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In Pseudomonas aeruginosa PAO1, the expression of several virulence factors such as elastase, rhamnolipids, and hydrogen cyanide depends on quorum-sensing regulation, which involves the lasRI and rhlRI systems controlled by N-(3-oxododecanoyl)-l-homoserine lactone and N-butyryl-l-homoserine lactone, respectively, as signal molecules. In rpoN mutants lacking the transcription factor σ54, the expression of the lasR and lasI genes was elevated at low cell densities, whereas expression of the rhlR and rhlI genes was markedly enhanced throughout growth by comparison with the wild type and the complemented mutant strains. As a consequence, the rpoN mutants had elevated levels of both signal molecules and overexpressed the biosynthetic genes for elastase, rhamnolipids, and hydrogen cyanide. The quorum-sensing regulatory protein QscR was not involved in the negative control exerted by RpoN. By contrast, in an rpoN mutant, the expression of the gacA global regulatory gene was significantly increased during the entire growth cycle, whereas another global regulatory gene, vfr, was downregulated at high cell densities. In conclusion, it appears that GacA levels play an important role, probably indirectly, in the RpoN-dependent modulation of the quorum-sensing machinery of P. aeruginosa.
Intercellular communication systems allow bacteria to monitor environmental conditions and to coordinate the expression of several genes, particularly those specifying extracellular products and virulence factors, in a cell density-dependent manner (44). This communication, termed quorum sensing, is based on the production of diffusible signal molecules (autoinducers) which accumulate in the environment during bacterial growth. Upon reaching a threshold concentration at high cellular densities, they can bind to and activate specific transcriptional regulators. In many gram-negative bacteria, quorum-sensing systems consist of pairs of genes in which a luxI-type gene encodes an N-acylhomoserine lactone (AHL; autoinducer) synthase and a luxR-type gene encodes a transcriptional regulator activated by the cognate autoinducer (15, 60, 67).
In the pathogen Pseudomonas aeruginosa, which is responsible for nosocomial infections (63) as well as for serious infections in patients suffering from cystic fibrosis, cancer, or burn wounds (6, 62), pathogenicity is due to the production of both cell-associated and extracellular virulence factors, most of which are regulated by quorum sensing (15, 60, 67). P. aeruginosa contains two interdependent quorum-sensing systems (26, 32, 45, 70). In the lasRI system, the LasI synthase catalyzes the biosynthesis of N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL). The LasR-OdDHL complex positively regulates the expression of virulence factors such as elastases (LasB and LasA), exotoxin A, alkaline protease, hydrogen cyanide (HCN), and pyocyanin and, moreover, induces lasI itself, forming an autoinduction loop. LasR activated by OdDHL also positively regulates the expression of the rhlR gene. RhlR is the transcriptional regulator of the second autoinducer system and functions with N-butyryl-l-homoserine lactone (BHL), whose biosynthesis requires the RhlI synthase. The rhlRI system enhances the expression of multiple exoproducts such as LasB elastase, HCN, pyocyanin, and rhamnolipids (14). Thus, there is a quorum-sensing hierarchy in which the las system is dominant.
Both quorum-sensing systems are positively regulated by the global regulator GacA; in particular, the response regulator GacA has a marked enhancing effect on BHL formation and BHL-dependent virulence factor production (48, 52). In addition, the CRP (cyclic AMP receptor protein) homologue Vfr specifically activates the lasR promoter (1, 66). Recently, the QscR protein, a homolog of LasR and RhlR, has been shown to act as a repressor of lasI and rhlI, leading to a downregulation of quorum-sensing-dependent virulence factors (8). Thus, LasR- and RhlR-controlled quorum-sensing mechanisms are embedded in global regulatory networks. Recent work on sigma factor σ54 (RpoN) in P. aeruginosa revealed that this alternative sigma factor is important for virulence in several models, e.g., infection of the respiratory epithelium in cystic fibrosis xenografts and of burned mice (9, 21). Reduced virulence of P. aeruginosa rpoN mutants may be explained, in part, by the fact that rpoN function is necessary for the expression of pili and flagella, two important adherence factors (25, 42, 61). However, considering the pleiotropic effects of rpoN mutations on metabolic functions in P. aeruginosa (5, 27, 40, 61), it has been postulated that the role of RpoN in the regulation of virulence factors could be quite complex, extending beyond positive control of pilin and flagellin synthesis (9). Here we show that RpoN exerts global negative control on the quorum-sensing machinery of P. aeruginosa. At least part of this effect appears to be mediated by GacA.
Bacterial strains and growth conditions.The strains and plasmids used in this study are listed in Table 1. Both Escherichia coli and P. aeruginosa strains were routinely grown in nutrient yeast broth (NYB) or on nutrient agar plates at 37°C (58). l-Glutamine was added at a concentration of 1 mM for growing the rpoN mutant strains in NYB or in minimal medium E (64). For β-galactosidase and autoinducer extraction experiments, P. aeruginosa strains were grown with aeration in NYB in 50-ml Erlenmeyer flasks. When required, antibiotics were added to media at the following concentrations: tetracycline (Tc), 25 μg ml−1 (E. coli) or 125 μg ml−1 (P. aeruginosa); ampicillin (Ap), 100 μg ml−1 (E. coli); gentamicin (Gm), 10 μg ml−1; kanamycin (Km), 400 μg ml−1; and spectinomycin (Sp), 1,500 μg ml−1 (P. aeruginosa).
To counterselect E. coli S17-1 donor cells in matings with P. aeruginosa for gene replacement, chloramphenicol (Cm) was used at a concentration of 10 μg ml−1; mutant enrichment experiments were performed with tetracycline at a final concentration of 20 μg ml−1 and carbenicillin at a final concentration of 2,000 μg ml−1. Flagellar swimming was tested as described by Rashid and Kornberg (51) on NYB containing 1 mM l-glutamine solidified with 0.3% (wt/vol) agar. Swarming was tested on plates containing 0.5% (wt/vol) agar, 8 g of nutrient broth per liter (Oxoid), 5 g of glucose per liter, and 1 mM l-glutamine (51). Twitching motility was assayed on 1% (wt/vol) agar supplemented with Luria broth and 1 mM l-glutamine (29).
DNA manipulation and cloning procedures.Small- and large-scale preparations of plasmid DNA were made by the cetyltrimethylammonium bromide method (13) and with JetStar columns (Genomed, Basel, Switzerland), respectively. Chromosomal DNA was purified from P. aeruginosa as described elsewhere (16). Restriction enzyme digestions, PCRs, ligation, electrophoresis, and electroporation were performed with standard procedures (52) and as described elsewhere (17, 46). Nucleotide sequences of PCR-derived constructs were determined on both strands with a dye terminator kit (Perkin-Elmer product 402080) and an ABI Prism 373 sequencer. Comparison of nucleotide and deduced amino acid sequences was performed with the Genetics Computer Group program GAP.
Plasmid constructions.Plasmid pME3829 was obtained by subcloning a 3.1-kb BamHI-HindIII fragment from pRP1-rpoN (kindly provided by Y. Itoh), containing genes PA4461 and rpoN, into pBBR1MCS (Fig. 1). Plasmid pME3836 was constructed by PCR-amplifying a 744-bp fragment (3 min at 95°C; 25 cycles of 1 min at 95°C, 30 s at 58°C, 1 min at 72°C; and 3 min at 72°C) with PAO1 chromosomal DNA as the template, with the primers qscR-3 (5′-AGGCCAGGATCCTGTTTATTGTCT-3′) and qscR-4 (5′-GACAAAATCTGCAGATATCCCTCT-3′). Artificial restriction sites (italic) for BamHI and PstI, respectively, were incorporated into these primers. The resulting 730-bp BamHI-PstI fragment, including a potential Lux box located 486 bp upstream of the ATG and the first eight codons of qscR, was fused in-frame with the ′lacZ reporter gene from pNM482 in vector pME6010.
To obtain plasmid pME3338, a 2.1-kb EcoRI-HindIII fragment containing the rpoN gene (Fig. 1) was excised from pME3829 and inserted into the SpeI site of pME3280b (S. Zuber and D. Haas, unpublished data). To construct plasmid pME3851, the rhlR promoter region and first nine rhlR codons were amplified by PCR as above with the primers RR1 (5′-CGCTTGCTCGAGACCCGGC-3′) and RR2 (5′-AAAACTGCAGCAGCAAAAAGCCTCCGTC-3′), which are XhoI and PstI tagged, respectively (italic); plasmid pME3840 was used as the template. The 0.3-kb PCR product was cut with XhoI, blunted, cut with PstI, fused in frame with the ′lacZ reporter gene of pNM482, and cloned into pME6010. To generate plasmid pME3858, a 0.4-kb SmaI-PstI fragment from pME3827, containing the lasR promoter region and the first 23 codons of lasR, was fused in frame with the ′lacZ reporter gene from pNM482 in vector pME6010.
A putative rpoN recognition site overlapping the 3′ end of the lasR (lux) box in the rhlI promoter was mutated as follows. Primer rhlR-1 (5′-CCGTGGATCCGGCGATCCTC-3′), which anneals around the BamHI site (italic) in the rhlR coding sequence, and primer Acl-2 (5′-GATGAACGTTTGGCAACCTGCCAGATCTGGT-3′), which is complementary to part of the lasR box in the rhlI promoter and carries an artificial AclI site (italic), were used to PCR-amplify a 551-bp fragment from pME3846. A second PCR fragment of 183 bp was amplified from pME3846 with primer Acl-3 (5′-GCCAAACGTTCATCCTCCTTTAGTCT-3′), which is tagged with an artificial AclI site (italic) and anneals immediately downstream of the putative rpoN box, and primer rhlI-4 (5′-AAAACTGCAGCGGAAAGCCCTTCCAGCG-3′), which is complementary to codons 8 to 13 of rhlI and carries an artificial PstI site (italic). The two PCR fragments were ligated to each other at their AclI sites, fused as a 0.71-kb BamHI-PstI fragment to the ′lacZ gene of pNM481, and cloned into pME6010. Sequence analysis of the resulting construct, pME6186, confirmed that in the putative rpoN recognition sequence GGCAGGTTGCCTGC, the last three bases were replaced by AAA, destroying the second half-site of the GG-N10-GC motif.
P. aeruginosa mutant constructions.Care was taken to construct all mutants and chromosomal reporter fusions in the same PAO1 background. In PAO6358, a 0.9-kb fragment was deleted in-frame in the rpoN gene. The deletion includes the helix-turn-helix motif and the rpoN box (Fig. 1) and was obtained as follows. A 604-bp KpnI-SacI fragment from pME3829, including the first 190 codons of rpoN, was linked to a 620-bp SacI-BamHI fragment containing the last six codons of rpoN, gene PA4463, and the beginning of pstN (Fig. 1), which had been amplified by PCR as above with primers KH4 (5′-AAAAGAGCTCCGCAAGCGACTGGTGTGA-3′) and KH5 (5′-AAAAGGATCCGGCGATGCCATTGCCGAA-3′) and cut at the artificial restriction sites for SacI and BamHI (italic). The resulting 1.22-kb fragment was cloned into the suicide plasmid pME3087 digested with KpnI and BamHI, giving plasmid pME3834. In conjugation with PAO1 as the recipient and S17-1/pME3834 as the donor, tetracycline-resistant transconjugants having a chromosomally integrated pME3834 plasmid were selected. After carbenicillin enrichment, glutamine-auxotrophic colonies were obtained and verified by PCR for their 0.9-kb deletion in rpoN with primers KH5 and KH10 (5′-TCCAGCAGGAAATCCAGGAAG-3′) (Fig. 1).
In strain PAO6359, the rpoN gene was interrupted by the insertion of a kanamycin resistance cassette (Ω-Km). We obtained this mutant by transducing the mutation from PAO4460 (40) into PAO1 with the temperate phage E79tv-2 (39) as described before (17). Kanamycin-resistant transductants were auxotrophic for glutamine. The double rpoN gacA mutant PAO6363 was obtained by transduction of the gacA mutation of PAO6281 into PAO6358 with phage E79tv-2.
In strain PAO6366, the qscR gene was interrupted by the insertion of a spectinomycin-streptomycin resistance cassette (Ω-Sp/Sm). The qscR gene was amplified by PCR with PAO1 chromosomal DNA as the template and primers QSCR1 (5′-AAAAGAGCTCATGGAGCGTGCGAGAAGAAC-3′) and QSCR2 (5′-TAAAGGATCCTATCCGGCCATTCGGTGAAT-3′), which contain artificial restriction sites for SacI and BamHI (italics). The 2-kb Ω-Sp/Sm resistance cassette from pHP45Ω was inserted as an EcoRI fragment into the EcoRI site located 30 codons downstream of the qscR ATG start codon. The resulting 3.15-kb fragment was cloned into the suicide plasmid pME3087, giving pME3828. This plasmid, carrying qscR::Ω-Sp/Sm, was mobilized into PAO1 and chromosomally integrated, with selection for tetracycline resistance. The chromosomal insertion in a tetracycline-sensitive, spectinomycin-resistant clone was verified by Southern blotting.
Construction of chromosomal insertions in the Tn7 attachment site.Complementation by a single copy of the rpoN+ gene was carried out with a Tn7-based system developed for gram-negative bacteria (3, 22). Chromosomal insertion of the mini-Tn7 construct pME3338 carrying rpoN+ (Fig. 1) was obtained via a triparental mating between the recipient PAO6358 (grown overnight at 43°C), E. coli SM10/λpir carrying the pUX-BF13 helper plasmid, and E. coli S17-1/pME3338, with selection for gentamicin and chloramphenicol resistance. The rpoN+ insertion in the resulting strain, PAO6360, was verified by the loss of the auxotrophy for glutamine and by Southern blotting analysis.
Chromosomal vfr′-′lacZ strains were constructed as follows. First, the vfr gene was amplified by PCR from chromosomal DNA of PAO1 with the XhoI (italic)-tagged primer pVFR1 (5′-CATCCTCGAGGAAGGCTTCGC-3′) and the EcoRI (italic)-tagged primer pVFR2 (5′-GGAATTCATGGGTGCTGTTCA-3′). The resulting 1.15-kb PCR fragment was cleaved with EcoRI and XhoI and inserted into pBluescriptII-SK to give pME6157. The blunted 0.6-kb XhoI-PvuI fragment of pME6157 was fused to ′lacZ in the SmaI site of pNM482, and the resulting vfr′-′lacZ fusion on a 3.7-kb EcoRI-DraI fragment was ligated to a 0.35-kb SphI (T4 DNA polymerase treated)-EcoRI fragment carrying the transcription stop signal of the Ω-Sp/Sm cassette, and cloned into the blunted HindIII site of the Tn7 delivery vector pME6313. The transcription stop signal upstream of the vfr′-′lacZ fusion in this construct, named pME6165, prevents potential readthrough from the gentamicin resistance gene. The vfr′-′lacZ fusion was delivered to the chromosome of PAO1 and PAO6358 by triparental mating with E. coli S17-1/pME6165 and E. coli SM10λpir/pUX-BF13 as donors. Gentamicin-resistant transconjugants were checked by Southern analysis.
A translational gacA′-′lacZ fusion excised from plasmid pME6118 as a 4.5-kb EcoRI-DraI fragment was ligated to the SphI (T4 DNA polymerase treated)-EcoRI fragment carrying the transcription stop signal of Ω-Sp/Sm and inserted into the blunted HindIII site of pME6313. The mini-Tn7 gacA′-′lacZ construct of the resulting plasmid, pME6166, was delivered to the chromosome of PAO1 and PAO6358 as described above. The resulting strains, PAO6320 and PAO6361, were checked by Southern analysis.
Semiquantitative determination of autoinducer concentrations by thin-layer chromatography.P. aeruginosa strains were cultivated with shaking in 20 ml of NYB amended with 1 mM glutamine in 50-ml Erlenmeyer flasks at 37°C to obtain an optical density at 600 nm (OD600) of 0.6 or 1.5. Cells were removed by centrifugation, and the pH of supernatants was adjusted to 5.0 prior to extraction with 3 volumes of dichloromethane in a separating funnel. Water was removed from the solvent phase with anhydrous Na2SO4, and dichloromethane was evaporated with a rotary evaporator. The extracts were concentrated 200-fold by dissolving them in aqueous 50% (vol/vol) acetonitrile. The presence of AHLs was tested by C18 reverse-phase (Merck) thin-layer chromatography, developed by elution in methanol-water (60:40, vol/vol), and revealed by overlaying either Chromobacterium violaceum CV026 (36), for BHL, or Agrobacterium tumefaciens NTL4/pZLR4 (7), for OdDHL. The amounts of BHL and OdDHL were estimated by comparison with standards, i.e., 4, 6, or 8 nmol of BHL and 50, 100, or 150 pmol of OdDHL.
β-Galactosidase assay.P. aeruginosa strains were cultivated with shaking in 20 ml of NYB with 1 mM glutamine in 50-ml Erlenmeyer flasks at 37°C. β-Galactosidase specific activities were determined by the method of Miller (55).
Growth characteristics of P. aeruginosa rpoN mutants.As the rpoN gene is the first gene in a cluster of five (27, 59), we constructed an in-frame deletion mutation in rpoN to avoid potential polar effects on the expression of the downstream genes. The resulting rpoN mutant, PAO6358, was complemented by a single rpoN+ copy inserted into the unique chromosomal Tn7 attachment site in strain PAO6360 (Fig. 1). An rpoN::Ω-Km insertion mutant, PAO6359, was also constructed (Fig. 1). Both rpoN-negative strains were auxotrophic for glutamine and unable to swim, swarm, and twitch, in agreement with previous studies showing that P. aeruginosa rpoN mutants are defective for flagella and type IV pili (21, 25, 29, 40, 61). In rich medium (NYB) amended with 1 mM l-glutamine, both rpoN mutants had a longer doubling time (about 70 min) than the wild-type PAO1 and the complemented mutant PAO6360 (about 40 min), indicating that substrate utilization was somewhat impeded by the loss of RpoN function. It is known that the utilization of several amino acids as C and N sources depends on RpoN (40, 61). However, both the wild type and the rpoN mutant reached stationary phase at similar levels (2.5 × 109 to 3 × 109 cells/ml). Under anaerobic conditions in a GasPak jar, the rpoN mutant PAO6358 and the wild-type PAO1 grew similarly on nutrient agar amended with 1 mM l-glutamine and either 100 mM KNO3 or 5 mM KNO2, suggesting that RpoN is not essential for denitrification.
Effects of rpoN null mutations on AHL production in P. aeruginosa.To investigate whether RpoN influences the production of the quorum-sensing signals OdDHL and BHL, we quantified AHL levels at cell densities corresponding to early exponential phase (OD600 = 0.6, i.e., about 6 × 108 cells/ml) and to late exponential phase (OD600 = 1.5) (Table 2). The rpoN mutants PAO6358 and PAO6359 produced about two times more OdDHL and about five times more BHL than did the parent strain during both early and late exponential phases of growth. As the rpoN deletion mutant was phenotypically similar to the insertion mutant, only the former was complemented. In the complemented rpoN mutant, PAO6360, autoinducer levels were close to those of the wild type (Table 2). A similar regulation phenomenon was also detected in overnight cultures of an rpoN mutant of strain PAK-N1 (PAK-SR rpoN::Ω-Tc [25]), which produced six to seven times more AHL than did the parental wild type, PAK-SR (data not shown). By comparison with strain PAO1, strain PAK-SR yielded fivefold-lower autoinducer levels (data not shown).
Effects of rpoN null mutations on expression of lasRI and rhlRI quorum-sensing genes.To confirm the regulatory effects of RpoN on AHL synthesis, we studied the expression of the lasR, lasI, rhlR, and rhlI genes with translational lacZ fusions in the wild-type PAO1, in both rpoN mutants PAO6358 and PAO6359, and in the complemented mutant PAO6360. Null mutation of the rpoN gene resulted in an approximately threefold derepression of lasR on pME3858 and lasI on pME3853 at cell densities below an OD600 of 1.0, but these effects were reversed at high cell densities (Fig. 2A and B). The complemented mutant PAO6360 showed lasR and lasI expression similar to that in the parent (Fig. 2A and B). RpoN was also found to control the second quorum-sensing circuit in that translational rhlR′-′lacZ and rhlI′-′lacZ fusions (on pME3851 and pME3846, respectively) were induced four- to fivefold at low and high cell densities (Fig. 2C and D). We additionally tested transcriptional lasR-lacZ and rhlR-lacZ fusions in an rpoN and in a wild-type background. The derepressing effect of the rpoN mutation was similar to that found with the analogous translational fusions (data not shown). In conclusion, the β-galactosidase activities of the lasRI and rhlRI fusion constructs closely paralleled AHL levels (Table 2).
Mutation in rpoN leads to increased expression of exoproduct genes.Quorum sensing controls the production of numerous extracellular enzymes and metabolites in P. aeruginosa; we chose to measure three of them, elastase, rhamnolipids, and HCN (67), by following the expression of some representative structural genes: lasB (for elastase), rhlA (the first of two genes for rhamnolipid synthesis), and hcnA (the first of three genes encoding HCN synthase). All three genes are most strongly induced by a synergistic action of both the las and rhl quorum-sensing systems (47, 68). Translational lacZ fusions of the lasB and rhlA genes on plasmids pTS400 and pECP60, respectively, showed a two- to fourfold increase of expression in the rpoN mutant PAO6358 compared to the levels in the wild type and in the complemented rpoN mutant. An hcnA-lacZ transcriptional fusion on pME3850.1 gave a similar result (data not shown). These experiments as well as the autoinducer determinations (Table 2) and the quorum-sensing expression studies (Fig. 2) were all conducted in the same growth medium, NYB amended with 1 mM glutamine. Growth reached a plateau at an OD600 of 2.5 to 3.0 under these conditions. The results obtained with the lasB, rhlA, and hcnA fusions are consistent with an overall negative effect of RpoN acting transiently on lasRI and constantly on rhlRI expression.
Mechanisms of quorum-sensing control by RpoN.We considered four hypotheses, which are not mutually exclusive, to explain the effects of RpoN on quorum-sensing in strain PAO1. (i) RpoN could directly repress the lasR, lasI, rhlR, and rhlI genes at their promoters, similar to the negative effect that σ54 can exert on alginate synthesis by binding directly to the algD promoter in P. aeruginosa (5). (ii) RpoN could repress Vfr, a positive regulator of lasR gene expression (1). (iii) RpoN could activate QscR, a negative effector of quorum sensing (8, 33). (iv) RpoN could repress GacA, a positive regulator of the expression of the lasR, rhlR, and rhlI genes (48, 52).
(i) RpoN recognizes a TGGCAC-N5-TTGCA consensus sequence in which the TGGC-N9-GC motif is most strongly conserved (4, 53). We searched for the presence of such a motif in the lasR, lasI, rhlR, and rhlI promoter regions; note that the transcription start sites have been determined experimentally for the lasR, lasI, and rhlI genes (1, 41, 48, 56). We did not find evidence for a conserved RpoN motif in the lasR, lasI, and rhlR promoter regions at positions that would be compatible with a repressive effect of RpoN, nor did we detect such a motif in the promoter of the rsaL gene, which exerts negative control on the lasI gene (11). By contrast, in the rhlI promoter, a potential RpoN recognition sequence, TGGCAG-N5-CTGCC, was found at positions −43 to −31 relative to the +1 site. This sequence overlaps a lasR (lux) box placed at −57 to −38. A 3-bp mutation replacing TGC at −33 to −31 with AAA was constructed in the rhlI promoter region. This mutation leaves the lasR box intact but would be expected to interfere with recognition of RpoN. However, the expression of an rhlI′-′lacZ fusion was not influenced by this mutation in the wild type or the rpoN mutant PAO6358 (data not shown).
(ii) In order to determine if the influence of RpoN on quorum sensing was mediated by Vfr, we measured the expression of a chromosomal translational vfr′-′lacZ fusion in a wild-type (PAO6304) and an rpoN mutant background (PAO6362). The expression of vfr was low throughout growth and significantly reduced in the rpoN background at high cell densities (Fig. 3). Inspection of the vfr promoter (54), however, did not reveal the presence of an RpoN recognition sequence, suggesting that the positive effect of RpoN on vfr expression may be indirect. The consequence of this regulation will be considered in the Discussion.
(iii) A qscR::Ω-Sp/Sm insertion mutant, PAO6366, was constructed and tested for expression of the lasR, lasI, rhlR, and rhlI genes with the lacZ fusion constructs pME3858, pME3853, pME3851, and pME3846, respectively. However, each fusion gave a similar expression profile in the wild-type PAO1 and in the qscR mutant PAO6366 (data not shown). A translational qscR′-′lacZ fusion (constructed as detailed in Materials and Methods) was expressed at a low level of 3 to 4 Miller units in the wild-type PAO1 as well as in the rpoN mutant PAO6358, at an OD600 of 2.0. These results suggest that RpoN does not modulate the quorum-sensing machinery via QscR.
(iv) In order to check if the influence of RpoN on quorum sensing was a result of RpoN-mediated control of gacA expression, we assayed a chromosomal translational gacA′-′lacZ fusion in the wild type (PAO6320) and the rpoN deletion mutant (PAO6361). A marked negative effect of RpoN on gacA expression occurred throughout growth (Fig. 4). In the wild type as well as in the complemented rpoN mutant, gacA expression was about three times lower than in the rpoN mutant (Fig. 4). In agreement with previous data (52), the expression levels of the lasB′-′lacZ, rhlA′-′lacZ, and hcnA-lacZ fusions were very low in a gacA mutant compared with those in the wild type and the rpoN mutant (Fig. 5A, B, and C).
If the repressive effects of RpoN on the quorum-sensing machinery were essentially a consequence of repression of GacA, then we might expect that a gacA rpoN double mutant would not differ substantially from a gacA mutant in terms of quorum-sensing-dependent expression. In the case of the lasB′-′lacZ fusion, this was indeed observed (Fig. 5A). However, in the case of the rhlA′-′lacZ and hcnA-lacZ fusions, the gacA rpoN mutant gave intermediate expression levels (Fig. 5B and C), indicating that a simple linear model (RpoN —| GacA → LasRI/RhlRI → RhlA/HcnA) would incompletely describe the situation and that RpoN may act on the expression of the rhlA and hcnA genes via GacA-independent pathways. The hcnA-lacZ fusion of pME3850.1 used is controlled tightly and probably exclusively by LasR and RhlR (47). It is likely that RpoN also acts on quorum sensing via regulators other than GacA, but these remain to be identified.
In P. aeruginosa, the alternative sigma factor RpoN has several roles. As in enteric bacteria, it is a vital component of nitrogen assimilation. rpoN mutants of strains PAO1 and PAK but not strain PA14 are auxotrophic for glutamine (21, 40, 61). In strain PAO, RpoN cooperates with the two-component systems NtrB-NtrC and CbrA-CbrB in the utilization of various carbon and nitrogen sources, e.g., arginine, histidine, and polyamines (40, 61). Moreover, PAO and PAK mutants that are defective for rpoN do not produce flagella and pili and therefore do not show swimming and twitching mobility (25, 61; this study). For the transcription of flagellar and pilus genes, σ54 needs the transcriptional regulators FleQ, FleR, and PilR (2, 35). Remarkably, the genomic sequence of strain PAO predicts >20 transcriptional regulators activating σ54; however, the function of most of these has not yet been discovered (59). Whether any of these is involved in the modulation of quorum-sensing activity reported here is also unknown. Although a direct repressive effect of RpoN on some quorum-sensing gene promoter has not been rigorously excluded, it seems more likely that RpoN, together with some transcriptional regulator(s), would transcribe one or several regulatory elements that control quorum sensing.
Certain transcriptional regulators that interact with σ54, e.g., NtrC and CbrB, are response regulators of two-component systems in P. aeruginosa. Since the cognate sensor kinases NtrB and CbrA are involved in the utilization of various N sources (40), we considered the possibility that the production of AHLs by P. aeruginosa might be influenced by the N source. However, we did not observe any significant differences in AHL levels produced aerobically by P. aeruginosa PAO1 cells when the growth medium contained either ammonium (a good N source) or nitrate (a poor N source) (data not shown).
One role of the postulated RpoN-dependent control element(s) is to downregulate the expression of the global regulator GacA. This downregulation occurs at both low and high cell densities (Fig. 4). By contrast, RpoN has a positive effect on the expression of another quorum-sensing regulator, Vfr, especially during late growth phases (Fig. 3). Vfr positively controls the expression of the lasR regulator (1). It therefore appears that the derepressing effect of an rpoN mutation on lasR expression (Fig. 2A and B) could be the result of the enhanced expression of GacA during early growth phases and at low cell densities. During later stages of growth and at higher cell densities, the downregulation of vfr may compensate for the upregulation of gacA in an rpoN mutant. This would explain the observation that lasR and, indirectly, lasI are not overexpressed in an rpoN mutant background at an OD600 of ≥1.5 (Fig. 2A and B).
The signal transduction pathways by which GacA acts on the expression of target genes, many of which are involved in the synthesis of exoproducts and biofilm formation (43), are incompletely understood at present (19). In P. aeruginosa, a detailed analysis of hcnABC expression revealed the existence of two GacA-dependent pathways (47, 48, 52). In the first pathway, GacA exerts a positive effect on the expression of the lasR and rhlRI genes and, as a consequence, also on the transcription of LasR- and RhlR-dependent genes, including hcnABC. In the second, AHL-independent pathway, GacA exerts a positive effect on target gene expression at a posttranscriptional level; in the case of the hcnABC genes, this effect requires a sequence surrounding the hcnA ribosome-binding site (48) and the RNA-binding protein RsmA (49). In both pathways, the DNA sequences directly recognized by the GacA protein, presumably in its phosphorylated form, have remained elusive. We considered the possibility that RpoN might regulate the expression of RsmA. However, we found no evidence for such an effect in experiments with an rsmA′-′lacZ fusion and Western blotting (our unpublished data).
It is interesting that another alternative sigma factor, the stress and stationary-phase sigma factor RpoS (σ38), is also involved in quorum-sensing regulation in P. aeruginosa. In an rpoS null mutant, BHL levels are elevated throughout growth, essentially due to derepression of rhlI expression, by comparison with the wild-type strain PAO1. As a consequence, expression of exoproduct genes, e.g., hcnB, is increased in an rpoS mutant (69). There is also evidence that the rhlRI system can positively control the RpoS level (31). Environmental conditions greatly influence the relative amounts of sigma factors in bacterial cells (24). These considerations led us to propose an empirical model in which GacA and some sigma factors (such as RpoN and RpoS) globally exert opposite effects on the quorum-sensing machinery in P. aeruginosa.
Interactions between quorum sensing and RpoN have also been proposed in E. coli. Addition of a signal molecule termed autoinducer 2 (a furanone compound) to E. coli cultures induces, among a large number of genes, the ybhH gene (corresponding to the gene PA4463 lying downstream of rpoN; Fig. 1) as well as the ygeV gene, encoding a σ54-dependent regulator homologous to LuxO, which is a component of the quorum-sensing cascade regulating bioluminescence in Vibrio harveyi (12, 34).
Considering the fact that inactivation of rpoN leads to the loss of two important adherence factors (pili and flagella) in P. aeruginosa on the one hand (21, 25, 61) and to an overexpression of several quorum-sensing-regulated virulence genes (lasB, rhlAB, and hcnABC) on the other hand, it would have been difficult to predict the virulence properties of a P. aeruginosa rpoN mutant in an animal model. The work of Hendrickson et al. (21) showed that an rpoN mutant of strain PA14 can manifest pathogenicity differently depending on the host. For nematodes and burnt mice, the rpoN mutant was less virulent than the wild type, whereas both strains were equally able to kill wax moth larvae (21).
RpoN is required in P. aeruginosa strain CHA for type III secretion of exotoxin S and exotoxin T, and thus RpoN makes a contribution to cytotoxicity in this strain (10). Moreover, since many pathogenicity models use relatively high infectious doses, the adherence properties of P. aeruginosa in these models may be less important than in most clinical situations. To some extent, the decreased virulence of P. aeruginosa rpoN mutants in some models might then be a consequence of a decreased ability to utilize a large number of organic substrates, not just N sources (40), and to produce secreted toxins (10).
We thank Christoph Keel for discussion, Yoshifumi Itoh for supplying plasmid pRP1-rpoN and strain PAO4460, and Nazife Beqa for technical help. We also thank Barbara Iglewski for providing plasmids pTS400 and pECP60, Dan Hassett for informing us of his work on RpoN prior to publication, and Glaxo-SmithKline for generously supplying carbenicillin.
This study was supported by the Swiss National Foundation for Scientific Research (project 31-56608.99) and the program Génie Biomédical.
- Received 24 July 2002.
- Accepted 13 January 2003.
- American Society for Microbiology


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Cocktail Making
Shake like the pros
At The Bay Bar in Newquay we love cocktails - it's something we take very seriously and we'd love to share our knowledge.
Perfect for a girls night, pre-dinner activity or birthday treat for a loved one, join us for a fun cocktail making masterclass by the beach as we teach you how to shake some of our Fistral favourites like a pro.
Our cocktail making classes are just £29 per cocktail lover. Why not make a night of it and add on dinner in Dune Restaurant too? Just £54 per person. for a three-course meal and cocktail making class.

subset: commoncrawl


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Only in England. A British energy company, Ecotricity, is promoting "vegan energy."
So called "green energy" is that produced by harnessing power from solar, wind, wave and tidal sources. Ecotricity claims that some green energy companies, mostly those that burn biomass, also burn animal byproducts. The horror. The Ecotricity website lists those offending British companies.
Ecotricity claims, "We're the only energy supplier in the UK that's registered with The Vegan Society for our green electricity. We've verified all of the energy sources that go into making our electricity to ensure we aren't inadvertently using animals in our energy production." Read more
See more madness here:


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Chinese Leaf-warbler (Phylloscopus yunnanensis) videos, photos and sound recordings | the Internet Bird Collection
Passeriformes > Leaf-warblers (Phylloscopidae)
Chinese Leaf-warbler Phylloscopus yunnanensisCheck out the full taxonomy and distribution of Chinese Leaf-warbler on HBW Alive.
HBW Alive contains information on Descriptive notes, Voice, Habitat, Food and Feeding, Breeding, Movements, Status and Conservation plus a list of bibliographical references for this species account.
videos • photos: list | gallery | slideshow • sound recordings
From 235 up to 3459 meters


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Donate now or read below for more info.
We are also working to make TPE an interactive and collaborative community through partnering with Engage 360 (a ministry founded by apologists seeking to bring more focus on equipping to church to be effective in believers sharing their faith). The goal is to create an online community where we can sharpen and encourage one another to share our faith and help equip others to do so effectively.
All of this takes time and resources. Would you prayerfully consider becoming a financial partner. Donations made through this page will be tax deductible and can be made on a one-time or recurring basis. Thank you in advance for your support!
-Greg West and the TPE Team
P.S. To begin to understand the impact The Poached Egg has already made you need to continue reading below.


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School district administrators in Glen Rock, Penn., are on the receiving end of a lot of criticism after one of their principals was seen wearing blackface.
In August, Lisa Boyer, the principal of Friendship Elementary School, dressed up like Steve Harvey in a game of "Family Feud" for a team building exercise with the staff. The photo just began getting a lot of attention late last month.
The students weren't in school at the time, because it was a teachers-only day, however many were still furious about the meeting. Besides using makeup to darken her face, Boyer also wore a do-rag on her head, plus a man's suit and phony mustache.
There wasn't any people of color in the room at the time, and on the same day a former food service contractor, Danny Boyce, posted a photo of the meeting and sent it to the school district.
On that same day, an investigation was launched and Boyer apologized to everyone at the meeting. She was also disciplined, but the details of how she was punished haven't been revealed. Because according to superintendent Sandra Lemmon, the matter is a personal one.
Lemmon also said that she didn't feel that Boyer attempted to make a racist statement, and many accused the district of trying sweep everything "under the rug," which they denied.
"After completing the investigation, the principal was disciplined and apologized to school staff who were present for the incident," read a statement from the school district.
"The central office administration also made clear to the principal that such conduct violated both the letter and the spirit of the School District's nondiscrimination policies. This matter was not 'swept under the rug.' It was promptly addressed, and it was made clear that this incident was inappropriate within the school setting."
Boyer released a statement as well.
"I deeply regret my decision and have learned from it," she wrote. "Even though I did not have ill intent, my poor choice was addressed by both the administrative team and school board. On top of those repercussions, I publicly apologized to staff and met with some parents as well."
For Boyce's part, he's been called a hero for exposing the photos and people continue to believe more should've been done to the principal.
"A principal that wasn't aware this was wrong should not be a principal," someone tweeted on Jan. 31


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Mira Loma Animal Hospital is proud to serve Mira Loma, CA and surrounding areas. We are dedicated to providing the highest level of veterinary medicine along with friendly, compassionate service.
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Is religious healing one thing that is achievable? It may well pretty properly be and all the actions necessary to tap into these kinds of spiritual therapeutic ayahuasca already exists within just you. You just are usually not mindful of it however! You can find experts that do fully grasp what on earth is needed to improve the probable for non secular therapeutic. Ron Roth is one these skilled. He has produced a piece of art from the form of The Spiritual Healing Intensive. Regardless of whether you read through the e-book variety or pay attention to the ten CDs that comprise the audio variation, you will explore this really is a tremendous method that may provide all of the necessary perception necessary to heal you regardless of what may be mentally or physically afflicting you.
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Make your holiday unforgettable
You are visiting us in Bonn and are looking for inspiration for a trip? Our tourists have put together some leisure tips for you. Whether action, nature, wellness or a trip with the children - here you will find exactly what you are looking for!
On narrow paths along the slope, fantastic viewpoints and a beautiful panorama of the Rhine as a highlight of this excursion. The hiking classic among the round trips in Königswinter.
Vera's tip
On the planetary path, our solar system was reproduced true to scale. If you walk the path at normal walking pace along. Are you already moving at about three times the speed of light? That sounds exciting? Let's go.
Florian's Tip
Just a day out of everyday stress and into the wellness oasis of the Kameha Spa & Power House. A relaxing massage, a healthy snack for lunch, sweat in the sauna and finally a few laps in the infinity outdoor pool.
Sophie's Tip
Zorbing, also called action ball, is a trend from New Zealand. You are inside an inflated, transparent, doppelhügligen ball and races with 50 km across the meadow. Nothing for weak nerves.
Julian's Tip


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AUCKLAND: Pakistan defeated New Zealand by 48 runs in the second T20 in Auckland Thursday to level the series and score their first win of the tour over the Black Caps.
The visitors set an imposing target of 202 thanks to half-centuries from Babar Azam and Fakhar Zaman, then bowled out New Zealand for 153 with nine balls to spare.
Pakistan made 201 for four in the second T20 against New Zealand at Auckland's Eden Park on Thursday.
Babar Azam and Fakhar Zaman jointly top-scored with 50 apiece for the tourists, while Ben Wheeler was New Zealand's best bowler with two for 36.
Earlier, Pakistan won the toss and elected to bat in the second Twenty20 against New Zealand at Auckland's Eden Park on Thursday.
The tourists are down 1-0 and looking to reverse a form slump to keep the three-match series alive.
"It looks a good batting pitch so hopefully we can come up with a good score then defend it," captain Sarfraz Ahmed said.
"I've just told the boys 'play your natural game, don't worry about the result, we have nothing to lose'."
Pakistan recalled opening batsman Ahmed Shehzad in a bid to make a strong start as they chase their first win on tour against the Black Caps.
New Zealand skipper Kane Williamson returns for the hosts after missing the first match with a thigh strain.
Trent Boult is also back alongside rookie paceman Ben Wheeler, while Tim Southee is rested.
The three-match series will finish in Tauranga on Sunday.
New Zealand: Martin Guptill, Colin Munro, Kane Williamson (capt), Tom Bruce, Glenn Phillips, Colin de Grandhomme, Mitchell Santner, Ben Wheeler, Ish Sodhi, Seth Rance, Trent Boult.
Pakistan: Fakhar Zaman, Ahmed Shehzad, Babar Azam, Sarfraz Ahmed, Umar Amin, Haris Sohail, Shadab Khan, Faheem Ashraf, Hasan Ali, Mohammad Amir, Rumman Raees.
Series: New Zealand lead the three-match series 1-0
Umpires: Shaun Haig (NZL), Chris Brown (NZL)
TV Umpire: Wayne Knights (NZL)
Match referee: Richie Richardson (WIS)


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About ISH Show in 2019 Frankfurt Germany
ISH is the world's leading trade fair focusing on the responsible management of water and energy in buildings. It sets trends for modern bathroom design, sustainable heating and air-conditioning technology as well as intelligent home systems.
Impressions from KUGE on the Exhibition
Main products: Stainless steel toilet, urinal, wash basin,shower tray,vacuum toilet, biological waterless toilet and all surroudning bathroom fittings and accessaries.
Website: www.cxkuge.com
Impressions from KUGE on the Exhibition
Kuge is a leading designer, manufacturer and supplier of stainless steel products: stainless steel urinal, stainless steel wash basin, stainless steel toilet bowl, stainless steel sanitary ware and so on.


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Taylor Swift and Calvin Harris's 'undeniable connection'
Taylor Swift and Calvin Harris have an "undeniable connection."
The 'Blank Space' singer was seen flirting with the Scottish DJ - who recently split from girlfriend Aarika Wolf - at last month's BRIT Awards and they have reportedly been in regular contact since then and were inseparable when the 25-year-old star visited Las Vegas, where Calvin has a residency, last week.
A source said: ''Taylor and Calvin are into each other in a big way. It's early days but there's an undeniable connection between them and they've been texting pretty much every day.
''They had a lot of fun in Las Vegas and she especially enjoyed hanging out after the show.
''Taylor has told friends she can't get him out of her head. Everyone thinks this could have serious potential.''
It is believed Taylor - who previously dated Harry Styles, and Conor Kennedy and was linked to singers Tom Odell and Matt Healy - and Calvin were introduced by mutual friends Ellie Goulding and Haim and all of Taylor's pals are hoping the romance works out.
The source added to Grazia magazine: ''They have so much in common. Her friends totally approve, which is a plus
''What's more, everyone knows she has a soft spot for British men. The fact Calvin is so well-mannered and creative is a bonus too.''
Daily Celebrity Watch


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My Relationship With Body Image and Why I Started Zen Ramen
Let me properly introduce myself. I am the founder of Zen Ramen and I started this business as a passion project to spread the message of positive body image and how Zen Ramen Nude Noodles have helped me achieve this.
But this world currently doesn't exist, and it is up to everyone to collectively stand up and shatter the illusion of physical perfection that women are held to. The 'last 5 kilos" that we all want to lose are the invisible shackles that stop us from pursuing our dreams and living our purpose. Instead we spend our time obsessed with diets and restricting what we eat, collectively ploughing billions of dollars into the weight loss / diet industry each year.
The birth of my daughter made me think back to my earlier years in university when I had a bout of anxiety which manifested into an unhealthy relationship with food. I was your typical Asian nerd in high school, ensconced in the pursuit of academic excellence. I went to an all girls private school, but didn't sit with the popular girls. My friends were all nerds, we blissfully discussed Harry Potter books at lunchtime and did extra Algebra after school. I didn't know any boys and nor was I interested, I just wanted to get over 99 on my HSC.
This blissful bubble devoid of exposure to poor body image (social media didn't really exist in 2002) was burst when I started university. Suddenly I was surrounded by new stimuli... and boys. The girls I hung out with didn't want to analyse the latest sci-fi fantasy novel, they wanted to go clubbing and pick up boys. The unknown world of attracting and interacting with the opposite sex made me turn to mass media to seek guidance on what makes a female "attractive". It told me it was size 0 Victoria Secret models, not loud spoken socially awkward ethnic girls who loved to read. I started to examine my own body in that light, I pinched bits of loose flesh on my body. My anxiety around loss of control in a new social environment manifested into an obsession to lose weight on an already naturally 'skinny fat' body. I tried to restrict what I ate but it never lasted and I would then inhale as much food as I could lay my hands on. This almost resulted in a full blown eating disorder. It took 6 months of going to a counsellor to reverse these behaviours and whilst it was nipped in the bud pretty early, I still feel this will forever leave a traumatic scar on my psyche and expose me to being 'triggered' in the future.
I started my side hustle Zen Ramen as a protest against restrictive eating behaviours, because eating in abundance and without guilt was the tool that helped me recover. By no means is it the underlying cure to poor body image, it's merely a band aid to help in the healing process. Zen Ramen Nude Noodles are shirataki Noodles from Japan and has been eaten in Asia for hundreds of years, it is made from a root vegetable not a carbohydrate so is extremely low in calories (only 28 calories per serve), which means you can pretty much eat as much as you want without gaining weight. My mum brought them for me from overseas and I ate this when I was recovering as I knew I wouldn't need to restrict my intake and they were the perfect base for delicious pasta dishes, stirfries or noodle soups. I continue to eat them today as a lighter substitute to pasta / noodles. Eventually as I tackled the underlying emotional issues I was able to recover fully.
This brings me back to today. I just had a baby, I'm carrying an extra 10 kilos. I didn't know that women tend to look 3 months pregnant for up to 6 weeks after giving birth as the uterus contracts back. But people don't want to see that in glossy magazines, people want to see "how Mrs WAG got her pre baby body in 30 days" because it makes for dramatic transformation photos. I don't plan on having a 6 pack next month but I will be eating my noodles more frequently along with an overall healthy diet and regular exercise to get back to a comfortable weight for my body. I will not obsess about my body but instead celebrate my body for what it just went through to create the most perfect, amazing little human I've ever met.


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Scientists Explore the Workings of Taste Buds
JOE PALCA, host:
This is TALK OF THE NATION/SCIENCE FRIDAY from NPR News. I'm Joe Palca, in for Ira Flatow.
People say there's no accounting for taste, but in the next hour, we're going to try and prove to you that that's just not true. Now we won't explain why some people actually like William Shatner's singing. We'll be talking about how your tongue lets your brain know whether you're eating chocolate cake or salt cod. The answer starts, at least, with your taste buds. Your taste buds let you know whether what you're eating is sweet, sour, salty or bitter. Exactly how taste buds works remains a bit of an enigma, and now scientists are one step closer to figuring it out.
In this week's Proceedings of the National Academies of Sciences, scientists demonstrated that a peptide called NPY is involved in the communication between taste cells in a taste bud, and that communication appears to be key to decoding taste. We'll talk with the author of the study to find out what he did and what it means.
If you'd like to join the discussion, give us a call. Our number is 1 (800) 989-8255; that's 1 (800) 989-TALK. For more information about taste, you can go to our Web site at sciencefriday.com, where you'll find links to our topic.
Now let me introduce my guest. Scott Herness is a professor in the College of Dentistry at Ohio State University in Columbus, Ohio, and was the senior author on the PNAS paper. He joins me now by phone.
Welcome to SCIENCE FRIDAY, Dr. Herness.
Dr. M. SCOTT HERNESS (College of Dentistry, Ohio State University): Hi, Joe. It's great to be here.
PALCA: So before we get into the details of what you did, I have to ask this question that's been bugging me. I mean, you know, taste buds--I thought that the taste buds were sort of all over the tongue and that you--I mean, that--but that there were special places where you could taste salty and more likely in one spot; there was that map that you learned in physiology class.
Dr. HERNESS: Oh, yeah. That's...
PALCA: Is that...
Dr. HERNESS: ...the famous taste map.
PALCA: Yeah. What about that? Is your work undercutting that at all? 'Cause I would hate to think that the foundations of my academic training are being shattered like that.
Dr. HERNESS: Well, I think you better grab something, Joe, because...
PALCA: Uh-oh.
(Soundbite of laughter)
Dr. HERNESS: ...might be a little rattling.
PALCA: Dang.
Dr. HERNESS: Actually, that famous taste map that we see in textbooks all the time, even in elementary school textbooks--it's a really nice little visual tool about the tongue, but there's only really one problem with it, and that's that it's completely wrong.
PALCA: Oh, I hate those little problems, you know?
Dr. HERNESS: Yeah.
PALCA: But surely there's--I mean, they didn't bring this--make this up out of whole cloth. What does that map...
Dr. HERNESS: Yeah.
PALCA: ...purport to show, and what are they--what are you disagreeing with it about?
Dr. HERNESS: Well, so the taste map--if you read it in the textbooks, it would tell you that you taste sweet in the front of your tongue and sour on the sides of your tongue, bitter in the back, and that part of it's actually not true at all. We taste all the taste qualities everywhere across the tongue, and even in places in the oral cavity that aren't on the tongue. So you can taste sweet, sour, salty and bitter just fine on the front of your tongue. What that map really means is where you're most sensitive to these taste qualities. You are more sensitive to bitter qualities in the back of your mouth and more sensitive to sweet on the front, and I don't have that many friends that are wine connoisseurs, but I've heard that wine tasters can pick up the sour taste on the sides of their tongue first, and that is where the tongue seems to be most sensitive to sour.
PALCA: So--OK. Before--I don't want to dwell on this, because it's not exactly on your topic, but why would you be more sensitive in different places? Is there a greater concentration of taste--particular kind of taste--well, you said all taste buds can taste all tastes, so is it...
Dr. HERNESS: That's true, but they don't do an equally good job at it. Recently in the field, we've finally cloned some of the receptors that are the actual molecules that interact with the taste molecules, like sucrose or quinine for sweet and bitter, and they're not distributed evenly across the tongue, so there are more bitter receptors in the taste buds in the back of the mouth, so those buds are a little more sensitive to bitter. And that helps to explain the regional differences that we see across the tongue.
PALCA: OK. So tell us a little bit about what a taste bud is. What we know now is it has a receptor, some kind of molecule on its surface, that attaches to flavorings, but what else do we know about it?
Dr. HERNESS: Well, one thing we know about taste buds is that they are unique across sensory systems, and they're highly conserved within the taste system, so that is to say, if you look at any kind of organism that has a backbone in it, whether it's a fish or a chicken or a person, they have a taste system; they're going to have taste buds. But if you look at other sensory systems, like vision or audition or even smell, you have the receptor molecules distributed across a wide array of the epithelium. So there's something unique about taste that it seems to require taste buds, 'cause that's--all systems that have taste have these taste buds. So we've known that there are these buds for hundreds of years, and we always thought there must be a good reason why you have to have taste buds in order to have a taste system that works well, and some of the research that we've been doing is beginning to address that question of why there are taste buds.
PALCA: And--but what's the story, then, with this peptide? I guess that's a string of amino acids called NPY.
Dr. HERNESS: That's right.
PALCA: What's it doing in the equation here?
Dr. HERNESS: Well, what we think it's doing--and we've looked at another peptide as well that's called cholecystokinin, or CCK for short, and these two peptides, CCK and NPY, seem to go hand in hand in the taste bud. And what we think that they're doing is they are a mechanism that these cells can use to talk to one other within the bud. It seems like the special structure that you have, a taste bud, where you pull all the receptors very close to one another into this cloistered arrangement, is really the perfect anatomical structure to allow the cells to begin to communicate with one another. And we think these peptides are agents that the buds are using for individual cells to talk to one another.
PALCA: Interesting. Well, I'd like to include my listeners--our listeners--on this. It's--our number's (800) 989-8255. And let's take a call now from--Is it Navandrea(ph)?...
NAVANDREA (Caller): Yes, that's it.
PALCA: ...in Tallahassee, Florida. Welcome to SCIENCE FRIDAY. Thanks for calling.
NAVANDREA: Thank you. I'm a longtime listener, first-time caller.
PALCA: Oh, great.
NAVANDREA: And I have a question for the speaker. This is kind of a weird question 'cause it's a strange phenomenon and maybe it's just for me, but when I eat something sweet and then I go back to drink maybe a sweet drink, the drink never really tastes quite as sweet before--as it did before I ate the sweet food. Is there a reason for that?
Dr. HERNESS: Oh, there sure is a reason for that. That's a phenomenon we call adaptation.
Dr. HERNESS: And it's interesting; all the animals that we've studied in the taste systems have adaptation, but one of the animals that has the greatest amount of adaptation is us, humans.
NAVANDREA: Oh, OK.
Dr. HERNESS: And if you taste something sweet and you continue to have it in your mouth, your taste receptor cells actually adapt to that stimulus, which is to say they don't respond as well to it as they did when you first put it in your mouth. And a good example of that is if you take a piece of hard candy and you put it in your mouth, it tastes sweet. And if you don't move it around, if you're very careful and leave it exactly in the same spot, pretty soon the sweet taste will go away.
NAVANDREA: Oh!
PALCA: Interesting.
NAVANDREA: Very, very. Well, thank you so much. I appreciate that.
PALCA: Navandrea, thanks for that question.
So I'm still a little--I need--I want to be a little clearer about exactly how the taste buds--I mean, they're not quite like the eyeball, where everything in the back of the retina connects straight to the brain. The taste buds don't all connect--or do all taste buds connect directly to the brain, or do all the cells in the taste bud connect to the brain?
Dr. HERNESS: Yeah. No, that's one of the puzzling features of taste buds that we've been really scratching our heads about, those of us who spend some time trying to figure out how taste buds operate. Actually, only a minority of the cells in the bud are actually connected to the brain by the nerve fiber, and the rest of the taste cells are not connected to the brain at all. But now we've learned that these taste cells that are not connected to the brain are actually the ones that express the bitter receptors. So it's a little puzzling, 'cause here you have a cell; it's going to interact with a bitter stimulus, it's going to get excited, but how is it going to relay that message to the brain that you're tasting something bitter when it's not connected to the brain?
PALCA: Huh.
Dr. HERNESS: And that's where we think these cells talking to one another within the bud is going to help us to explain how the bud operates.
PALCA: I see. So it's this communication that's essential for the taste bud to work properly.
Dr. HERNESS: It seems to be...
PALCA: Seems to be.
Dr. HERNESS: ...turning out that way, yes.
PALCA: OK. And we should emphasize here that your work was done primarily in rodents, isn't that right?
Dr. HERNESS: This is correct. Yeah.
PALCA: Right. But we're making the assumption, since this taste is so highly conserved across species, that what's true for rodents at least is going to be reasonably close to being true for humans.
Dr. HERNESS: Well, we have looked at these peptides in a number of different species, such as hamsters and rabbits. We haven't looked at them in primates yet, but we are finding them in the other species that we look at, so we're going to make an assumption that something like this could be operating in the human system as well.
PALCA: OK. Let's go to Kim in Brooklyn. I suppose that's Brooklyn, New York. Kim, welcome to the program.
KIM (Caller): Hi. Thank you.
PALCA: What's your question?
KIM: Well, my question is about a taste that my Japanese students have told me about that Americans and, I guess, all other cultures don't consider. You know how we have salty tastes and bitter and sweet? They have one other taste. I think it's called umami and...
Dr. HERNESS: That's correct.
KIM: Yeah. And I don't know what that taste is in our cuisine.
PALCA: Oh, I--well, go ahead. I've heard of examples, but maybe you can tell us, Scott Herness.
Dr. HERNESS: Sure. I can give it a shot. Umami has been studied in Japan for many, many years, and I've been in the field over 20 years myself. When I first started out in this field, we talked about four taste qualities: sweet, sour, salty and bitter. And now we talk about five, and that fifth one is umami, which is--the most common example of that would be glutamate, MSG, that's used as a flavor enhancer. But it seems to be a little more general than that, and it's the flavor of amino acids. So a good common example of that would be sort of that brothy taste that you might have in a very--in a consomme or a very bland soup--the taste of the amino acids from the proteins of the meat.
PALCA: Huh. And someone told me that eel has an umami (pronounced ooMAmee), umami (pronounced yooMAmee) taste quality to it. Is that possible?
Dr. HERNESS: Oh, I'm sure that it does.
PALCA: Hmm.
KIM: OK. Thank you very much.
PALCA: Thanks very much.
Well, so how--you know, are we going to get the complete picture, or is there a--still ways to go here, do you think?
Dr. HERNESS: Well, I think that we still have a ways to go. I've been doing this for 20 years. I've got a little bit to go before retirement, but I don't think I'm going to run out of things to do.
PALCA: OK. Excellent. Well, unfortunately, you may have things to do, but we have no time to hear about them 'cause we've run out of time for this segment. So thanks very much for joining us today.
Dr. HERNESS: It's been my pleasure, Joe. Thanks for having me.
PALCA: Scott Herness is a professor in the College of Dentistry at Ohio State University in Columbus, Ohio.
We're going to take a short break now, and when we come back, we're going to talk about the science of bicycling. What makes one bicyclist better than another? What makes one bicycle faster than another? And a simple question: How do you keep a bicycle upright? All that when we get back. Stay with us.
This is TALK OF THE NATION from NPR News.
Copyright © 2005 NPR. All rights reserved. Visit our website terms of use and permissions pages at www.npr.org for further information.
NPR transcripts are created on a rush deadline by Verb8tm, Inc., an NPR contractor, and produced using a proprietary transcription process developed with NPR. This text may not be in its final form and may be updated or revised in the future. Accuracy and availability may vary. The authoritative record of NPR's programming is the audio record.


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Communication distance:400M(visible distance , it has some different base on environment)
Work frequency section:2.4G ISM,126 channels.frequency section
Max transmitting power rate: 20dBm
Receiver sensitive: -94dBm
DMX connector: 3pin female plug
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Establishing Communication:
1. Power on DMX512 wireless received board and transmitted board.
2. Press "Key" button to set same ID value of transmitted board and received board. Please use different ID value if you need use more than 1 group wireless universals at same time in same place.
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4. Communication was established correctly.
Tong Chuang Technology Park, Shijing Town, Baiyun District, Guangzhou, 510430 China
EMAIL: email@example.com


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Angie's List Answers is the trusted spot to ask home improvement and health questions and get answers from service companies, health providers and consumers. For ratings and reviews on companies in your area, search Angie's List.
*Updates every 4 hours
This can be a wide-open question - by the time you have gas service installed (which your utility might or might not do for free), the oil tank removed in accordance with UST / AST (Underground Storage Tank or Above Ground Storage Tank) regulations, and furnace and hot water heater (if that is oil fired or you will be converting from electric to gas to save cost) replaced, I seriously doubt you can do it for under $5000, and probably closer to $8-13,000 if gas service will be new.
Google this search string for various blogs and organizations that compare pricing - "convert from oil to gas heat cost".
You local gas company, state energy efficiency agency, and the EPA all have booklets and computation guides to help assist you in estimating cost and potential savings, though of course till you get actual bids you will not know for sure what it willl cost. Do NOT forget to check on the value and timeframe availability of federal and state tax credits, and any local energy efficiency rebates available - if you milk those for all they are worth, in some locales it can add up to 100% of the cost though typically more like 15-25%.
If this is new gas service, consider for resale value or additional savings for you having gas run to stove, clothes dryer, maybe fireplace as well at the same time.
Hopefully Todd or BayAreaAC will jump in here with their thoughts and cost estimates.
Answered 6 years ago
by LCD
Report It
It will cost from 5 to 10 grand the better the quality, 97percent efficient the more the unit will cost. also run a gas line for A BBQ .
Source: firstname.lastname@example.com
Answered 6 years ago
by plumber2007


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Kit Carson County Health Service District's Specialty Clinic is pleased to announce the addition of Dr. Robert Hoyer.
Dr. Hoyer is board-certified in internal medicine, medical oncology, and hematology. He received his medical degree from Georgetown University School of Medicine, and went on to complete his residency in internal medicine. He completed his fellowship in hematology and oncology at Mayo Graduate School of Medicine.
Dr. Hoyer is dedicated to patient-centered care and he strongly believes in the value of comprehensive, multidisciplinary cancer treatment. He specializes in gastrointestinal cancers and lung cancer.
Dr. Hoyer's goal is to bring the highest level of oncology care to the community of Burlington.
See more information in this week's Record.


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Motozone Auto Group is a pre-owned vehicle dealership conveniently located at 333 North Road, Coquitlam that specializes in all makes and models of used cars for sale. A dealership with various customer service of experience, Motozone Auto Group is your ultimate shopping destination for pre-owned cars, sport utility vehicles and vans. We meet the automotive needs of car buyers and owners in all over the BC. Whether you need a new car or pre-owned vehicle, financing & Leasing, we are here for you!
Motozone Auto Group, we have a business philosophy that is clear-cut and straightforward; deliver the highest quality vehicles at the most competitive prices, all in an honest, relaxed and pressure-free atmosphere. We strive to carry on a long-standing tradition of quality and excellence with every vehicle that we showcase. Every effort and care is made to deliver to our customers a vehicle that meets or exceeds their expectations.


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Returns Policy
You may return most new, unopened items within 30 days of delivery for a full refund. We'll also pay the return shipping costs if the return is a result of our error (you received an incorrect or defective item, etc.).
You should expect to receive your refund within four weeks of giving your package to the return shipper, however, in many cases you will receive a refund more quickly. This time period includes the transit time for us to receive your return from the shipper (5 to 10 business days), the time it takes us to process your return once we receive it (3 to 5 business days), and the time it takes your bank to process our refund request (5 to 10 business days).
If you need to return an item, please Contact Us with your order number and details about the product you would like to return. We will respond quickly with instructions for how to return items from your order.
If you need to exchange an item, we do things a little differently. Because we are a small shop, we can't guarantee that the item you might want to exchange for will be in stock by the time your returned item gets to us. Because of that, if you want a different product, please order the product, then contact us for a a return of your original item. We will ship out your new item and refund your return in accordance with our Return policy.
Due to the nature of our business and International Trafficking in Arms Regulations, we will only ship to U.S. addresses. In addition, we will not ship to Massachusetts, New York, New Jersey, Maryland, or California. The lawmakers in those states have made it too difficult for a small business like myself to keep up with their regulations on what can/cannot be shipped to their state.
We charge a flat rate of $10 for shipping, with the exception of custom uppers. Custom uppers ship for $20.
Drop Shipping


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The ultimate, most unique, extraordinary, amazing downtown Live/Work Dream Space on the market! Two meticulously restored, attached, 3-story buildings feature some of the most fabulous residential space we've seen (and we sold most of the condos in the Conrad!). The property includes approx. 900 SF of commercial space + 5,300 SF of incredible "loft" space not yet finished. Huge 4-car built-in garage. Unbelievable 700 SF+ exercise area. Storage for the ages. Extraordinary custom lighting, sound & detailing throughout. Amazing woodwork. Multiple heating & cooling systems. Incredible views of downtown & a stone's throw from the Canal, IUPUI & the IU Health campus. This magnificent property is an architectural tour de force in every respect.
View Tax Assessment & SF Info | View Tax Records
F.C. Tucker Company, Inc. | 442 Massachusetts Avenue, Indianapolis, IN 46204 | 317.634.6676 | Licensed brokers in Indiana.


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The current hottest female brand in the Ghanaian music industry, Wendy Shay, real name Wendy Addo has stated that she does not believe in falling in love, at first sight.
In an interview after her performance on Tv3's relaunch of Music Music, Saturday, December 15, the musician who has released six hit songs in six months was asked among other questions if she believed in love at first sight by the host of the show, Nana Kwame and she answered in the negative.
"No, I do not believe in love at first sight. I think love should be a process, it should grow",she said
In other news, Wendy Shay has finally apologised to Ghanaians and Multimedia following her dumb question statement.
Recently, Wendy insulted a journalist from Multimedia called MzGee by referring a question she asked as dumb.
MzGee asked "Who are you as Wendy Shay" and she replied "Who am I as Wendy Shay, Ok can we cut the questions, this is a dumb question…Who is Wendy Shay? No, I don't want to do the interview anymore please", and walked out.
However, the Uber Driver hitmaker has, therefore, showed remorse and has rendered an unqualified apology to the whole Multimedia crew and to Ghanaians as well.


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Basic Strides to Finding the Best Enemy of Anti-aging Face Creams From Home
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Honestly, the best enemy of anti-aging face cream may not be a well-known brand; this is on the grounds that the majority of the brands out there including the prevalent brands don't contain viable fixings that truly take care of business. Along these lines, this article will advise the fixings to search for in the event that you truly need the best enemy of anti-aging face cream.
It isn't sufficient to search for compelling fixings; you ought to likewise guarantee that you go for items that don't contain cruel synthetic concoctions that are destructive to your body. Dominant part of the counter anti-aging face creams available contain brutal synthetic concoctions like aromas, parabens, mineral oil, alcohols and sulfates; in all actuality these synthetic compounds accomplish more damage than anything else. They strip your skin of common oil and dampness Nathan Peterman Womens Jersey , along these lines causing dryness, bothering and unfavorably susceptible responses.
Consequently, the best enemy of anti-aging face cream is found among the common brands. Nonetheless, all common items are not made equivalent; thus, you should search for regular brands that contain fixings demonstrated to make your skin look lovely.
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BEIJING, Feb. 21 (Xinhua) -- As bloodshed and tensions rise in Ukraine, analysts said all political parties, facing tempting invitations from Russia and Europe, need to reach a common ground on the country's future direction and steer away from a possible civil war.
Kiev has witnessed the bloodiest outburst of violence since demonstrations began last November Dion Dawkins Womens Jersey , with 67 people killed and 551 others injured in clashes starting Tuesday, marking the culmination of confrontation between the opposition and the Yanukovych government.
The East European country, sitting at the strategically important juncture between Russia and Europe, with which it also shares the Black Sea coast, has long been under competing influence from both powers for geopolitical interests, said Liu Fenghua Zay Jones Womens Jersey , an expert at the Chinese Academy of Social Sciences.
"The current demonstrations were fuelled by Yanukovych's sudden suspension of the Free Trade Zone (FTZ) talks with the European Union last November, which was seen in Ukraine as a landmark for joining the European integration process," Liu said in an interview with Xinhua.
Noting that Yanukovych had long promoted closer ties with Europe in his previous speeches, Liu, vice director of the Institute of Russian, East European and Central Asian Studies at the academy Tre'Davious White Womens Jersey , said the president must have felt troubled before making the decision.
"Ukraine's economy is largely depending on Russia's market and natural gas import," he said, adding joining the Russia-initiated customs union will bring "immediate interests to the country, including a cut on gas price," while joining the FTZ risks Russia's counter measures, "which the FTZ agreement with Europe cannot equally compensate."
"Russia paused the import of Ukrainian dairy and steel products in 2013 Kyle Williams Womens Jersey , and used gas price as leverage, which have caused mounting deficit and losses of over 6.5 billion dollars on the Ukrainian side," Liu said.
Meanwhile, support for the Ukrainian opposition leaders demonstrated by Western countries was an "immediate cause" of the recent violence in Kiev, Timofei Bardachev, director of the Center for European Studies at the High School of Economics in Russia LeSean McCoy Womens Jersey , told Xinhua in an exclusive interview.
The Ukrainian opposition leaders, for example, were invited by senior officials to Berlin, where they were openly told Germany favored them in the recent confrontation, he said.
"That recognition provoked the radical opposition to increase their aggressive actions against authorities, what we are witnessing right now Tremaine Edmunds Womens Jersey ," Bardachev said.. Cheap Old Skool Shoes Cheap Old Skool Black Cheap Old Skool Cheap Old Skool Cheap Old Skool Black Air Max 95 Sale Nike Air Max 2017 Sale Cheap Nike Air Max 90 Cheap Air Max 2018 Shoes Cheap Air Max 90 Online


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I've been trying to get a good picture of this set since I made them. We have a sunny day today, so I took them outside and snapped this picture.
They are truly a gorgeous organic set of beads. I made them pretty much the same way as the big one I made into a pendant a few weeks ago because I knew they would be beautiful in a bracelet.
If you look at the enlargement of the third bead from the left, shown here, you can see how complex and amazing they are.
The little silver dots add just the right sparkle too!
I'll be getting back to the redware beads again as soon as I have a chance to torch again.
BUT! The other huge thing I really need to try VERY soon is to MAKE CHEESE!
Last evening, Tina & I went to a class taught by Betsy May, one of Tina's regular herb writers, at the Rosemary House in Mechanicsburg.
Ever since I read "Animal, Vegetable, Miracle" by Barbara Kingsolver, I've felt that I have to learn to make cheese, so when we heard about the class, we signed up.
Betsy showed us, step by step, how to make a variety of soft cheeses including goat cheese (with none of the goaty aftertaste that has kept me from eating it for years) and Neufchatel which is very similar to cream cheese.
But the Pièce de résistance was the 30 minute mozzarella!
Here's a picture of Betsy. I think she's just poured the whey off the curds for the mozzarella.
Even in the less than ideal conditions of the lovely dining room of the Sweet Remembrances Tea Room (next door to the Rosemary House) where the class was actually held, she made it look easy enough that I logged on to the computer this morning and ordered the hard to find ingredients (like Rennet and Lipase) to make our own.
Watching her make the soap - I mean cheese - reminded me so much of our soapmaking adventures. Mixing up the rennet ahead of time, heating the milk, waiting for the curds to form ... it just seemed vaguely familiar.
There were 16 people there and we ate every bite of the (about 2 pounds of) mozzarella! I wish I could have brought some home for DH to try, but I'm sure he'll be trying some soon enough.
I just can't wait to get everything we need together and make some. Wow! That and a homemade loaf of bread - I'm golden!


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Jon's DSLR Buying Guide: Part 3 – Accessories
November 10, 2011
This is part of a multipart series on buying a DSLR. For the camera, see Part 1 or the Lens, see Part 2. Once you've bought your camera and your lens, you'd think you were done, but like anything remotely expensive and/or complex — there's more! Of course, cameras have a thousand different accessories, bits, boggles, and doodads that you can buy. Just because you can, doesn't mean you should (unless of course you've got money to burn, in which case, skip this guide and just buy whatever strikes your fancy). As with the previous segments, I'm targeting the first time photographer here.
Jon's DSLR Buying Guide: Part 2 – The Lens
November 1, 2011
This is part of a multipart series on buying a DSLR. For the camera, see Part 1. Once you've selected a camera, you need to get a lens (or two, or three ^_^ ). If you're buying a Nikon, you don't need to worry about them fitting. All Nikon cameras that we care about (and most of the lenses) since 1959 use their F-mount system. Canon isn't so lucky, but they've been pretty good about standardization too (from what I understand). As with the camera, lenses come in a variety of shapes and sizes with their own classifications of "types" and their own set of numbers/features/doodads.
Jon's DSLR Buying Guide: Part 1 – The Camera
October 25, 2011
Today, digital cameras are ubiquitous — everyone who has a cellphone has a camera. Even with this proliferation of cameras, if you want a camera that is a step above the rest, you need an SLR or more correctly a DSLR. Fortunately for intrepid amateur photographers, DSLRs can be had on the cheap (Nikon D3100s start under $600), but what features do you need? What lenses should you get? What accessories? These are all questions I was recently asked and I thought I'd share.


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The Ugly Side of Making Fake Pay Stubs.
If you are applying for a loan, the lender will require some proof that you do get regular income and pay stubs can do that but you ought to make sure you are not breaking the law to make your point. No matter how bad you want the loan to be approved, remember that if you are caught in a fake pay stub scam you will be answerable to the court. In most cases, the fake pay stubs are used when applying for car loans. The people committing the fraud are usually people who are paid in cash which means they do not have a payslip to confirm that they earn what they say they or they are receiving tips which hike their earning. Those who have lost their employment or have just started working will face a pay stub problem too which can send them to the extreme edge.
Even though you might think that the fake pay stubs will solve your problems, the hope you are promised is usually false. Remember that the people who make them alter information regarding your education level and the grades your scores, the company you are working with, the salary you are getting and how long you have been employed. This might seem like a small price to pay considering the benefits you expect if the loan application goes through but once the lender realizes this, your relationship will crumble. Remember that you want to borrow from the same lender again in the future and if you do not maintain the relationship then you ought to find another institution to borrow from. The loan application contract requires the borrower to be forthcoming about the information asked for and submitting a forged pay stub goes against the agreement which means the lending institution can sue you for that.
If you were going through a middleman to get the loan, you will be putting their business in jeopardy not to mention the relationship they have with the lender. When the case is out, you will not just be charged with a felony but remember that it can destroy your reputation in the lending field such that no financial institution will ever touch you. When the lender does not have the time to haggle with you, the application will be declined and you will have to find another way to get the money you needed.
How I Achieved Maximum Success with Paystubs


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