with complementary sequences in a target RNA to impact transcription, translation, or degradation of the target. Control of conjugation of pCF10, a pheromone response plasmid of Enterococcus faecalis, is a well-characterized system that serves as a model for the regulation of gene expression in bacteria by intercellular signaling. The prgQ operon, whose products mediate conjugation, is negatively regulated by two products of the prgX operon, Anti-Q, a small RNA, and PrgX, the transcriptional repressor of the prgQ promoter. Here we show that Qs, an RNA from the 5' end of the prgQ operon, represses expression of PrgX by targeting prgX mRNA for cleavage by RNase III. Our results demonstrate that the prgQ and prgX operons each use RNAs to negatively regulate gene expression from the opposing operon by different mechanisms. Such reciprocal regulation between two operons using RNAs has not been previously demonstrated. Furthermore, these results show that Qs is an unusually versatile RNA, serving three separate functions in the regulation of conjugation. Understanding the potential versatility of RNAs and their various roles in gene regulatory networks will allow us to better understand how cells regulate complex behavior. IMPORTANCE: Bacteria use RNA to regulate gene expression by a variety of mechanisms. The prgQ and prgX operons of pCF10, a conjugative plasmid of Enterococcus faecalis, have been shown to negatively regulate one another by a variety of mechanisms. One of these mechanisms involves Anti-Q, a small RNA from the prgX operon that prevents gene expression from the prgQ operon. In this work, we find that Qs, an RNA from the prgQ operon, negatively regulates gene expression from the prgX operon. These findings have a number of implications. (i) The Anti-Q and Qs RNAs act by different mechanisms, highlighting the variety of ways in which bacteria can regulate gene expression using RNAs. (ii) Reciprocal regulation between operons mediated by small RNAs has not been previously described, deepening our understanding of how bacteria regulate complex behavior. (iii) Additional roles for Qs have been described, demonstrating the versatility of this RNA.

A multiresistance megaplasmid pLG1 bearing a hylEfm genomic island in hospital Enterococcus faecium isolates.

Enterococcus faecium is considered to be a nosocomial pathogen with increasing medical importance. The putative virulence factor, hyl(Efm), encoding a putative hyaluronidase, is enriched among the hospital-associated polyclonal subpopulation of E. faecium.. The hyl(Efm) gene is described to be part of a genomic island and was recently identified to be plasmid-located. Here, we present a description of the structure, localization, and distribution of the putative pathogenicity factor hyl(Efm) and its putative island among 39 clinical isolates and elucidate the composition and host range of pLG1, a hyl(Efm) multiresistance plasmid of approximately 281.02kb. The hyl(Efm) gene was located within a 17,824-bp element highly similar to the putative genomic island (GI) structure that had been previously described. This genomic region was conserved among 39 hyl(Efm)-positive strains with variation in a specific region downstream of hyl(Efm) in 18 strains. The putative hyl(Efm) was located on large plasmids (150-350kb) in 37 strains. pLG1 could be horizontally transferred into four different E. faecium recipient strains (n=4) but not into E. faecalis (n=3). Sequencing of pLG1 resolved putative plasmid replication, conjugation, and maintenance determinants as well as a pilin gene cluster, carbon uptake and utilization genes, heavy metal and antibiotic resistance clusters. The hyl(Efm) transferable plasmid pLG1 bears additional putative pathogenicity factors and antibiotic resistance genes. These findings suggest horizontal gene transfer of virulence factors and antibiotic resistance gene clusters by a single genetic event (conjugative transfer) which might be triggered by heavy antibiotic use common in health care units where E. faecium is increasingly prevalent.

Structural analysis of the Anti-Q-Qs interaction: RNA-mediated regulation of E. faecalis plasmid pCF10 conjugation.

Conjugation of the E. faecalis plasmid pCF10 is triggered in response to peptide sex pheromone cCF10 produced by potential recipients. Regulation of this response is complex and multi-layered and includes a small regulatory RNA, Anti-Q that participates in a termination/antitermination decision controlling transcription of the conjugation structural genes. In this study, the secondary structure of the Anti-Q transcript and its sites of interaction with its target, Qs, were determined. The primary site of interaction occurred at a centrally-located loop whose sequence showed high variability in analogous molecules on other pheromone-responsive plasmids. This loop, designated the specificity loop, was demonstrated to be important but not sufficient for distinguishing between Qs molecules from pCF10 and another pheromone-responsive plasmid pAD1. A loop 5' from the specificity loop which carries a U-turn motif played no demonstrable role in Anti-Q-Qs interaction or regulation of the termination/antitermination decision. These results provide direct evidence for a critical role of Anti-Q-Qs interactions in posttranscriptional regulation of pCF10 transfer functions.

Direct evidence for control of the pheromone-inducible prgQ operon of Enterococcus faecalis plasmid pCF10 by a countertranscript-driven attenuation mechanism.

The mating response of Enterococcus faecalis cells carrying the conjugative plasmid pCF10 is controlled by multiple regulatory circuits. Initiation of transcription of the prgQ conjugation operon is controlled by the peptide receptor protein PrgX; binding of the pheromone peptide cCF10 to PrgX abolishes PrgX repression, while binding of the inhibitor peptide iCF10 enhances repression. The results of molecular analysis of prgQ transcripts and genetic studies suggested that the elongation of prgQ transcripts past a putative terminator (IRS1) may be controlled by the interaction of nascent prgQ mRNAs with a small antisense RNA (Anti-Q) encoded within prgQ. Direct evidence for interaction of these RNAs, as well as the resulting effects on readthrough of prgQ transcription, has been limited. Here we report the results of experiments that (i) determine the inherent termination properties of prgQ transcripts in the absence of Anti-Q; (ii) determine the direct effects of the interaction of Anti-Q with nascent prgQ transcripts in the absence of complicating effects of the PrgX protein; and (iii) begin to dissect the structural components involved in these interactions. The results confirm the existence of alternative terminating and antiterminating forms of nascent prgQ transcripts in vivo and demonstrate that the interaction of Anti-Q with these transcripts leads to termination via inhibition of antiterminator formation. In vitro transcription assays support the major results of the in vivo studies. The data support a model for Anti-Q function suggested from recent studies of these RNAs and their interactions in vitro (S. Shokeen, C. M. Johnson, T. J. Greenfield, D. A. Manias, G. M. Dunny, and K. E. Weaver, submitted for publication).

Identification and characterization of a family of toxin-antitoxin systems related to the Enterococcus faecalis plasmid pAD1 par addiction module.

The par locus of the Enterococcus faecalis plasmid pAD1 is an RNA-regulated addiction module encoding the peptide toxin Fst. Homology searches revealed that Fst belongs to a family of at least nine related peptides encoded on the chromosomes and plasmids of six different Gram-positive bacterial species. Comparison of an alignment of these peptides with the results of a saturation mutagenesis analysis indicated regions of the peptides important for biological function. Examination of the genetic context of the fst genes revealed that all of these peptides are encoded within par-like loci with conserved features similar to pAD1 par. All four Ent. faecalis family members were demonstrated to produce the expected toxin-encoding and regulatory RNA products. The locus from the Ent. faecalis plasmid pAMS1 was demonstrated to function as an addiction module and Fst was shown to be toxic to Staphylococcus aureus, suggesting that a plasmid-encoded module in that species is performing the same function. Thus, the pAD1-encoded par locus appears to be the prototype of a family of related loci found in several Gram-positive species.

An overlap between the control of programmed cell death in Bacillus anthracis and sporulation.

The Staphylococcus aureus cid and lrg operons have been shown to control cell death and lysis in a manner thought to be analogous to programmed cell death (apoptosis) in eukaryotic organisms. Although orthologous operons are present in a wide variety of bacterial species, members of the Bacillus cereus group are unique in that they have a total of four cid-/lrg-like operons. Two of these operons are similar to the S. aureus cid and lrg operons, while the other two (designated clhAB(1) and clhAB(2)) are unique to this group. In the present study, the functions and regulation of these loci were examined. Interestingly, the Bacillus anthracis lrgAB mutant displayed decreased stationary-phase survival, whereas the clhAB(2) mutant exhibited increased stationary-phase survival compared to the parental and complementation strains. However, neither mutation had a dramatic effect on murein hydrolase activity or autolysis. Furthermore, a quantitative analysis of the sporulation efficiency revealed that both mutants formed fewer spores than did the parental strain. Similar to S. aureus, B. anthracis lrgAB transcription was shown to be induced by gramicidin and CCCP, agents known to dissipate the proton motive force, in a lytSR-dependent manner. Northern blot analyses also demonstrated a positive role for lytSR in the clhAB(2) transcription. Taken together, the results of the present study demonstrate that B. anthracis lrgAB and clhAB(2) play important roles in the control of cell death and lysis and reveal a previously unrecognized role of this system in sporulation.

The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids.

The pheromone-responsive conjugative plasmids of Enterococcus faecalis and the multiresistance plasmids pSK1 and pSK41 of Staphylococcus aureus are among the best studied plasmids native to Gram-positive bacteria. Although these plasmids seem largely restricted to their native hosts, protein sequence comparison of their replication initiator proteins indicates that they are clearly related. Homology searches indicate that these replicons are representatives of a large family of plasmids and a few phage that are widespread among the low G+C Gram-positive bacteria. We propose to name this family the RepA_N family of replicons after the annotated conserved domain that the initiator protein contains. Detailed sequence comparisons indicate that the initiator protein phylogeny is largely congruent with that of the host, suggesting that the replicons have evolved along with their current hosts and that intergeneric transfer has been rare. However, related proteins were identified on chromosomal regions bearing characteristics indicative of ICE elements, and the phylogeny of these proteins displayed evidence of more frequent intergeneric transfer. Comparison of stability determinants associated with the RepA_N replicons suggests that they have a modular evolution as has been observed in other plasmid families.

An intramolecular upstream helix ensures the stability of a toxin-encoding RNA in Enterococcus faecalis.

The par stability determinant is required for the stable inheritance of the plasmid pAD1 in its native host, Enterococcus faecalis. It is the only antisense RNA-regulated addiction module identified to date in gram-positive bacteria. It encodes two small, convergently transcribed RNAs, RNA I and RNA II. RNA I encodes the Fst toxin and RNA II acts as the antitoxin by interacting with RNA I posttranscriptionally. As the toxin-encoding component of the system, it is important that RNA I is more stable than RNA II. This study reveals that a helix sequestering the 5' end of RNA I plays a crucial role in maintaining the stability of the RNA I. An adjacent structure previously determined to regulate Fst translation was not required to enhance stability. Results indicated that endoribonuclease J2 contributes significantly to the degradation of a mutant disrupting the upstream helix (UH) of RNA I in Bacillus subtilis. Finally, it was shown that interaction with RNA II stabilized the UH mutant of RNA I.