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MOLECULAR BIOLOGY OF PATHOGENS
A Novel Mutation within the Central Listeria monocytogenes Regulator PrfA That Results in Constitutive Expression of Virulence Gene Products
Kendy K. Y. Wong, Nancy E. Freitag
Kendy K. Y. Wong
Seattle Biomedical Research Institute, and Departments of Pathobiology and Microbiology, University of Washington, Seattle, Washington
Nancy E. Freitag
For correspondence: nancy.freitag@sbri.org
DOI: 10.1128/JB.186.18.6265-6276.2004
The PrfA protein of Listeria monocytogenes functions as a key regulatory factor for the coordinated expression of many virulence genes during bacterial infection of host cells. PrfA activity is controlled by multiple regulatory mechanisms, including an apparent requirement for either the presence of a cofactor or some form of posttranslational modification that regulates the activation of PrfA. In this study, we describe the identification and characterization of a novel PrfA mutation that results in constitutive activation of the PrfA protein. The PrfA L140F mutation was found to confer high-level expression of PrfA-regulated genes and to be functionally dominant over the wild-type allele. The presence of the PrfA L140F mutation resulted in the aggregation of L. monocytogenes in broth culture and, unlike previously described prfA mutations, appeared to be slightly toxic to the bacteria. High-level PrfA-dependent gene expression showed no additional increase in L. monocytogenes strains containing an additional copy of prfA L140F despite a >4-fold increase in PrfA protein levels. In contrast, the introduction of multiple copies of the wild-type prfA allele to L. monocytogenes resulted in a corresponding increase in PrfA-dependent gene expression, although overall expression levels remained far below those observed for PrfA L140F strains. These results suggest a hierarchy of PrfA regulation, such that the relative levels of PrfA protein present within the cell correlate with the levels of PrfA-dependent gene expression when the protein is not in its fully activated state; however, saturating levels of the protein are then quickly reached when PrfA is converted to its active form. Regulation of the PrfA activation status must be an important facet of L. monocytogenes survival, as mutations that result in constitutive PrfA activation may have deleterious consequences for bacterial physiology.
Listeria monocytogenes is a ubiquitous gram-positive bacterial pathogen that can cause serious food-borne infections in immunocompromised individuals, pregnant women, and neonates (15, 18). This facultative intracellular pathogen is able to invade a wide variety of host cells, gain entry to the cytosol, and subsequently replicate and spread to adjacent host cells (6, 7, 9, 10, 23, 24, 32, 34, 35, 46). Several virulence factors important for the different steps of L. monocytogenes infection have been identified (reviewed in references 26, 41, and 48). The majority of these virulence determinants are located within a 10-kb chromosomal region and are regulated by the positive transcriptional regulator PrfA. The prfA gene is also located within this cluster, which is commonly referred to as the PrfA regulon (29, 30). The prfA gene product is a key factor for L. monocytogenes pathogenesis, and bacterial strains lacking functional PrfA are essentially avirulent in mouse models of infection (13, 30, 31). PrfA is a 27-kDa site-specific DNA-binding protein that recognizes a 14-bp palindrome (PrfA box) within the −40 region of PrfA-dependent promoters (1, 8, 13, 40).
Multiple mechanisms exist to regulate prfA expression and protein activity. Three promoters contribute to the transcriptional regulation of prfA. The promoters prfAp1 and prfAp2 are located immediately upstream of the prfA coding region and are important for providing the initial levels of PrfA protein required for the activation of gene products essential for bacterial escape from host cell vacuoles (12, 13). These promoters are functionally redundant in vivo and appear to contribute to both positive and negative regulation of prfA expression (12, 13, 19). The third promoter contributing to regulation of prfA is located upstream of the plcA gene and increases prfA expression via the generation of a bicistronic plcA-prfA transcript; this promoter is PrfA dependent and represents a positive feedback loop for prfA expression (5, 13). The 5′ untranslated region of prfA mRNA has recently been shown to function as a thermosensor that regulates the translation of prfA mRNA in response to changes in temperature (22).
In addition to the regulation of prfA expression and mRNA translation, the activation state of PrfA appears to be regulated either through the binding of a cofactor or through some mode of posttranslational modification. The PrfA protein is a member of the Crp/Fnr family of transcriptional activators (25, 27, 41) and has significant structural and functional homology to the Escherichia coli cyclic AMP (cAMP) receptor protein (Crp) (17, 39, 41). Crp requires the binding of the cofactor cAMP for full activity, and cAMP-independent, constitutively active Crp* mutants have been described (14). A PrfA mutant analogous to Crp* has been identified in L. monocytogenes (37); a glycine-to-serine substitution at position 145 in PrfA resulted in constitutively high expression of all PrfA-regulated genes (37). The phenotype of the PrfA G145S mutant suggests that PrfA, like Crp, requires cofactor binding or modification for full activity (37). Two additional mutations in PrfA leading to activation of the protein have recently been identified (PrfA E77K and PrfA G155S); both were found to result in increased levels of PrfA-dependent gene expression, but strains containing the mutant alleles were phenotypically distinct, suggesting that the mutations altered different aspects of PrfA function (43).
In this study, we describe a fourth PrfA mutation (PrfA L140F) that results in the constitutive activation of PrfA but is also functionally distinct from the three previously described constitutive PrfA mutations.
Bacterial strains and plasmids.The bacterial strains used in this study are listed in Table 1. E. coli DH5α was used as a host strain for recombinant plasmids. E. coli strain SM10 (44) was used as the donor strain for conjugation of pPL2 plasmid constructs. L. monocytogenes 10403S (serotype 1/2) is resistant to streptomycin and has a 50% lethal dose for mice of 2 × 104 CFU/ml (13). L. monocytogenes wild-type (WT) strain 10403S containing a chromosomal actA-gus-plcB transcriptional fusion (NF-L476) and NF-L758, a derivative of NF-L476 containing an erythromycin resistance gene (erm) inserted between orfZ and orfB downstream of the PrfA regulon, have been described (43). A 10403S L. monocytogenes strain containing a 339-bp in-frame deletion in prfA was generously provided by Hao Shen (University of Pennsylvania) and Jeff Miller (UCLA); this strain was transduced with a phage U153 lysate (20) obtained from NF-L758, and the resulting erythromycin-resistant transductant was designated NF-L1003. NF-L1003 therefore contains an in-frame deletion of prfA with a downstream copy of erm located between orfZ and orfB in the presence of a chromosomal actA-gus transcriptional fusion. L. monocytogenes strain NF-L879 was isolated as a mutant with high in vitro actA-gus expression following chemical mutagenesis of NF-L476 with ethylene methylsulfonate (EMS) (43). The integration vector pPL2 has been described (28) and was used for the construction of the recombinant pPL2prfA constructs listed in Table 1 (described below). L. monocytogenes and E. coli were grown in brain heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.) and Luria broth (LB) (Invitrogen Corp., Grand Island, N.Y.), respectively. For selection of pPL2 or its recombinant plasmids, chloramphenicol at 25 μg/ml was used for E. coli; chloramphenicol at 7.5 μg/ml was used for L. monocytogenes, and streptomycin at 200 μg/ml was used to select for L. monocytogenes following conjugation. For solid media, 2.5% (wt/vol) agar was included.
Bacterial strains used in this study
Preparation of bacteriophage U153 lysates.High-titer U153 lysates were prepared as described previously with minor modifications (20). Dilutions of a starting lysate were prepared in TM buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgSO4), and 100 μl of diluted lysate was added to sterile 13- by 100-mm glass tubes containing 100 μl of mid-log-phase L. monocytogenes grown in LB with shaking at 30°C. Bacterial cultures were incubated with U153 lysate dilutions at room temperature (RT) for 40 min, and then 3 ml of molten LB plus 0.75% [wt/vol] agar-10 mM MgSO4-10 mM CaCl2 was added to each tube, followed by gentle mixing and immediate pouring of the tube contents onto LB-10 mM MgSO4-10 mM CaCl2 plates, which were then incubated at RT overnight. Plates containing U153 plaques just touching one another (giving the lawn of L. monocytogenes a lacy appearance) were selected for the preparation of fresh bacteriophage lysates. TM buffer (2 ml) was added to the selected plates, and the plates were incubated for 20 min at RT. The soft top agar layer was then broken up with a sterile glass spreader, and the agar and liquid were transferred into a sterile centrifuge tube and vortexed. The supernatant was recovered after centrifugation at 7,000 × g for 10 min, and chloroform was added to 10% (vol/vol) final volume. After the chloroform was mixed in and allowed to settle, bacteriophage lysates in the supernatant were transferred to sterile tubes and mixed with equal volumes of 100% glycerol. The lysates obtained were generally on the order of 108 to 109 PFU/ml.
U153 bacteriophage-mediated transduction.High-titer U153 lysates (50 to 200 μl) prepared from strain NF-L758 (NF-L476 with an erm insertion downstream of the PrfA regulon) were mixed with 200 μl of mid-exponential-phase L. monocytogenes mutant strain NF-L879 (grown at 30°C with shaking) in the presence of 10 mM MgSO4 and 10 mM CaCl2, and the mixture was incubated at RT for 1 h with occasional gentle mixing. An aliquot of 3 ml of warm BHISA (BHI plus 0.75% [wt/vol] agar-10 mM sodium citrate, pH 7.5) containing 50 μg of 5-bromo-4-chloro-3-indolyl-β-d-glucuronide salt (XG; Inalco)/ml was added with gentle mixing to the bacteriophage-L. monocytogenes suspension, erythromycin at 1.3 μg/ml was included to induce erm expression, and the mixture was immediately poured onto BHI plates containing 10 mM sodium citrate (pH 7.5) and 50 μg of XG/ml. After incubation at 37°C for 2 h, another layer of BHISA containing 50 μg of XG/ml and 13 μg of erythromycin/ml was poured onto the plates, which were again incubated at 37°C. Bacterial transductants were usually visible within 1 to 2 days, and Ermr transductants were scored for blue or white colony color. Mutants containing mutations closely linked to the erm gene produced white transductants on indicator plates at a frequency that correlated with the distance of the mutation from the erm marker [distance = (1 − the cube root of the transduction frequency) × phage size (40.8 kb for U153)] (20). Transductants isolated after phage infection of mutant NF-L879 were either dark blue or white. The dark-blue ones retained the mutation that conferred a high level of in vitro actA-gus expression, whereas the white colonies contained the WT copy of prfA from NF-L758.
Construction of pPL2 recombinant plasmids.Genomic DNA was isolated from the L. monocytogenes WT strain NF-L476 and the EMS mutant strain NF-L879 for splicing by overlap extension PCR (21) to clone the WT or mutant prfA genes with the prfA and plcA promoters and their PrfA-binding palindromes into the integrative vector pPL2. The primers 5′SacI-pPlcA (5′-GGGAATTCGGTATCAAATAAAACG-3′; the added SacI site is underlined) and 3′SalI prfA tx term (5′-AATCGCTTTCTTTACTGCAGGA-3′; the added SalI site is underlined) (sequences of both primers were provided by D. Higgins, Harvard Medical School, Boston, Mass.) were used as the external primer pair for splicing by overlap extension PCR with the internal primer pairs delplcAR (5′-TACTTTGTTGTTTAATGCTGCATTAAAATAAATTGG-3′) and delplcAF (5′-CCAATTTATTTTAATGCAGCATTAAACAACAAAGTA-3′). The internal primers generated a 453-bp in-frame deletion in plcA. An ∼500-bp product was obtained from PCR with 5′SacI-pPlcA and delplcAR, and a 1.2-kb fragment was amplified with delplcAF and 3′SalI prfA tx term. The two overlapping products formed were purified with a QIAquick gel extraction kit (Qiagen Inc., Valencia, Calif.) and subjected to a second PCR containing only the two external primers. The final product (∼1.7 kb) was gel purified, digested with SacI and SalI, and ligated to pPL2 cut with the same enzymes for transformation into E. coli DH5α. After the inserted fragments were checked by sequencing, each of the recombinant plasmids was electroporated into E. coli SM10 for conjugation into recipient L. monocytogenes strains.
Conjugation and plasmid integration.Conjugation of the pPL2 recombinant plasmids from E. coli SM10 into recipient strains was performed as previously described (28) with slight modifications. Briefly, the bacterial strains were grown at 30°C with shaking to mid-log phase (optical density at 600 nm [OD600], ∼0.6). E. coli donor strains were grown in LB containing 25 μg of chloramphenicol/ml, and L. monocytogenes recipient strains were grown in BHI. The donor culture (250 μl) was mixed with 150 μl of recipient culture, put onto a 0.45-μm-pore-size HA-type filter (Millipore, Billerica, Mass.) on a BHI plate without antibiotics, and incubated at 30°C for 2 h. The bacterial cells were then resuspended in 2 ml of BHI, and aliquots (50 to 200 μl) were mixed with 3 ml of LB top agar and overlaid onto BHI plates with 7.5 μg of chloramphenicol/ml and 200 μg of streptomycin/ml. The plates were then incubated at 30°C overnight and shifted to 37°C for 2 to 3 days. Individual colonies were screened by PCR. The colonies were picked with sterile toothpicks, streaked onto a fresh selection plate, and then boiled at 100°C for 10 min in 75 μl of sterile water. PCR was performed with 5 μl of the boiled bacterial suspension. The primer pair NC16 (5′-GTCAAAACATACGCTCTTATC-3′) and PL95 (5′-ACATAATCAGTCCAAAGTAGATGC-3′) amplified a 499-bp product in strains that are lysogenic or contain the integration vector at tRNAArg-attBB′ (28). The primer pairs 5′SacI-pPlcA and 3′SalI prfA tx term amplified a single 2.1-kb product from NF-L476 or when the vector alone (pPL2) was integrated into the chromosome; a single 1.8-kb fragment was amplified from NF-L1003 (ΔprfA) and its pPL2 integrant. An additional band at ∼1.7-kb was obtained if a pPL2 recombinant plasmid carrying a prfA allele was integrated into the chromosome. Potential mutant insertion strains were confirmed by PCR using isolated genomic DNA.
Determination of hemolytic activity.Overnight cultures of L. monocytogenes grown without shaking in BHI at 37°C were diluted 1:10 in fresh BHI broth and grown with shaking at 37°C. The culture supernatants were assayed for hemolytic activity with sheep erythrocytes as previously described (4). Hemolytic activity was determined as the reciprocal of the supernatant dilution at which 50% lysis of erythrocytes was observed and then normalized to the OD595 of the bacterial cultures.
Measurement of GUS activity.Overnight cultures of L. monocytogenes grown without shaking in BHI at 37°C were diluted 1:20 into fresh BHI and grown with shaking at 37°C. Chloramphenicol at 7.5 μg/ml was included in the medium for L. monocytogenes strains integrated with the pPL2 vector or a pPL2 recombinant plasmid. At various time points, the OD595 was determined for each culture by using a spectrophotometer (UV-1201 UV-VIS spectrophotometer; Shimadzu Scientific Instruments, Inc., Columbia, Md.). Bacterial pellets were harvested from 1-ml culture suspensions by centrifugation, washed once with ABT buffer (1 M potassium phosphate [pH 7.0], 0.1 M NaCl, 1% Triton), and resuspended in 100 μl or 1 ml of the same buffer. β-Glucuronidase (GUS) activity was measured as described by Youngman (51) with the substitution of 4-methylumbelliferyl-β-d-glucuronide (Sigma, St. Louis, Mo.) in place of 4-methylumbelliferyl-β-d-galactoside.
Motility assays.Swimming motility was evaluated on semisolid (0.3% [wt/vol] agar) BHI medium treated with 0.2% [wt/vol] activated charcoal as previously described (43). The plates were inoculated with 2 μl of mid-log-phase (OD600, ∼0.7) bacterial cultures grown in BHI at 37°C; chloramphenicol at 7.5 μg/ml was included in the media for strains with the pPL2 vector or a pPL2 recombinant plasmid integrated into the chromosome. The plates were incubated overnight at 37°C. Motility was quantified as the diameter of the swimming colony minus the diameter of a nonmotile ΔflaA L. monocytogenes deletion mutant (43). Results were obtained from duplicate samples of two independent experiments for each strain.
Plaque formation in murine L2 fibroblasts.Plaque assays were performed with murine L2 fibroblasts as described previously (45), with a multiplicity of infection of ∼1:3. Plaque size was measured using a comparator (Finescale, Orange, Calif.). Chloramphenicol at 5 μg/ml was included in the tissue culture media for L2 cells infected by L. monocytogenes strains integrated with the pPL2 vector or a pPL2 recombinant plasmid. The average diameter of 10 plaques from at least three independent experiments was determined for each strain.
PlcB activity assay.PlcB activities were assayed by streaking bacterial colonies onto BHI medium overlaid with molten agar containing activated-charcoal-treated (0.2% [wt/vol]) BHI and a 5% ([vol/vol]) concentration of a 1:1 egg yolk-phosphate-buffered saline (PBS) solution. The plates were then incubated at 37°C or room temperature for 1 to 2 days.
PrfA monoclonal antibodies.Monoclonal antibody MAB-prfA55 derived against recombinant PrfA protein containing an N-terminal six-residue histidine tag was generated by the Hope Heart Institute (Seattle, Wash.).
SDS-PAGE and Western immunoblotting.To isolate cell lysates, overnight cultures of L. monocytogenes strains were diluted 1:20 in 30 ml of fresh BHI broth and grown to an OD595 of ∼0.7 (chloramphenicol at 7.5 μg/ml was included in the media for strains with the pPL2 vector or a pPL2 recombinant plasmid integrated into the chromosome). Each culture was centrifuged at ∼5,000 × g for 10 min, washed in 1× PBS (Invitrogen, Grand Island, N.Y.), and centrifuged again. Each pellet was resuspended in 400 μl of lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0.1% [vol/vol] Triton X-100). Bacterial suspensions were mixed with 200 mg of glass beads (Sigma) and disrupted using a Mini-Beadbeater (Biospec Products, Barttesville, Okla.) at maximum speed for 20 s. Samples were kept on ice and then centrifuged at top speed in a microcentrifuge for 1 min to recover the supernatants. Protein concentrations were determined using the DC Protein Assay kit (Bio-Rad Laboratories, Hercules, Calif.) following instructions provided by the manufacturer. Equivalent amounts of total protein from each culture lysate were mixed with 4× sample buffer (0.25 mM Tris-HCl, pH 6.8, 8% [wt/vol] sodium dodecyl sulfate [SDS], 40% [vol/vol] glycerol, 4% [vol/vol] 2-mercaptoethanol, 0.04% bromophenol blue). Samples were heated to 100°C for 5 min prior to SDS- polyacrylamide gel electrophoresis (SDS-PAGE) on 15% polyacrylamide gels. The proteins were transferred to nitrocellulose membranes at 100 V for 1.5 h. Immunobloting for PrfA or PrfA derivatives was performed at RT. The membranes were briefly washed with 1× PBS, blocked with 5% (wt/vol) nonfat dry milk in 1× PBS for 1 h, and then incubated overnight with a 1:100 dilution of the monoclonal antibody MAB-prfA55 in 1× PBS with 5% (wt/vol) nonfat dry milk and 0.05% (vol/vol) Tween 20. After three 10-min washes in 1× PBS, the membranes were incubated with a 1:2,000 dilution of an alkaline phosphatase-conjugated goat anti-mouse antibody (Sigma, St. Louis, Mo.) for 30 min. Following three 10-min PBS washes and equilibration of the membranes for 5 min in detection buffer (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5), PrfA proteins were detected by using Sigma Fast 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium tablets, a colorimetric alkaline phosphatase substrate. For quantitative comparison of PrfA protein levels visualized by Western analysis, serial dilutions of protein extracts were compared to determine the relative amounts of PrfA produced by different strains.
Intracellular growth in macrophage-like J774 cells.To examine intracellular growth, cell monolayers were grown on circular coverslips (12-mm diameter) in tissue culture dishes. Bacterial cultures were grown overnight without shaking in BHI at 37°C; chloramphenicol at 7.5 μg/ml was included in the media for strains with the pPL2 vector or a pPL2 recombinant plasmid integrated into the chromosome. The cultures were washed with PBS and then used to infect J774 cells at a multiplicity of infection of ∼1:20. After infection for 30 min, the monolayers were washed three times with 37°C PBS prior to the addition of 5 ml of 37°C medium, and gentamicin was added to a final concentration of 10 μg/ml at 1 h postinfection to kill any remaining extracellular bacteria. At various time points, the coverslips were removed to determine the number of intracellular bacteria. The cell monolayers on each coverslip were lysed by vortexing them for 10 s in 5 ml of sterile water, and dilutions of the cell lysate were plated on LB plates. Bacterial CFU were determined for each strain in triplicate after overnight incubation at 37°C on LB plates.
Identification of a novel prfA mutation that results in constitutive activation of PrfA.Previous experiments using EMS chemical mutagenesis resulted in the isolation of 43 L. monocytogenes mutants that exhibited high-level in vitro expression of gene products normally induced within the cytosol of infected host cells (43). These mutants were identified by screening for isolates exhibiting increased expression of actA, a gene normally expressed at low levels in bacteria grown outside of host cells, through the use of a transcriptional actA-gus reporter gene fusion located within the bacterial chromosome. Mutants with increased GUS activity were identified by the dark-blue color of the colonies on solid media containing the indicator substrate XG. One mutant, NF-L879, exhibited stable high-level in vitro actA expression based on the dark-blue color of the colonies and was selected for further study.
To determine whether the mutation in NF-L879 was associated with a gene located within or near the prfA locus, the L. monocytogenes-transducing bacteriophage U153 (20) was used to transduce an erythromycin resistance (erm) gene marker linked to the PrfA regulon from strain NF-L758 to the NF-L879 mutant strain. NF-L758 is a derivative of the WT strain NF-L476 (actA gus), which contains a copy of erm inserted downstream of orfXYZ at the 3′ end of the PrfA regulon. If the mutation in NF-L879 was closely linked to the PrfA regulon, then transduction of NF-L879 with a phage lysate derived from NF-L758 would result in a population of white transductants with WT levels of actA-gus expression on XG medium, as well as a population of dark-blue transductants that retained the NF-L879 mutation. The frequency of white versus blue transductants would reflect how closely linked the NF-L879 mutation was to the erm marker. If the mutation in NF-L879 was not linked to the PrfA regulon, then transduction of NF-L879 with the NF-L758-derived phage lysate would result in only dark-blue transductants. Transduction of NF-L879 resulted in the isolation of white colonies on XG medium at a frequency of 47% (31 white colonies identified from a total of 66 transductants), indicating that the mutation in NF-L879 conferring high-level in vitro actA expression was closely linked (within ∼9 kb) to the PrfA regulon. DNA sequence analysis of NF-L879 prfA revealed the presence of a CTT-to-TTT mutation within the prfA coding region, which would result in the replacement of a leucine with a phenylalanine at amino acid 140 of the protein sequence (PrfA L140F) (Fig. 1). This mutation maps near the site of another prfA mutation that has been described (37), PrfA G145S (or PrfA*), which is believed to lock the protein into a constitutively activated state (37, 49). No other mutations were found within the 9-kb region encompassing the prfA regulon of NF-L879.
Locations of PrfA mutations with respect to predicted protein secondary-structure motifs and functional regions. The previously described PrfA* mutation (G145S) is included for comparison (37). AR, activation region that may form contacts with RNA polymerase; Leu-zip, leucine zipper-like motif. Depictions of structural motifs and functional regions are adapted from Goebel et al. (17).
Several attempts were made to confirm that the PrfA mutation was responsible for the high actA expression phenotype via the introduction of the PrfA L140F mutation into the parent strain, NF-L476, by allelic replacement. These attempts proved unsuccessful, despite the fact that other PrfA mutations resulting in increased virulence gene expression (PrfA G145S, PrfA E77K, and PrfA G155S) have been successfully introduced into NF-L476 using this approach (43 and data not shown). The NF-L879 mutant had no obvious growth defect in comparison to the WT parent strain, NF-L476 (data not shown), but interestingly, cultures of NF-L879 grown overnight in broth culture without shaking were repeatedly found to settle to the bottoms of the culture tubes rather than to remain in suspension (Fig. 2), suggesting that the mutant cells possessed altered surface structure or cell density. A comparative Gram stain of WT and mutant bacteria taken from settled broth cultures revealed no obvious changes in cell septation or cell shape (data not shown); thus, the bacteria were apparently not settling as a result of cell filamentation.
Overnight cultures of L. monocytogenes strains grown in BHI broth at 37°C without shaking. The strain numbers and relevant genotypes are shown underneath the respective culture tubes. 476, NF-L476 (WT); 879, NF-L879 (EMS L140F mutant); 1041, NF-L1041 (ΔprfA + WTi); 1011, NF-L1011 (ΔprfA + L140Fi); 1067, NF-L1067 (L140F + L140Fi); 1006, NF-L1006 (WT + pPL2i); 1008, NF-L1008 (WT + L140Fi).
As an alternative approach to introduction of the PrfA L140F mutation by allelic exchange, an L. monocytogenes site-specific phage integration vector, pPL2 (28), was used to introduce a single copy of a WT or mutant prfA allele into strain NF-L1003, which contains an in-frame deletion within prfA in addition to the actA-gus reporter gene fusion. Recombinant pPL2 plasmids integrated at the phage attachment site of the PSA prophage within the tRNAArg gene that is present only once in the genome of L. monocytogenes (16, 28). To provide all promoters known to contribute to prfA expression, coding sequences for WT PrfA or PrfA L140F were introduced in the presence of both of the prfAp1 and prfAp2 promoters, as well as the upstream plcA promoter. During the cloning process, another PrfA mutation (PrfA L147P) (Fig. 1) was fortuitously generated, and this mutation was also independently introduced for comparison into pPL2 for integration into the chromosome of NF-L1003. The three integrants generated were NF-L1041 (containing an integrated copy of WT prfA [WTi]), NF-L1011 (containing an integrated copy of prfA L140F [L140Fi]), and NF-L1042 (containing an integrated copy of prfA L147P [L147Pi]) (Table 1). The L140Fi integrants were isolated at the same frequency and with the same colony growth characteristics as prfA, L147Pi, and pPL2 integrants on agar plates (data not shown); however, unshaken overnight cultures of NF-L1011 (ΔprfA + L140Fi) settled to the bottoms of tubes in a manner identical to that of the original EMS mutant strain, NF-L879 (Fig. 2). In addition, in contrast to NF-L1041 (ΔprfA + WTi) or NF-L1042 (ΔprfA + L147Pi), the NF-L1011 (ΔprfA + L140Fi) integrant was found to be unstable in the absence of drug selection when grown in BHI broth culture (data not shown), suggesting that the presence of the L140Fi allele may adversely affect some aspect of L. monocytogenes growth.
Analysis of PrfA protein levels in ΔprfA strains containing integrated copies of WT or mutant prfA.To compare the levels of PrfA present in prfA integrant strains, total cell extracts derived from broth cultures grown to similar mid-log-phase densities were examined by Western analysis using a mouse monoclonal antibody directed against PrfA. No PrfA was detected in the ΔprfA parent strain NF-L1003 or in the vector control integrant NF-L1009 (ΔprfA + pPL2i) (Fig. 3A). The EMS mutant NF-L879 produced increased levels of PrfA protein in comparison to the WT strain NF-L476 (Fig. 3A). Similarly, larger amounts of PrfA protein were detected in the integrant strain NF-L1011 (ΔprfA + L140Fi) than in the control strain NF-L1041 (ΔprfA + WTi) (Fig. 3A). Interestingly, no PrfA was detected from the PrfA L147P integrant NF-L1042 (Fig. 3A), even when a rabbit polyclonal antibody against PrfA was used for detection (data not shown). The PrfA L147P mutation therefore appeared to result in an unstable, rapidly degraded form of the protein. For quantitative comparison of PrfA protein levels, serial dilutions of protein extracts were compared (Fig. 3B and C). The EMS mutant NF-L879 produced about two to four times more PrfA protein than the WT strain NF-L476 (Fig. 3B), and the integrant strain NF-L1011 (ΔprfA + L140Fi) produced at least four times more PrfA protein than the control strain NF-L1041 (ΔprfA + WTi) (Fig. 3C). These results demonstrated that the introduction of the integrated prfA mutant and WT alleles into L. monocytogenes ΔprfA strains resulted in levels of PrfA protein that were comparable to WT and mutant protein levels produced by NF-L476 and NF-L879 strains.
Western analysis of PrfA protein levels produced by the various L. monocytogenes strains. Soluble bacterial whole-cell lysates were prepared from mid-log-phase cultures. The strain numbers and relevant genotypes are shown above the lanes. PrfA was detected by using a monoclonal antibody against PrfA and an alkaline phosphatase-conjugated goat anti-mouse secondary antibody. (A) PrfA protein levels from L. monocytogenes integrant strains in the NF-L1003 (ΔprfA) background. Equal amounts of total protein (23 μg) solubilized in SDS-PAGE sample buffer were loaded for each sample. (B) Quantitative comparison of PrfA protein levels from WT strain NF-L476 (476) and original EMS mutant strain NF-L879 (879), with the amount of total proteins loaded in each lane indicated. (C) Quantitative comparison of PrfA protein levels from integrant strains NF-L1041 (ΔprfA + WTi) (1041) and NF-L1011 (ΔprfA + L140Fi) (1011), with the amount of total proteins loaded in each lane indicated.
In vitro expression of virulence genes by prfA integrant strains.PrfA is the major regulatory element controlling the transcription of many L. monocytogenes virulence genes (30, 31); thus, the effects of the PrfA mutations on the expression of several PrfA-dependent genes were examined. Listeriolysin O (LLO), encoded by hly, is important for L. monocytogenes to escape from primary host cell vacuoles and is normally expressed during bacterial growth in standard broth culture (3, 4, 11, 33). LLO activity can be readily detected in culture supernatants. In contrast to hly, actA is expressed at low-to-undetectable levels in broth-grown cultures but is highly induced following entry of L. monocytogenes into the host cell cytosol (2, 3, 11, 24, 33). Following bacterial spread into adjacent host cells, the activity of the phospholipase PlcB is important for bacterial escape from double-membrane vacuoles, and similar to actA, plcB is normally expressed at low-to-undetectable levels in bacteria grown in standard broth culture (38).
Relative amounts of secreted LLO were determined by measuring bacterial supernatants derived from mid-log-phase cultures for hemolytic activity against sheep red blood cells. Supernatants derived from the ΔprfA strain NF-L1003, the vector control integrant NF-L1009 (ΔprfA + pPL2i), and the PrfA L147P integrant strain NF-L1042 (ΔprfA + L147Pi) exhibited very little hemolytic activity (Table 2). The EMS mutant strain NF-L879 produced significantly larger amounts of LLO activity than the WT strain NF-L476 (Table 2), indicating that the PrfA L140F mutation is capable of elevating the expression of PrfA-dependent gene products distinct from actA. The integrated WTi allele in NF-L1041 (ΔprfA + WTi) complemented the prfA deletion in the NF-L1003 background, and similarly, the integrated L140Fi allele conferred levels of hemolytic activity on NF-L1011 (ΔprfA + L140Fi) that were comparable to those observed for NF-L879 (Table 2).
Phenotypic analysis of L. monocytogenes strains with prfA mutations
The PrfA L140F mutation was originally identified based on its apparent ability to confer high levels of actA expression in bacteria grown outside of host cells. Quantitative analysis of levels of actA expression was performed for prfA WT and mutant strains by measuring levels of GUS activity in strains containing actA-gus transcriptional fusions. As mentioned previously, the PrfA L140F integration appeared to be unstable under nonselective conditions, and all integrant strains were therefore grown in the presence of chloramphenicol. The inclusion of selective concentrations of chloramphenicol did not affect the growth rates of integrant strains compared to the rate observed for NF-L476, NF-L879, or NF-L1003 (ΔprfA) grown in BHI alone (Fig. 4A). The ΔprfA control strains NF-L1003 and NF-L1009 (ΔprfA + pPL2i) had almost undetectable levels of GUS activity, and the same was observed for the integrant NF-L1042 (ΔprfA + L147Pi) (Fig. 4B); all of these strains did not produce detectable PrfA protein as observed by Western analysis (Fig. 3A). The integration of a WTi copy into the prfA deletion strain NF-L1003 completely restored actA expression levels in NF-L1041 (ΔprfA + WTi) to those observed for the WT strain NF-L476 (Fig. 4B). actA expression of the original EMS mutant NF-L879 was observed to peak during exponential growth, and levels of expression were up to 37-fold higher than the expression levels observed for strains containing a single copy of the WT prfA allele (NF-L476 and NF-L1041) (Fig. 4C). The actA expression profile of integrant NF-L1011 (ΔprfA + L140Fi) was very similar to that of NF-L879 (Fig. 4C), indicating that the L140F mutation in PrfA was responsible for the high in vitro actA induction in the original EMS mutant. NF-L1011 (ΔprfA + L140Fi) produced increased levels of PrfA protein in comparison to the original NF-L879 mutant strain (Fig. 3A); the similar levels of actA expression observed for NF-L879 and NF-L1011, therefore, suggest that the mutant PrfA L140F reached a level of PrfA saturation beyond which there was no further increase in the induction of actA expression despite increased production of PrfA.
actA expression of PrfA mutant strains and corresponding control strains during growth in broth culture. Units of GUS activity were determined at the indicated time intervals as described in Materials and Methods and were normalized for bacterial culture OD595 (51), using 4-methylumbilliferyl-β-d-glucuronide as the substrate. The data shown are from duplicate samples and are representative of at least three independent experiments, expressed as average ± standard error. (A) Growth of WT and PrfA mutant strains in BHI broth at 37°C as measured by OD595 of cultures. (B) actA expression from strains NF-L476 (WT), NF-L1003 (ΔprfA), NF-L1009 (ΔprfA + pPL2i), NF-L1041 (ΔprfA + WTi), and NF-L1042 (ΔprfA + L147Pi). (C) actA expression from strains NF-L476 (WT), NF-L879 (EMS L140F), and NF-L1011 (ΔprfA + L140Fi).
The expression of plcB is coregulated with that of actA (42). PlcB activity was not observed for the ΔprfA control strains NF-L1003 and NF-L1009 (ΔprfA + pPL2i) or for the integrant NF-L1042 (ΔprfA + L147Pi) (Fig. 5). No PlcB activity was observed for WT strain NF-L476 and the integrant NF-L1041 (ΔprfA + WTi) (Fig. 5). In contrast, NF-L1011 (ΔprfA + L140Fi) exhibited increased levels of PlcB activity similar to that observed for the original EMS mutant NF-L879 (Fig. 5), even in media not treated with activated charcoal (treatment of BHI broth with activated charcoal has been reported to induce the expression of plcB [38]) (data not shown).
PlcB (lecithinase) phenotypes of L. monocytogenes mutants and corresponding control strains on BHI medium overlaid with molten agar containing activated-charcoal-treated (0.2% [wt/vol]) BHI and 5% (vol/vol) 1:1 egg yolk-PBS solution. The plates were incubated at 37°C overnight. 476, NF-L476 (WT); 879, NF-L879 (EMS L140F mutant); 1003, NF-L1003 (ΔprfA); 1006, NF-L1006 (WT + pPL2i); 1039, NF-L1039 (WT + WTi); 1040, NF-L1040 (WT + L147Pi); 1008, NF-L1008 (WT + L140Fi); 1009, NF-L1009 (ΔprfA + pPL2i); 1041, NF-L1041 (ΔprfA + WTi); 1042, NF-L1042 (ΔprfA + L147Pi); 1011, NF-L1011 (ΔprfA + L140Fi); 1067, NF-L1067 (L140F + L140Fi).
In summary, the introduction of a WT copy of prfA via vector integration at the tRNAArg-attBB′ site was sufficient to fully complement PrfA-dependent gene expression in the prfA deletion strain NF-L1003. The introduction of the PrfA L140F mutation demonstrated that this mutation was sufficient for generating the high-level in vitro actA-gus expression observed for the EMS mutant NF-L879; production of LLO and PlcB was also increased in both strains. In contrast, the introduction of the PrfA L147P mutation did not lead to the induction of PrfA-dependent gene expression, indicating that this mutation results in an inactive or unstable form of the protein.
The PrfA L140F mutation is phenotypically dominant to WT PrfA.The PrfA protein is thought to be active as a multimer based on its similarity to E. coli Crp and on the recently solved PrfA crystal structure (27, 49; http://www.rcsb.org/pdb/). It seemed possible, therefore, that strains producing both the PrfA WT and PrfA L140F proteins might exhibit altered patterns of PrfA-dependent gene expression as a result of PrfA heterodimer formation. The PrfA L140F and PrfA L147P mutations, with the two prfA promoters and the plcA promoter, were therefore introduced into the chromosome of WT strain NF-L476 to examine the potential effects on PrfA-dependent gene expression. prfA merodiploid integrants NF-L1008 (WT + L140Fi) and NF-L1040 (WT + L147Pi) were generated, along with strain NF-L1006 with an integrated vector alone (WT + pPL2i) and a WTi merodiploid strain, NF-L1039 (WT + WTi), as controls. All strains exhibited apparently normal growth characteristics in broth cultures (data not shown). Similar to the EMS mutant NF-L879 and the NF-L1011 (ΔprfA + L140Fi) integrant, unshaken overnight cultures of NF-L1008 (WT + L140Fi) were observed to settle to the bottoms of culture tubes (Fig. 1).
Western analysis of PrfA protein levels in the vector control integrant NF-L1006 (WT + pPL2i) and the merodiploid NF-L1040 (WT + L147Pi) demonstrated levels of PrfA protein comparable to those of the WT strain NF-L476 (Fig. 6A). This result suggested that mutant PrfA L147P protein was not produced in NF-L1040 (WT + L147Pi) and that the presence of the mutant allele did not affect the expression or stability of WT PrfA. Increased amounts of PrfA protein were detected in extracts derived from NF-L1008 (WT + L140Fi) (Fig. 6A), with protein levels at least fourfold higher than in the vector control integrant NF-L1006 (WT + pPL2i) (Fig. 6B). Two copies of the WTi allele in NF-L1039 (WT + WTi) also led to a >4-fold increase in PrfA synthesis (Fig. 6A and C).
Western analysis of PrfA protein levels produced by the various L. monocytogenes strains. Soluble bacterial whole-cell lysates were prepared from mid-log-phase cultures. The strain numbers and relevant genotypes are shown above the lanes. PrfA was detected by using a monoclonal antibody against PrfA and an alkaline phosphatase-conjugated goat anti-mouse secondary antibody. (A) PrfA protein levels from L. monocytogenes integrant strains in the WT strain NF-L476 background. Equal amounts of total protein (23 μg) solubilized in SDS-PAGE sample buffer were loaded for each sample. (B) Quantitative comparison of PrfA protein levels from integrant strains NF-L1006 (WT + pPL2i) (1006) and NF-L1008 (WT + L140Fi) (1008). (C) Quantitative comparison of PrfA protein levels from integrant strains NF-L1006 (WT + pPL2i) and NF-L1039 (WT + WTi) (1039). (D) Quantitative comparison of PrfA protein levels from EMS mutant strain NF-L879 (879), NF-L1003 (ΔprfA) (1003), and integrant strain NF-L1067 (L140F + L140Fi) (1067). The amounts of total proteins loaded in each lane in panels B, C, and D are indicated.
The various prfA merodiploid strains were then examined for PrfA-dependent gene expression. The vector control integrant NF-L1006 (WT + pPL2i) had actA expression levels similar to those observed for WT strain NF-L476, as did the NF-L1040 (WT + L147Pi) integrant (Fig. 7B). Despite the high level of PrfA protein produced by NF-L1039 (WT + WTi) (Fig. 6A and C), this strain had only a twofold increase in actA expression in comparison to WT strains at both 3 and 5 h of growth (Fig. 7B). actA expression levels from the NF-L1008 (WT + L140Fi) integrant were the same as those from the original EMS mutant NF-L879, indicating the apparent dominance of PrfA L140F over WT PrfA (Fig. 7C).
actA expression of PrfA mutant strains and corresponding control strains during growth in broth culture. Units of GUS activity were determined at the indicated time intervals as described in Materials and Methods and were normalized for bacterial culture OD595 (51) using 4-methylumbilliferyl-β-d-glucuronide as the substrate. The data shown are from duplicate samples and are representative of at least three independent experiments, expressed as average ± standarderror. (A) Growth of WT and PrfA mutant strains in BHI broth at 37°C as measured by OD595 of cultures. (B) actA expression from strains NF-L476 (WT), NF-L1003 (ΔprfA), NF-L1006 (WT + pPL2i), NF-L1039 (WT + WTi), and NF-L1040 (WT + L147Pi). (C) actA expression from strains NF-L476 (WT), NF-L879 (EMS L140F), and NF-L1008 (WT + L140Fi). (D) actA expression from strains NF-L476 (WT), NF-L879 (EMS L140F), and NF-L1067 (L140F + L140Fi).
Similar results were obtained when the strains were examined for LLO activity. NF-L1006 (WT + pPL2i) and NF-L1040 (WT + L147Pi) produced levels of LLO that were the same as those produced by WT NF-L476, and NF-L1039 (WT + WTi) LLO activity was approximately twofold higher than that of strains with only one copy of WTi (Table 2). Integrant NF-L1008 (WT + L140Fi) retained high hemolytic activity, confirming the dominance of the PrfA L140F mutation over the WT (Table 2). An increase in PlcB activity was observed only for the NF-L1008 strain (WT + L140Fi) (Fig. 5). If there was increased PlcB activity in NF-L1039 (WT + WTi), it was too low to be detected using this assay.
Construction of an L. monocytogenes strain containing two copies of L140Fi.To examine the potential effects on PrfA-dependent gene expression that would result from the presence of multiple copies of the PrfA L140F mutation, the L140Fi allele was introduced via integration into the EMS mutant NF-L879 to generate NF-L1067 (L140F + L140Fi). Unshaken overnight cultures of NF-L1067 were observed to settle to the bottoms of culture tubes (Fig. 1), although NF-L1067 had no obvious growth defects in broth culture (data not shown). Approximately two- to fourfold more PrfA protein was produced in NF-L1067 than in its EMS mutant parent strain, NF-L879 (Fig. 6D), but hly, actA, and plcB expression levels were not found to differ significantly between the two strains (Table 2 and Fig. 5 and 7D). These data indicate that the induction of PrfA-dependent gene expression was already saturated with the amount of activated PrfA protein synthesized from one L140Fi allele, and therefore, the introduction of a second copy resulted in no additional increase.
Intracellular growth and cell-to-cell spread of prfA mutant strains in tissue culture cells.The capacity of L. monocytogenes to replicate within the cytosol and spread to adjacent cells can be assessed by visualizing zones of cell clearing (plaques) in monolayers of infected tissue culture cells (45). Strains that are deficient in intracellular growth and/or cell-to-cell spread fail to form plaques or form plaques of reduced size. It has been demonstrated that strains lacking functional PrfA do not escape from host cell vacuoles and are unable to replicate within cells or spread to adjacent cells; these strains do not form plaques in monolayers of mouse L2 fibroblasts (13, 30). As anticipated, strains NF-L1003 (ΔprfA), NF-L1009 (ΔprfA + pPL2i), and NF-L1042 (ΔprfA + L147Pi) failed to form plaques in monolayers of L2 fibroblasts (Table 2). The EMS mutant NF-L879 formed plaques that were ∼79% the size of those formed by WT NF-L476 (Table 2). In the presence of chloramphenicol, integrant strain NF-L1041 (ΔprfA + WTi) formed plaques smaller than those formed by NF-L476 (63.5% ± 6.4% [average ± standard deviation with NF-L476 set at 100%]); however, in the absence of the antibiotic, plaque formation was comparable to that of the WT (data not shown). Chloramphenicol therefore appeared to reduce the efficiency of plaque formation, despite the presence of the cat gene in the pPL2 integrant strains. The inclusion of chloramphenicol was necessary to ensure the stability of the integrated L140Fi allele; therefore, the drug was included for all pPL2 integrants, and the plaque size of NF-L1041 (ΔprfA + WTi) was set at 100% for comparison with mutant prfA integrant strains. The integrant strain NF-L1011 (ΔprfA + L140Fi) was still found to form reproducibly smaller plaques than strain NF-L1041 (ΔprfA + WTi); however, the plaques were only slightly (∼10%) smaller than those formed in the presence of the WTi allele (Table 2). L. monocytogenes strains containing the L140Fi allele may therefore be slightly deficient in invasion, intracellular replication, and/or cell-to-cell spread. No significant differences were observed in plaque size or frequency for any of the various prfA integrant strains in the WT background or, interestingly, for NF-L1067 containing two copies of L140Fi (Table 2). Experiments examining bacterial infection of the macrophage-like cell line J774 indicated that all integrant strains had similar intracellular growth for at least 7 h postinfection (data not shown).
L. monocytogenes strains containing L140Fi exhibit decreased swimming motility.Previous work has demonstrated that two strains of L. monocytogenes that contained constitutively activated forms of PrfA protein (PrfA E77K and PrfA G155S) exhibited decreased swimming motility in soft agar (43). We therefore examined the swimming motility of the PrfA L140F strains. The EMS mutant NF-L879 and integrants NF-L1008 (WT + L140Fi), NF-L1011 (ΔprfA + L140Fi), and NF-L1067 (L140F + L140Fi) were all strikingly deficient in swimming motility, while the other integrants were similar in motility to WT strain NF-L476 (Table 2). Strains containing constitutively activated forms of PrfA (such as PrfA L140F) may thus be deficient in nutrient acquisition and exhibit decreased fitness for survival in environments outside of host cells.
The transcriptional regulator PrfA of L. monocytogenes tightly coordinates the expression of many virulence determinants to facilitate bacterial survival both inside and outside of host cells. Regulation of prfA expression and PrfA activity occurs on multiple levels and includes transcriptional, posttranscriptional, and posttranslational regulatory mechanisms, all of which are necessary for pathogenesis (5, 12, 13, 19, 22). In this study, we have identified a novel mutation within PrfA (PrfA L140F) that results in a constitutively activated form of the protein and high-level virulence gene expression in bacteria grown outside of host cells. The discovery of the PrfA L140F mutation brings the total number of PrfA-activating mutations defined to four, and each mutation appears to have its own distinguishable effect on PrfA function. Deciphering how specific structural alterations in the PrfA protein alter distinct functional aspects of this key virulence regulator will further illuminate how L. monocytogenes controls virulence gene expression in response to environmental cues.
In contrast to the three previously described mutations within PrfA that lead to the protein's constitutive activation (PrfA G145S, PrfA G155S, and PrfA E77K), the PrfA L140F mutation conferred some degree of toxicity upon bacterial cells. Multiple attempts to reconstruct the PrfA L140F mutation in WT L. monocytogenes by allelic replacement proved unsuccessful. In addition, when the mutant allele was integrated into an ectopic location on the L. monocytogenes chromosome (as in strain NF-L1011 [ΔprfA + L140Fi]), the L140Fi allele could not be stably maintained in the absence of selective pressure (unlike the WT allele, which was stable without selection). The observation that unshaken overnight cultures of L. monocytogenes strains harboring the L140Fi allele settled to the bottoms of culture tubes suggests that the presence of PrfA L140F results in an altered bacterial surface structure or cell density (Fig. 1). Altered bacterial cell physiology or morphology probably reflects changes in the spectrum of gene products normally produced by the bacteria during growth in broth culture; these products are likely to be either directly or indirectly regulated by PrfA. Preliminary analysis of L. monocytogenes membranes and secreted proteins of PrfA L140F strains using denaturing PAGE indicates readily detectable changes in the abundance of several polypeptide species (K. K. Y. Wong, unpublished data). The PrfA-directed alterations in L. monocytogenes bacterial surfaces or cell physiology may function to promote bacterial survival in the cytosol of infected host cells, an environment demonstrated to induce PrfA activation (42). It is notable that, in contrast to the pPL2-prfA L140F reconstructed L. monocytogenes strains, the PrfA L140F mutation is stably maintained in the original NF-L879 mutant isolate; it is possible that this strain contains additional mutation(s) that alleviate the toxicity associated with this PrfA mutant allele. NF-L879, however, formed plaques in fibroblast monolayers that were significantly smaller than those formed by WT strains and smaller than those formed by the isogenic NF-L1011 (ΔprfA + L140Fi) mutant (79% ± 3% for NF-L879 versus 90% ± 2% for NF-L1011). It is possible, therefore, that the mutation in NF-L879 that compensates for the presence of the L140Fi allele adversely affects the ability of L. monocytogenes to spread to adjacent cells. Alternatively, NF-L879 may contain an independent mutation, unrelated to the presence of L140Fi, that reduces cell-to-cell spread. We do not believe that the L140Fi suppressor mutation is present in either NF-L1011 or NF-L1008, as these strains do not stably maintain the L140Fi allele in the absence of drug selection, and multiple L. monocytogenes isolates obtained from independent pPL2-prfA L140F vector transformations were indistinguishable from NF-L1011 and NF-L1008 in independent assays (K. K. Y. Wong, unpublished data). Additionally, L. monocytogenes integrants containing the pPL2-prfA L140F plasmid were isolated at the same frequency and with the same colony growth characteristics on agar plates as integrants containing the pPL2-prfA or pPL2 vector, a result that suggests that the toxicity associated with the presence of L140Fi is not severe.
The relative levels of PrfA protein have been shown to be important for optimal induction of virulence gene expression (5, 12, 13, 36). PrfA synthesis increases upon entry of L. monocytogenes into host cells (36), and this increase is necessary to direct the expression of gene products required for bacterial spread to adjacent cells (5, 12, 13). The data presented here indicate that saturating levels of virulence gene expression are more rapidly achieved by activated forms of PrfA. For example, the introduction of a second WT copy of prfA into L. monocytogenes doubled the production of LLO and ActA (Table 2 and Fig. 7). However, no significant increase in either actA or hly expression was observed for L. monocytogenes strains containing two L140Fi alleles (NF-L1067 [L140F + L140Fi]) versus those with one copy (NF-L879 and NF-L1011 [ΔprfA + L140Fi]) (Table 2 and Fig. 4 and 7), even though the presence of a second copy increased the levels of PrfA protein produced in these strains (Fig. 6). These results suggest that the expression of PrfA-dependent genes is most sensitive to variations in PrfA protein levels when PrfA is present in its nonactivated state.
The apparent phenotypic dominance of the L140Fi allele over the WT allele is interesting in light of predictions that PrfA is active as a multimer. These predictions are based upon the homology PrfA shows with Crp and upon the recently solved crystal structure of PrfA (37, 49; http://www.rcsb.org/pdb/). The merodiploid integrant NF-L1008 (WT + L140Fi) presumably produces both WT PrfA and PrfA L140F, but this has not been definitely demonstrated. The PrfA L140F protein would be predicted to activate expression not only from its own upstream plcA promoter but also from that of the WT copy, thus leading to high-level expression of both proteins. It is possible that PrfA L140F has sufficiently high promoter binding affinity to outcompete the WT protein at all promoters examined; it has been reported that WT PrfA could compete and neutralize the activation of virulence genes by an activated PrfA form (PrfA G145S) if the WT PrfA was produced in trans from a multicopy plasmid (37). We favor the possibility that PrfA L140F may induce conformational changes in the WT protein upon multimerization that lead to activation of both PrfA molecules. Alternatively, the PrfA L140F mutation may serve to stabilize the formation of active PrfA dimers.
As mentioned above, the crystal structure of PrfA has recently been solved and released (Protein Data Bank accession code 1OMI [http://www.rcsb.org/pdb/]). The structure obtained indicates that PrfA is structurally similar to the cAMP receptor protein (Crp) from E. coli (50). Figure 8 depicts the locations of the PrfA L140F and the PrfA L147P mutations within the WT structure. Also depicted is the original PrfA G145S mutation, the first mutation identified that resulted in a constitutively active form of PrfA (PrfA*) (37). The PrfA L140F and L147P mutations are located very close to the DNA-binding helix-turn-helix (HTH) domain of PrfA. The phenylalanine side chain of PrfA L140F, based on its location within the WT structure, might possibly alter the positioning of the HTH domain to facilitate the interaction of this region with DNA; confirmation of this hypothesis awaits further structural and functional analyses of PrfA L140F. In contrast, proline substitutions often introduce dramatic turns into protein chains, and thus, the PrfA L147P substitution might result in improper folding and protein degradation, consistent with the failure of this strain to produce detectable PrfA protein.
Locations of PrfA mutations in the crystal structure of PrfA (http://www.rcsb.org/pdb/). The two diagrams illustrate two different views of the PrfA dimer. The blue and yellow traces are the Cα traces of the two monomers of PrfA. The HTH motifs are shown in red. The previously described PrfA* mutation (G145S) is included for comparison (37).
Finally, it has been proposed that L. monocytogenes is a bacterium that must mediate a balance between virulence for mammalian hosts and survival in environments outside of host cells (43). L. monocytogenes strains containing the prfA E77K or prfA G155S allele are fully virulent (or, in the case of PrfA G155S, hypervirulent) in murine models of infection; however, these mutants are severely compromised in swimming motility, a behavior that is likely to be important for nutrient acquisition outside of host cells (43). Similarly, strains containing the PrfA L140F mutation were found to be defective for swimming motility (Table 2). Elucidating how the activation status of PrfA contributes to the survival of L. monocytogenes in its varied environments will aid our understanding of how L. monocytogenes is able to adapt and flourish in such a wide diversity of habitats.
We thank Darren Higgins for the pPL2 plasmid, the SM10/pPL2 strain, and the sequences for primers 5′SacI-pPlcA and 3′SalI prfA tx term. We thank Hao Shen and Jeff Miller for the gift of the L. monocytogenes prfA deletion strain and Daniel Portnoy and Richard Calendar for the gift of the bacteriophage U153. We also thank members of the Freitag laboratory for helpful discussions and the reviewers of the manuscript for insightful comments.
This work was supported by Public Health Service grant AI41816 (N.E.F.) from the National Institutes of Health and by the M. J. Murdock Charitable Trust.
Received 30 January 2004.
Accepted 9 June 2004.
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Gray, M. L., and A. H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacteriol. Rev. 30:309-382.
Greene, S. L., and N. E. Freitag. 2003. Negative regulation of PrfA, the key activator of Listeria monocytogenes virulence gene expression, is dispensable for bacterial pathogenesis. Microbiology 149:111-120.
Hodgson, D. A. 2000. Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes. Mol. Microbiol. 35:312-323.
Horton, R. 1993. In vitro recombination and mutagenesis of DNA, p. 251-260. In B. White (ed.), PCR protocols: current methods and applications. Humana Press Inc., Totowa, N.J.
Johansson, J., P. Mandin, A. Renzoni, C. Chiaruttini, M. Springer, and P. Cossart. 2002. An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110:551-561.
Kathariou, S., P. Metz, H. Hof, and W. Goebel. 1987. Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes. J. Bacteriol. 169:1291-1297.
Kocks, C., E. Gouin, M. Tabouret, P. Berche, H. Ohayon, and P. Cossart. 1992. L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. Cell 68:521-531.
Körner, H., H. J. Sofia, and W. G. Zumft. 2003. Phylogeny of the bacterial superfamily of Crp-Fnr transcription regulators: exploiting the metabolic spectrum by controlling alternative gene programs. FEMS Microbiol. Rev. 792:1-34.
Kreft, J., and J. A. Vazquez-Boland. 2001. Regulation of virulence genes in Listeria. Int. J. Med. Microbiol. 291:145-157.
Lampidis, R., R. Gross, Z. Sokolovic, W. Goebel, and J. Kreft. 1994. The virulence regulator protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and both belong to the Crp-Fnr family of transcription regulators. Mol. Microbiol. 13:141-151.
Lauer, P., M. Y. Chow, M. J. Loessner, D. A. Portnoy, and R. Calendar. 2002. Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors. J. Bacteriol. 184:4177-4186.
Leimeister-Wachter, M., W. Goebel, and T. Chakraborty. 1989. Mutations affecting hemolysin production in Listeria monocytogenes located outside the listeriolysin gene. FEMS Microbiol. Lett. 53:23-29.
Leimeister-Wachter, M., C. Haffner, E. Domann, W. Goebel, and T. Chakraborty. 1990. Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of Listeria monocytogenes. Proc. Natl. Acad. Sci. USA 87:8336-8340.
Mengaud, J., S. Dramsi, E. Gouin, J. A. Vazquez-Boland, G. Milon, and P. Cossart. 1991. Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated. Mol. Microbiol. 5:2273-2283.
Mengaud, J., M. F. Vicente, J. Chenevert, J. M. Pereira, C. Geoffroy, B. Gicquel-Sanzey, F. Baquero, J. C. Perez-Diaz, and P. Cossart. 1988. Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes. Infect. Immun. 56:766-772.
Moors, M. A., B. Levitt, P. Youngman, and D. A. Portnoy. 1999. Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes. Infect. Immun. 67:131-139.
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Portnoy, D. A., T. Chakraborty, W. Goebel, and P. Cossart. 1992. Molecular determinants of Listeria monocytogenes pathogenesis. Infect. Immun. 60:1263-1267.
Renzoni, A., P. Cossart, and S. Dramsi. 1999. PrfA, the transcriptional activator of virulence genes, is upregulated during interaction of Listeria monocytogenes with mammalian cells and in eukaryotic cell extracts. Mol. Microbiol. 34:552-561.
Ripio, M. T., G. Dominguez-Bernal, M. Lara, M. Suarez, and J. A. Vazquez-Boland. 1997. A Gly145Ser substitution in the transcriptional activator PrfA causes constitutive overexpression of virulence factors in Listeria monocytogenes. J. Bacteriol. 179:1533-1540.
Ripio, M. T., G. Dominguez-Bernal, M. Suarez, K. Brehm, P. Berche, and J. A. Vazquez-Boland. 1996. Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition. Res. Microbiol. 147:371-384.
Sheehan, B., A. Klarsfeld, R. Ebright, and P. Cossart. 1996. A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence. Mol. Microbiol. 20:785-797.
Sheehan, B., A. Klarsfeld, T. Msadek, and P. Cossart. 1995. Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator. J. Bacteriol. 177:6469-6476.
Sheehan, B., C. Kocks, S. Dramsi, E. Gouin, A. D. Klarsfeld, J. Mengaud, and P. Cossart. 1994. Molecular and genetic determinants of the Listeria monocytogenes infectious process. Curr. Top. Microbiol. 192:187-216.
Shetron-Rama, L. M., H. Marquis, H. G. Bouwer, and N. E. Freitag. 2002. Intracellular induction of Listeria monocytogenes actA expression. Infect. Immun. 70:1087-1096.
Shetron-Rama, L. M., K. Mueller, J. M. Bravo, H. G. Bouwer, S. S. Way, and N. E. Freitag. 2003. Isolation of Listeria monocytogenes mutants with high-level in vitro expression of host cytosol-induced gene products. Mol. Microbiol. 48:1537-1551.
Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vitro genetic engineering: transponson mutagenesis in Gram negative bacteria. Bio/Technol. 1:784-791.
Sun, A. N., A. Camilli, and D. A. Portnoy. 1990. Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread. Infect. Immun. 58:3770-3778.
Tilney, L. G., P. S. Connelly, and D. A. Portnoy. 1990. Actin filament nucleation by the bacterial pathogen, Listeria monocytogenes. J. Cell Biol. 111:2979-2988.
Tilney, L. G., and D. A. Portnoy. 1989. Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J. Cell Biol. 109:1597-1608.
Vazquez-Boland, J. A., M. Kuhn, P. Berche, T. Chakraborty, G. Dominguez-Bernal, W. Goebel, B. Gonzalez-Zorn, J. Wehland, and J. Kreft. 2001. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 14:584-640.
Vega, Y., C. Dickneite, M. T. Ripio, R. Böckmann, B. Gonzalez-Zorn, S. Novella, G. Dominguez-Bernal, W. Goebel, and J. A. Vazquez-Boland. 1998. Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA. J. Bacteriol. 180:6655-6660.
Weber, I. T., and T. A. Steitz. 1987. Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 Å resolution. J. Mol. Biol. 198:311-326.
Youngman, P. 1987. Plasmid vectors for recovering and exploiting Tn917 transpositions in Bacillus and other gram-positive bacteria, p. 79-103. In K. Hardy (ed.), Plasmids: a practical approach. IRL Press, Oxford, England.
Journal of Bacteriology Sep 2004, 186 (18) 6265-6276; DOI: 10.1128/JB.186.18.6265-6276.2004
You are going to email the following A Novel Mutation within the Central Listeria monocytogenes Regulator PrfA That Results in Constitutive Expression of Virulence Gene Products


This song is said to have been written by Henry VIII, King of England, in the 16th century. It's a beautiful melody, although, it's a questionable listening activity for non-English speakers because the language is so old. Still, that's the beauty of having short songs – they aren't going to frustrate learners too much. By the way, the song has many verses; I only sing one here.
Give out all the words to the song, and let students do a few sing-alongs and learn the melody.
Tell students they are to write new words to the same melody.
That's the number of syllables. There are eight. Actually, there are nine because "love" is sung like "luh-uhv," a two syllable word.
Your students might re-write the song using these opening lines as starting points. Or even better – they can make an entirely new song. It's, however, a good idea to write out the blanks on the board to give them a pattern. It doesn't have to be nine syllables: you can add or subtract a few.
If you just want to do a listening activity, you're welcome to copy the text below and remove some words before supplying it to the students.


We will set up an initial 15 minute call to assess what your goals are and receive your initial swing video. We will provide tips, drills and video analysis.
What do online lessons consist of?
Online lessons start with an initial assessment call to understand your goals. We then discuss the video angles we need for you to send us. We work with you to make the video transfer easy for you. The remaining lessons are done online.
We will utilize the V1 Sports app. We'll upload your swings, create an account for you, and use the app to record instruction as well as provide side-by-side analysis.
We have various forms of payment and will work with you to use the best form for you.


1- Department of Horticulture, Damghan branch, Islamic Azad university, Damghan, Iran.
Abstract: One of the main problems of almond producers in Iran is the irregular and fluctuating production rates. This is the result of early flowering of native genotypes and coincidence of their flowering times with a cold spring.An experiment was done on three major commercial of Monagha, Sh- 12 and , Sh-18 through a double-factor factorial in the form of random blocks(cultivar, cold and phenological phase at 3, 6 and 3l levels respectively) with three replications with ages around 15 years. The results showed that the highest rate of proline was found at -6° C in Sh-18 (3.43μmol/g). While the highest rate of carbohydrates at -6 °C were found in cultivar Monagha (0.305mg/g). The rate of calcium, magnesium, potassium and sodium at different stages of bud development of the experimental cultivars under cold stress was diverse significantly. According to this research results, Sh- 12 of almond was observed more resistant than other cultivars.


Concorde at the Fleet Air Arm Museum.
War to use the area as a rehearsal base for the D-Day landings.
are allowed to visit the village.
road from Bovington where the Tank Museum is situated.
'Glasgow Haiku'. An exhibition by Group 13 Artists.
This is a collection of paintings with Haiku's based on Glasgow.
on the island of Evvia in Greece.


text:
Ken Niumatalolo
Officer shifts duties with Mids
By Bill Wagner
| bwagner@capgaznews.com |
Oct 17, 2015 | 4:51 PM
Coach Robert Green gives signals from the sideline during football practice on The Yard at USNA on Thursday afternoon. (By Matthew Cole / Capital Gazette)
Navy football was confronted with a coaching staff crisis just one week into August training camp.
Head coach Ken Niumatalolo determined that defensive coordinator Buddy Green needed to take a sabbatical this season in order to fully focus from health issues. It was an easy decision for Niumatalolo to promote Dale Pehrson, the longest-tenured member of the staff with 20 years in Annapolis, to interim defensive coordinator.
Finding another assistant coach to fill the vacancy created by Green's departure was not nearly as simple. Fortunately for the Midshipmen, Lt. Col. Robert B. Green was ready, willing and able to step into the role.
In a span of less than 24 hours, Niumatalolo had to make the difficult decision to transition Buddy Green into a consultant's role for this season then figure out a way to replace Navy's 14th-year defensive coordinator. He shifted Dan O'Brien into the secondary to fill in for Buddy Green, who always worked with the defensive backs. He then asked Lt. Col. Green to trade administrative duties for an on-field coaching role working with the outside linebackers.
"I had to find another assistant and it had to happen quickly," Niumatalolo said. "I thought about the whole person of RB Green and what he brings to our program. I couldn't have found anyone else better under the circumstances."
Green spent the previous two years as director of player development for Navy football. In that role, the Marine Corps officer was charged with mentoring the Midshipmen and helping prepare them to assume future leadership positions in the Navy and Marine Corps.
Green, an Atlanta native, was a starting defensive back at Navy from 1994-96 – earning All-Independent honors as a senior standout on a squad that won the Aloha Bowl. During his 15-year career in the Marine Corps, Lt. Col. Green has worked with the 9th Communication Battalion, Marine Aviation Logistics Squadron 36, Recruiting Station Indianapolis and 4th Marine Logistics Group among other duties.
What Green has never done is coach football at any level. So suddenly being thrust onto a Division I staff is a daunting responsibility.
"I'm a rookie coach in a room full of professionals, but they've been very, very kind to me over the last few months," Green said. "They accept all my dumb questions and comments. They take the time to sit down with me and help me understand things. All the coaches have taken me under their wing and helped me learn this job step-by-step."
Outside linebackers coach Justin Davis is about four years younger than Green, but has 13 years of collegiate coaching experience. Davis has been on the front line of getting Green up to speed since the newcomer is helping him with the outside linebackers.
"RB jumped in with both feet and has hit the ground running. He's been very diligent taking notes in meetings and asking questions of all the coaches. It didn't take long for him to really get going in the job," Davis said. "Anyone who knows Navy football knows that RB was a darn good player when he was here. You take a good football player and you know he understands the game. Factor in the leadership and character that RB brings as a Marine Corps officer and you have the makings of a good coach."
Davis laughed when asked if Green was tentative and unsure of himself at first.
"He's a Marine now. He's not afraid to do anything. RB did a really did a nice job of catching himself up to speed," Davis said. "Like I said, the man is a Marine. You ask him to take on a role and he's going to get the job done."
Green has discovered coaching college football is a lot more time-consuming and entails far more effort than he ever imagined.
"As a player you think you know everything. You think the coaches are working three hour days; that they are hanging out while you are working out; that they yell and scream at you and that's all they do," he said. "I have learned in a very short time that coaching is a lot of work. You spend a great number of hours in the office here preparing for games. And it's a lot tougher than folks know. There are a lot of man hours that go into preparing for a football game."
By far the biggest challenge for Green has been indoctrinating himself in the language of football coaches, who have a lingo all their own. Green admits his head was spinning for a few weeks as he listened to the other assistants in defensive meetings.
"They're talking about all these different fronts and spitting out numbers because this stuff is all second nature to them," he said. "I'm having to learn everything from scratch, starting with the terminology. What fronts go with what coverages, why you don't want to run certain coverages against specific offensive formations. It's a lot to process and digest."
Actually, the knowledge Green gained as a defensive back at Navy has proven quite helpful when working with the outside linebackers, who often must drop into coverage. During the individual drills session of practice, Davis has Green tutoring the strikers since they are required to cover slot receivers and tight ends a bit more than the raiders.
"I actually really like having Colonel around. He played defensive back when he was here and the strikers are doing a lot more coverage stuff this year than we've been doing so it's good to have him around tell us how to make certain reads, how to react in certain situations. It's really helpful," junior outside linebacker Josiah Powell said.
Niumatalolo praised Green for working hard to learn the defensive package, but isn't overly concerned about his coaching expertise at this point. The eighth-year head coach didn't tab Green to join the coaching staff so he could break down spread offenses.
"More than the Xs and Os, what RB brings to the table right now is our culture. Just understanding The Brotherhood and who we are, nobody is more qualified," Niumatalolo said. "RB is a great leader of men. He was one of the best cornerbacks we've ever had here at Navy. He went on to serve in the Marine Corps and reach the rank of Lieutenant Colonel. I just think the wealth of knowledge and experience that he brings to the military side of it are going to pay great dividends for our players."
In some respects, it's like having a young, eager and energetic graduate assistant on the staff. However, this graduate assistant brings quite a bit of wisdom, not to mention military pedigree.
"It's exciting for me to see RB out here on the field. You can tell he is enjoying the job and having fun," Niumatalolo said. "Plus, it's kind of cool for me to be able to boss around a Lieutenant Colonel in the Marine Corps."
Green joked that when he first started most of the players knew more about the X's and O's than he did. However, that hasn't stopped the rookie coach from putting in extra time studying film and reviewing the playbook.
"I was warned very early when Coach Niumatalolo presented me with this opportunity that I would catch the bug, and I surely have. So much so that my wife is like 'Enough of the film work. It's time to get to bed.' It is addictive and captures you and wants you to do more," Green said. "I am fully invested into the coaching program and I'm going to work my butt off to do a good job for Coach Niumatalolo and the players."
Green thinks his previous role as director of player development has helped with the transition to assistant coach.
"Because I had a relationship with the players that went far beyond what the other coaches are able to do," he said. "I had relationships with players that were personal. They told me stuff they might not have told their position coaches. So even though I am now a coach, they still respect me as a Marine and know that I am always there for them."
Powell agreed with that assessment: "I always saw Colonel as a mentor, somebody I could look up to and learn from. Having him as a coach hasn't really changed much."
About the only problem so far is that some players have struggled with how to address the new assistant, whether to call him Colonel Green or Coach Green.
Green has no intentions of abruptly retiring from the Marine Corps and getting into the coaching profession. Most likely, the father of twin boys will resume his role of director of player development next year.
"It's been an awesome experience. This is a once-in-a-lifetime deal and I am taking full advantage of it. I'm having a ball!" Green said.
TULANE @ NAVY
Saturday, 1 p.m.
TV: CBSSN
RADIO: 1430 AM; 1090 AM

redpajama_set_name: RedPajamaCommonCrawl


Every spring, a predictable and almost scripted conversation will happen at least once in at a breakfast shack somewhere in rural America. The conversation will take place amongst a group of camo-clad spring turkey hunters, who vent their frustration stemming from a morning in which "all of their turkeys" were on "Jim Bob the neighbors" property. The common accusation is that Jim Bob has illegal piles of chopped corn and milo spread around his property that the turkeys simply cannot resist. They contemplate calling the local game warden, but refrain because they have no hard evidence that Jim Bob is feeding. They choose to simply seethe in their disdain for the situation they'll likely face for the remainder of the spring season.
One poet of the spring once said that "an outlaw baits, and a gentleman feeds." There may be no topic more debated amongst turkey hunters than the issue of feeding. Two people that have opposing views on the issue of feeding are more likely to use expletives than cordial handshakes while discussing the issue. It is certainly a hot topic amongst turkey hunters and opinions are found up and down the spectrum.
Certain states allow year-round and unconditional feeding, and a turkey hunter can harvest a gobbler over artificial feed if he so chooses (which to me personally sounds like no sport at all). However, the majority of the states do not allow feeding during the spring turkey season or have stipulations in place that make it almost impractical. The issue in question here is not whether feeding should be allowed or not, it is if you should feed your turkeys before the season starts. Let's dive into this issue.
After deer seasons come to a close, the wild turkey hunter instinctively shifts into prep mode for the upcoming spring season. One of the first things the majority of turkey hunters and managers do is head to their local feed store, stock up on chopped corn, wheat, or grain sorghum, and spread it out in areas turkeys frequent. The intent is to not only draw turkeys from adjoining properties onto yours, but to also hold the turkeys currently calling your property home, all with the hopes of imprinting them and convincing them to stay long after the food source is cut off. A fat turkey is a happy turkey, and this strategy seems to work in most occurrences.
Just as a whitetail manager uses summer supplemental feeding to take the pressure off his corn or soybean crop (and benefit the herd's health), feeding your turkeys can help save some of your chufa patches or other crops for the season. The issue lies with when you cut the food source off. If turkeys have taken a liking to the "artificially provided" food sources, and natural food sources do not exist, they may traverse your property lines in search of food sources elsewhere. If you have a neighbor who feeds through the season, this becomes a big problem and a risk you take by giving them a taste of a readily available artificial food source. If you are not careful, you'll hear "your turkeys" from afar.
In many states, providing supplemental food sources for the whitetail deer has been banned due to outbreaks of Chronic Wasting Disease (CWD), which has decimated portions of the deer herd in certain pockets of states such as Wisconsin. While CWD has not been known to affect turkeys, my point here is that feeding sites congregate animals and may put them at a higher risk of disease than naturally occurring food sources. With numerous diseases such as Avian pox and histomoniasis (commonly referred to as "blackhead disease) floating around the country, every step should be taken to prevent an outbreak from occurring. If you choose to feed, make sure it is spread out over a large area.
Also, certain grains such as corn have the possibility of developing a fungus called aflatoxin, which can cause cancer in the animals that come in contact with them. Special care should be taken to ensure the feed does not get wet while in storage, as this is the time it is most susceptible to developing aflatoxin.
With the previous concerns in mind, the wise move may be to manage the naturally occurring or planted food sources on your property versus preseason feeding. You cannot control the actions of both the turkeys and your neighbors, so providing your resident turkeys all they need by way of food plots, prescribed burning, and other management practices could be the best long term fix. If you are pressed for time, do not own the land you hunt, or are a weekend warrior, feeding before the season may be your best route. However, if you want to hold your turkeys year round, make sure you have plenty of food for them once you cut them off from the bait.
People forget that turkeys need to be managed just as whitetails need to be managed. In many ways, turkeys require more tender loving care than whitetails. For instance, wild turkeys are not as tolerant of thick understory as is the whitetail deer, so you need to control the underbrush as well as provide them wide travel corridors to use. To get a feel for what life is like as a turkey, get on your knees and see if traveling through a timber stand would be possible. If not, then you may want to either consider prescribed burning or an herbicide treatment to reduce some of the understory.
As mentioned above, food plots for turkeys are a crucial piece of the management puzzle. Wild turkeys need crops like Biologic's Clover Plus, Whistleback, and Turkey Gold Chufa, all of which can be planted in tandem with your whitetail crops. Because a flock of wild turkeys does not require the tonnage that a deer herd would, you have some flexibility in where you plant these crops. Remember how I said turkeys needed wide roads? A 100 yard long strip of Whistleback on the side of a road will produce millions of tiny seeds that wild turkeys and other upland game can enjoy. Two or three half-acre patches of Turkey Gold Chufa in the sandy back corners of your deer patches can make a huge impact on holding turkeys year-round. Planting and maintaining Clover Plus can benefit both wild turkeys and whitetails; clover patches are especially important in the poult-rearing summer months, because they hold millions of insects that the poults feast on.


text:
II is the second album recorded by Seven Thorns. It was released on 19 January 2013 independently, and was later re-released on 1 February 2014 by the record label Sonic Revolution. Despite being the band's lead singer, Gustav Blide performed only backing vocals for the album as he was not yet ready to cover the lead singing duties at the time. Lead vocals are performed by Erik Blomkvist like on the band's previous album.
The chorus melody of the song Justice is based on a Danish church psalm with music composed by C. Chr. Hoffmann in 1878.

Track listing

Personnel
Lars Borup - Drums
Gabriel Tuxen - Guitars, backing vocals
Christian B. Strøjer - Guitars
Asger  W. Nielsen - Keyboards, backing vocals
Nicolaj Marker - Bass
Erik "EZ" Blomkvist - Lead vocals
Gustav Blide - Backing vocals
Mik Holm - backing vocals

Guest musicians
André Andersen (Royal Hunt) - Keyboard solo on Eye of the Storm.
David Henriksson (Heel, ex-Insania) - Additional lead vocals on Redemption.

Production
Peter Brander - Recording and mixing
Tommy Hansen - Mastering

2014 albums
Seven Thorns albums
Power metal albums by Danish artists

redpajama_set_name: RedPajamaWikipedia


Did you know you can make a contribution to PVUMC-supported charities and receive a dollar-for-dollar Arizona state tax credit in 2016? A single individual can receive a tax credit of up to $400 and a married couple can receive up to $800. And you don't need to itemize deductions to claim the tax credit. Just mail your check directly to one of the following charities (not to PVUMC) or go to their websites (see links below) by April 18, 2017 to make your contribution online.
The Wesley Community Center and UMOM's Next Step Housing Program are recipients of our annual Christmas missions offering, and we sent volunteers to serve at all three places on Change the World Day, November 13, 2016. PVUMC supports all three through volunteer hours and our missions ministry.


text:
`Meta Component` that parses the parent `Component` from a [JSON](https://github.com/nlohmann/json) object and attaches it to a given `Entity`.

## Prototype

```cpp
void (const nlohmann::json & json, Entity & e);
```

### Parameters

* `json`: JSON object for `e`, NOT specifically for the parent `Component`
* `e`: `Entity` which the new `Component` should be attached to

## Usage

It is up to the user to implement this `meta Component` for the `Component` types they wish to be able to parse.

A helper [registerLoadFromJSON](../../helpers/meta/impl/registerLoadFromJSON.md) function is provided that takes as a template parameter a `Component` type and implements the `LoadFromJSON` `meta Component` for it.

Note that the implementation provided in `registerLoadFromJSON` is only a sample, and users may freely replace it with any other implementation they desire.

redpajama_set_name: RedPajamaGithub


text:
Q: How to Pass Object from android webview to c# to load html page  WebView web_view = root.FindViewById<WebView>(Resource.Id.IVChartsViews);

 String API = "http://Test.com/Sample/MobileApp?Userdetails=" + str + "&id=" + -9999;

 string obj1 = JsonConvert.SerializeObject(GlobalParams.ivs);
         HttpContent content = new StringContent(obj1.ToString());
         content.Headers.ContentType = new System.Net.Http.Headers.MediaTypeHeaderValue("application/json");
         web_view.PostUrl(API, content.ReadAsByteArrayAsync().Result);

I am trying to load html page by passing object but it not working.
  public ActionResult MobileApp(string Userdetails, int? id, InfoViewSettings ivssettings){}

here ivsettings object taking default data. can you please tell me where am i doing mistake.

A:
Yes want to use WebView to parse JsonObject and show the Html page.

Just from your code, I still believe your JsonObject is parsed to a string and the content of this string should be a html content, you want your WebView to load this html content. If so, you can simply use LoadData method of WebView for example like this:
web_view.LoadData(htmlContent, "text/html; charset=utf-8", "UTF-8");

The htmlContent in the code above should be a string format of html content, for more information, you can refer here. And please make sure you've pass the right html content into this LoadData method.

redpajama_set_name: RedPajamaStackExchange


Every day I ask myself and my entire firm, "What can we do for our advisors today?" Spire Investment Partners' culture and the reason for existing is to be there for financial professionals! We're in their corner. The goal as I see it is to balance our now 22 years of hybrid platform experience to support an entirely open architecture platform while simultaneously keeping them and their client protected.
It might be surprising, in this era where the majority of RIAs are corporately owned, for you to learn that I've walked in your shoes. I was a successful wealth manager – top 1% of the industry, after a legal career in the Air Force. I know what it means, and more importantly what it feels like to win and lose million-dollar engagements. I never wanted to live in a "box," and neither should you. That is why Spire is an independent, boutique, and flexible resource for you. You envision the puzzle pieces of your practice, and we'll work with you to build that perfect vision you have for you and your clients.
The future is and will remain independent. Leading Spire, I will continue to invest in our effectiveness and efficiency to continually grow our advisor-centric platform whether it be in human capital, technology or products. My mission is to create a more prominent future which makes it exciting for each of us to get up and go to work every day.
Wouldn't it be great to associate with a firm that takes steps every day to ensure you have a platform that's relevant and compliant that serves you as the financial champion of your clients. If this level of passion resonates with you, we're always here for an open and candid conversation.
This is my commitment to all my current advisors those yet to join us in our movement over the coming years.


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Over-65s are the biggest savers of any age group - and a quarter have had a savings account for more than 50 years
Over-65s still put away an average of £270 a month or £3,240 a year
Experts say this is because saving becomes a habit that is hard to break
28 per cent of over-65s have had savings accounts for more than 50 years
By Charlie Moore For Thisismoney.co.uk
Published: 04:31 EST, 30 June 2017 | Updated: 04:31 EST, 30 June 2017
Two thirds of pensioners are still saving in retirement, putting away more than any other age group, according to new research.
Rather than just spending all the money they have built up over their working lives, over-65s put away an average of £270 a month or £3,240 a year on average.
Experts say this is because saving becomes a habit that is hard to break, especially when retirees want build up a pot to pass on to their loved ones.
Two thirds of pensioners are still saving in retirement, research shows today
The research by Charter Savings Bank found that 28 per cent of over-65s have had savings accounts for more than 50 years - and 59 per cent wish they had started saving even earlier.
This figure is slightly less than the 64 per cent of all adults who regret not starting to put money aside earlier in life.
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The survey of 1,000 adults also showed those aged between 35 and 54 save the least, largely due to financial pressures of supporting a family.
They put away around £200 a month compared with £234 for 18 to 34 year-olds and £240 for those aged over 55 as the pressure on family finances starts to ease.
The research by Charter Savings Bank found that 28 per cent of over-65s have had savings accounts for more than 50 years
Paul Whitlock, Director of Savings, Charter Savings Bank said: 'Many people keep saving even if the amount they're able to put aside seems too small to matter.
'Judging from the number of older people with over a half century of saving behind them, once the habit is established it continues well into retirement. In our experience many pensioners are still depositing sizeable amounts every month.
'Savers of all generations shop around in order to seek out the best accounts for their needs, whether it's the certainty that comes with a fixed rate bond, or having an easy access account in case of emergency.
'Our research shows that one thing that all people, regardless of age, had in common is their regret at not having started saving earlier.'
Levels of saving have fallen across the board in recent months as wage growth fails to keep up with inflation and robust levels of consumer spending.
The saving ratio - which compares how much people are saving compared to their level of disposable income - fell to its lowest rate on record of 3.3 per cent at the end of 2016.
The ratio has been declining since autumn 2015, after rising sharply following the financial crisis.
Households are saving less, while simultaneously spending more on credit, leading to concerns that many would struggle if they suffered a shock to their finances, such as higher interest rates or job loss.
Pensioners save more than any other age group