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Enzymology and Protein Engineering
Identification and Functional Characterization of a Xenorhabdus nematophila Oligopeptide Permease
Samantha S. Orchard, Heidi Goodrich-Blair
Samantha S. Orchard
Heidi Goodrich-Blair
DOI: 10.1128/AEM.70.9.5621-5627.2004
The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA1, oppA2, or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.
The gram-negative bacterium Xenorhabdus nematophila has two types of eukaryotic hosts: the nematode Steinernema carpocapsae and various species of larval-stage insects (for a review of X. nematophila and its host interactions, see reference 11). The nematode acts as a mutualistic vector to allow X. nematophila access to the hemocoel (circulatory system) of insects. Once there, the bacterium is expelled from the nematode, the two organisms collaborate to kill the insect, and the invaders multiply and consume the insect corpse. Inside the insect, the bacterium is thought to produce exoenzymes including proteases, lipases, and lecithinases (11), and X. nematophila-secreted protease II has been shown to promote the digestion of some proteins of insect hemolymph (circulatory fluid) (8). The identities of the breakdown products of the protease activities are not yet known, but some bacteria, such as Salmonella enterica, are able to utilize peptides or free amino acids as nitrogen and carbon sources (15). Thus, oligopeptides could be a source of nutrients for X. nematophila in insect hosts.
As the food supply within the insect runs low, the bacterium and the nematode reassociate, the nematode develops into its vector stage (called the infective juvenile [IJ]), and the bacterium-nematode pair leaves the spent insect carcass in search of a new host. The bacterium resides in a specialized compartment of the IJ nematode intestine called the vesicle, within which X. nematophila multiplies to a population of 30 to 200 cells after an initial colonization by 1 to 2 cells (22). Even though IJ nematodes are nonfeeding, X. nematophila bacteria can survive within them for over 4 months in laboratory storage (21). At present, it is not known what nutrients support bacterial growth in the vesicle or how X. nematophila survives nutritionally during long-term IJ nematode quiescence. It is possible that X. nematophila bacteria residing in the nematode vesicle use nutrients released from dying bacterial siblings, which could include peptides. Alternatively, or perhaps additionally, the nonfeeding nematode may provide X. nematophila bacteria with nutrients to its own detriment. Consistent with the latter possibility, uncolonized nematodes survive longer in storage than X. nematophila-colonized nematodes (25).
Bacterial ABC (ATP-binding complex)-type oligopeptide permeases bring short peptides (two to six amino acid residues in enteric bacteria) into the bacterial cell. The oligopeptides to be transported are first bound by a periplasmic (in gram-negative bacteria) oligopeptide-binding protein (e.g., OppA), which directs the oligopeptides to the permease. The major known functions of oligopeptide transport in bacteria are the salvaging of cell wall components (using MppA instead of OppA) (26) and the uptake of nutrients (14, 27). Additionally, the peptide-binding proteins of some bacteria play important roles in host interactions, participating in intracellular survival (6) and binding to host cells (9), host proteins (10), or other bacterial cells in the host environment (18, 19, 23). Consistent with a specific role for oligopeptide-binding proteins in host interactions, several pathogens have been shown to have multiple oppA gene homologues. For example, Borrelia burgdorferi, which has two types of eukaryotic hosts (ticks, which act as its vector, and mammals), has five oppA homologues—three immediately upstream of the permease-encoding genes, and two individually carried on separate plasmids (5, 20). It was proposed that B. burgdorferi utilizes multiple oligopeptide-binding proteins to broaden substrate recognition for different oligopeptides encountered in each host environment (20). Indeed, the various B. burgdorferi oppA genes are differentially expressed as the bacterium traverses between host environments (32). However, to our knowledge, this hypothesis has not been tested further with B. burgdorferi.
In this work, we identify an X. nematophila opp locus consisting of two oppA homologues and putative permease-encoding genes oppB, oppC, oppD, and oppF. We constructed strains with mutations in the opp locus, which allowed us to directly examine the role of these genes in bacterial uptake of peptides, growth, and host interactions. We demonstrate that the permease is not essential for X. nematophila-host interactions, even though loss of the permease resulted in an altered growth phenotype in a peptide-rich laboratory medium and in hemolymph isolated from Manduca sexta insects.
Bacterial strains and standard growth conditions.Table 1 shows a list of bacterial strains used. Cultures were grown in a tube roller at 30°C in Luria-Bertani (LB) broth (24) that had been stored in the dark (34). LB agar (20 g liter−1) plates were supplemented with 1 g of sodium pyruvate liter−1 (34) and, when appropriate, kanamycin (KAN; 20 mg liter−1 for X. nematophila or 50 mg liter−1 for Escherichia coli) or chloramphenicol (CHL; 20 mg liter−1). Solid lipid agar (LA) medium was prepared as described elsewhere (30). For peptide-feeding experiments, defined medium consisted of 22 mM KH2PO4, 40.2 mM K2HPO4, 15.1 mM (NH4)2SO4, 0.41 mM nicotinic acid, 9.1 mM sodium pyruvate, 50 mM glucose, 10 ml of SL4 salts liter−1 (3), 50 mg each of all amino acids except leucine (the amino acid being tested) liter−1, and glutamate, which was added at 250 mg liter−1 (50 mg of glutamate liter−1 was subsequently determined to be sufficient for growth of X. nematophila), and 15 g of noble agar liter−1. Leucine or peptides (Leu-Leu, Tyr-Leu, Leu-Leu-Leu, and Tyr-Tyr-Tyr; Sigma, St. Louis, Mo.) suspended in water were spread on defined medium plates to give a final concentration of 70 μM (17). Permanent stocks of the cultures were stored at −80°C in LB broth supplemented with 10% dimethyl sulfoxide (Sigma, St. Louis, Mo.).
Conjugation of plasmids into X. nematophila.E. coli S17-1 λpir was used as the donor strain to conjugate plasmids for gene knockout constructions into X. nematophila as described previously (13), except that each strain was washed and resuspended in phosphate-buffered saline (PBS; pH 7.5) (28), and the conjugation plates were incubated at room temperature to allow X. nematophila to kill and/or outcompete the E. coli donor strain. Following selective growth, exconjugant cells were verified to be X. nematophila by their negative catalase reaction when hydrogen peroxide was applied and by their characteristic smell.
DNA manipulations and sequence analysis.DNA manipulations and transformation of E. coli were performed using standard protocols (reference 4 and product literature). Integrated DNA Technologies (Coralville, Iowa) synthesized all oligonucleotide primers. All compared sequences were obtained from the GenBank database (2) through the National Center for Biotechnology Information.
Identification of the opp locus.oppA1 and part of oppA2 were identified on an X. nematophila chromosomal fragment used in unrelated studies on tdk (S. S. Orchard and H. Goodrich-Blair, unpublished data). PCR with arbitrary primers (7) was used to sequence downstream of the oppA2 gene in its chromosomal location, leading to the discovery of an oppB homologue. To determine if the oppC, oppD, and oppF genes were further downstream, we designed a degenerate primer (oppFdeg1; NARNGGRTCNACYTTNARRCA, where N is A+C+T+G, R is A+G, and Y is C+T) based on the C-terminal OppF amino acid sequence, CLKVDPL, of E. coli K12, S. enterica serovar Typhimurium LT2, and Yersinia pestis. This degenerate primer was used in a PCR with primer oppXnrev1 (AAAACAGATGAAGAGCGTGC) to amplify and clone a 4.1-kb fragment from the X. nematophila chromosome, which was sequenced to reveal oppB, oppC, oppD, and oppF homologues.
Identification of an X. nematophila leucine auxotroph.HGB081 was conjugated with S17-1 λpir containing mini-Tn5 (CHLr) donor plasmid pBSL203 (1). CHLr exconjugants were tested for leucine auxotrophy on defined medium lacking leucine. The transposon insertion site of one leucine auxotroph, designated HGB731, was sequenced using primer P6 (16). The sequence appears to encode the first 34 amino acids of a LeuB homologue with 71% identity to E. coli LeuB (3-isopropylmalate dehydrogenase), the enzyme responsible for the penultimate step of leucine biosynthesis.
Construction of X. nematophila opp and leuB opp mutant strains.To construct oppA1 and oppA2 mutants, a 4.7-kb fragment of the X. nematophila chromosome containing oppA1, oppA2, and part of oppB was PCR amplified using primers KpnIoppA (GGTACCATTGGCCCTTTAGTGATTCC) and SacIoppB (GAGCTCAGTCCCATTTGGTATTCTGG). To create an oppB mutant strain, a 1.6-kb fragment of the X. nematophila chromosome including most of oppB (lacking the 5′ 89 bp) and oppC (lacking the 3′ 287 bp) was PCR amplified using primers KpnIopp5for (GGTACCACCTGAATGACCCGATG) and SacIoppAflIIrev (GAGCTCCTTAAGAAAGATTCAAACAG). These PCR products were cloned independently into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) to create plasmids poppA-B′ and popp′B-C′, respectively. Each insert was subcloned into pKR100 (Camr oriR6K suicide vector; K. Visick, Loyola University) using KpnI and SacI digests to create plasmids pKRoppA-B′ and pKRopp′B-C′, respectively. The GeneJumper primer insertion kit (Invitrogen) was used to insert GeneJumper KANr transposons into these plasmids, and the mutagenized plasmids were transformed into E. coli S17-1 λpir. Strains containing transposed pKRoppA-B′ or pKRopp′B-C′ were selected on the basis of KANr and CHLr. pKRoppA1::Tn5-12, pKRoppA2::Tn5-15, and pKRoppB::Tn5-20-4, chosen for suitable transposon location, were conjugated into X. nematophila, and exconjugants were selected on LB plates containing KAN. Strains that had undergone double recombination events with concurrent loss of the plasmid (KANr CHLs) were chosen for further study: X. nematophila oppA1::Tn5-12 (HGB585), oppA2::Tn5-15 (HGB586), and oppB::Tn5-20-4 (HGB717). The insertion in oppA1 disrupts the predicted encoded protein at amino acid 169 of 546. oppA2 is disrupted at predicted encoded amino acid 192 of 548, and oppB is disrupted at predicted encoded amino acid 235 of 306. To construct X. nematophila leuB oppA1, leuB oppA2, and leuB oppB mutant strains, HGB731 (leucine auxotroph) was conjugated with E. coli S17-1 λpir containing plasmid pKRoppA1::Tn5-12, pKRoppA2::Tn5-15, or pKRoppB::Tn5-20-4 as described for making the oppA1, oppA2, and oppB mutants, respectively. KANr exconjugants were screened for double recombination by PCR analysis.
Nematode maintenance and cocultivations with bacteria (noncompetitive).S. carpocapsae All (Weiser) was propagated by passage through Galleria mellonella larvae (Vanderhorst Wholesale, Inc., St. Marys, Ohio), harvested in White traps (33), and stored in water. Culture of nematodes on bacterial lawns for colonization assays was performed on LA plates as described previously (30). Axenic nematode eggs were isolated from gravid adult females as described previously (31), except that the eggs were washed and resuspended in LB broth. The eggs were left in LB broth at room temperature for 1 to 2 days to assess sterility and to enable hatching into larval-stage nematodes, which were used to inoculate coculture plates. IJ nematodes from the cocultures were harvested by allowing their migration to water. The IJ nematodes were surface sterilized by using a 3-min incubation in a 2% HOCl solution followed by three rinses with sterile distilled water. To release the bacteria from the nematode vesicles, a suspension of 104 nematodes in 0.5× LB broth was sonicated in a water bath sonicator (Branson Ultrasonics, Danbury, Conn.) for 1 min. The sonicated nematode suspension was vortexed for 3 s and then diluted and plated on LB agar to determine average IJ CFU counts.
Competitive bacterial colonization assays.Overnight cultures of mutant and wild-type strains (200 μl each) were mixed and inoculated onto 6-cm-diameter LA plates. After overnight incubation, each plate was inoculated with 2,000 to 4,000 early-larval-stage nematodes and incubated for 8 days at 25°C. KAN resistance of individual colonies was used to assess the proportion of opp mutant strains in the bacterial lawn at day 9 (plate input) and in the resulting nematode progeny (plate output). The competitive index was calculated with the ratio proportionplate output/proportionplate input. To determine if the mutant strains are competitive during a natural infection process, these IJ nematodes were then used to infect G. mellonella insects (insect input). G. mellonella was selected for use due to the ease of recovering IJ nematodes. The insects were infected with 100, 500, or 1,000 IJ nematodes, and all insects died within 72 h. After 2 weeks, the proportion of colonization by the opp mutant strains of the IJ nematode progeny from the insect infections (insect output) was determined, and the competitive index during infection was calculated with the ratio proportioninsect output/proportioninsect input. A ratio of 1 in either assay would indicate that the mutant strain competes equally as well as the wild-type strain for colonization of nematodes, and a ratio of <1 would indicate that the mutant has a defect in competitive colonization. Six independent experiments were conducted for all competition assays, except that only three experiments were performed for the insect infections with the oppB mutant strain. The oppA2 mutant strain was outcompeted by the wild-type strain on plates in four of the six experiments, and competitive indices for those experiments could not be reported. Competitive indices were calculated for each experiment, and the values were averaged and used to calculate standard errors of the means (SEM).
M. sexta insect rearing and injection assays.M. sexta (tobacco hornworm) insect eggs were obtained from W. Goodman (University of Wisconsin—Madison), and larvae were reared as described previously (30). After 18 to 24 h of growth in LB broth from −80°C freezer stocks, strains were subcultured at a concentration of 1:100 into fresh LB broth and grown for an additional 24 h. The strains were washed and resuspended in PBS, serially diluted, and plated on LB agar for subsequent CFU determination. One to one thousand CFU in 10 μl of PBS were injected behind the left first proleg of fourth-instar M. sexta. The insects were fed and monitored, and deaths in each set of 10 insects were recorded to provide a value for percent mortality at 72 h postinjection. Previous observations indicated that only rarely do additional insects die in response to injection beyond 72 h postinjection.
Analysis of growth profile in hemolymph.M. sexta larvae were chilled on ice for 30 min and cleaned with 95% ethanol. An incision was made in the left first proleg, and hemolymph was drained into a microcentrifuge tube. Extracted hemolymph was centrifuged at 10,000 × g at 4°C for 10 min, and glutathione (Sigma, St. Louis, Mo.) was added to a final concentration of 5 mM to retard the phenoloxidase reaction. Hemolymph, typically from ∼10 to 20 insects per experiment, was filtered through a 0.8-μm-pore-size filter and then through a 0.2-μm-pore-size filter (Millipore, Bedford, Mass.) and used the day of harvest or following a single freeze and less than 1 week of storage at −20°C. Bacteria from isolated colonies were cultured overnight in LB broth, washed, and resuspended in PBS and diluted 1:100 in hemolymph. Samples were placed in non-tissue-culture-treated, 96-well, flat-bottom polystyrene plates (Becton Dickinson Labware, Franklin Lakes, N.J.) and incubated at 30°C with shaking in a Molecular Devices SPECTRAmax 190 plate reader. Absorbance data was collected using SOFTMax PRO 3.1.2 software (Molecular Devices, Sunnyvale, Calif.).
Growth profile in LB broth and relative oppA1 and oppA2 transcript analyses.Bacteria from isolated colonies were cultured overnight in LB broth, and 400 μl of the cultures was used to inoculate 40 ml of fresh LB broth into 125-ml flasks. Because overnight cultures of the mutant and wild-type strains did not differ in viable cell counts (see Results), no adjustment was made for the difference in optical density of the starter strains. Three independent cultures for each strain were shaken at 250 rpm in a water bath at 30°C. Samples of the triplicate wild-type cultures were harvested to analyze relative levels of oppA1 and oppA2 transcripts. Total RNA from the wild-type strain was isolated, DNase treated, and used to make cDNA with random hexamer primers (Integrated DNA Technologies, Coralville, Iowa) and avian myeloblastosis virus reverse transcriptase (Promega, Madison, Wis.). As a control to detect DNA contamination of the DNase-treated RNA, samples with no added avian myeloblastosis virus reverse transcriptase were analyzed by PCR for amplification of oppF, and, as expected, none showed a product. Reactions (25 μl) for real-time PCR were performed in duplicate with iQ SYBR Green Supermix (Bio-Rad, Hercules, Calif.), cDNA template, appropriate primers, and a three-step cycling protocol on a Bio-Rad iCycler and analyzed with Bio-Rad iCycler iQ software. The amount of oppA1 and oppA2 transcript was measured by amplification with primers oppAIprfor (ATGTGCAGAACGCCGATG) and opp12rev (AGTTTTCCGGCAACGTCC) for oppA1 and primers oppfor8 (CAACATCGCCCGTGATTTG) and oppAIIprrev1 (TATGGACAAATAACTTGACC) for oppA2. As a negative control, water was used in place of cDNA template. Cycle threshold results for each sample were adjusted according to recA levels (amplified with the following primers designed from the X. nematophila recA sequence published under GenBank accession no. AF127333: recaminfor, TGTCCGTTTGGATATCCGCC, and recaminrev, CCCAGAGTATTAATACCTTCCCCAT) and then converted to arbitrary units, factoring in a twofold change in PCR product per cycle. The X. nematophila recA gene is not expressed at constant levels over a growth curve (K. N. Cowles and H. Goodrich-Blair, unpublished data), and we therefore chose to present the data as oppA1 transcript level as a proportion of the oppA2 transcript level at each sampling point.
Statistical analysis.One-way analysis of variance (ANOVA) with Dunnett's posttest was used to analyze the colonization and pathogenesis data. It was used to compare each mutant strain to the wild-type strain, or to a value of 1 for the competitive nematode colonization assays, at the 95% confidence interval. One-way ANOVA with Tukey's posttest was used to analyze the relative oppA1 and oppA2 transcript data. All statistical analyses were performed using Prism version 3.0a for Macintosh (GraphPad Software, San Diego, Calif.).
Nucleotide sequence accession numbers.All opp genes in the described X. nematophila locus have been sequenced from PCR-amplified fragments, and a 15.08-kb sequence including the opp genes has been deposited in GenBank (accession no. AY363171). A region of DNA including a 3′ portion of the putative leuA gene and a 5′ portion of the putative leuB gene has been sequenced from a cloned copy, and the 734-nucleotide sequence has been deposited in GenBank (accession no. AY443349).
Construction of X. nematophila oppA1, oppA2, and oppB mutant strains.We identified an opp locus putatively encoding an oligopeptide permease (OppB, OppC, OppD, and OppF) and two OppA peptide-binding protein homologues, OppA1 and OppA2 (see Materials and Methods). The entire operon was sequenced, and the genetic organization of the opp operon is presented in Fig. 1. The amino acid sequences of the predicted X. nematophila peptide-binding proteins, OppA1 and OppA2, had 58% identity to each other and 57 and 67% identity, respectively, to OppA of E. coli. The predicted permease proteins, OppB, OppC, OppD, and OppF, had 83 to 88% identity to the corresponding Opp permease proteins in E. coli. Insertions were made in oppA1, oppA2, and oppB to examine the role of the putative oligopeptide permease and associated peptide-binding proteins in the host interactions of X. nematophila. The mutations were confirmed by PCR and Southern hybridization analysis (data not shown). Insertions in oppA2 and oppB reduced transcription of oppF to levels similar to that of the negative control (no template) as measured by real-time PCR (data not shown), indicating that these insertions completely abolish transcription of the downstream opp genes under the tested conditions. However, the oppA2-oppF genes were still expressed in the oppA1 mutant strain, suggesting the presence of a promoter downstream of the insertion in oppA1 (data not shown).
opp operon organization in X. nematophila and locations of Tn5 insertions. Triangles indicate approximate locations of insertions in opp mutant strains used in these studies. Gene designations are indicated above the arrows, which represent open reading frames. The direction of the arrows indicates the direction of transcription. This region spans 8.35 kb.
The X. nematophila opp operon encodes a functional oligopeptide permease.To determine if the opp homologues encode a functional oligopeptide permease, we tested the ability of an X. nematophila leucine auxotroph to acquire leucine through peptide uptake. Insertions were made in oppA1, oppA2, and oppB in a leuB background. These double mutant strains were tested for growth on the dipeptides Tyr-Leu and Leu-Leu and the tripeptides Leu-Leu-Leu and Tyr-Tyr-Tyr (Fig. 2). Tyr-Tyr-Tyr was included as a negative control because it contains no leucine, and, as expected, none of the leuB strains grew on this tripeptide. The leuB (Opp+) auxotroph grew on all tested peptides that contain a leucine residue, while the leuB oppA2 and leuB oppB strains only grew on leucine-containing dipeptides. In contrast, the leuB oppA1 strain, like the leuB (Opp+) strain, grew on Leu-Leu-Leu, indicating that OppA2 and the permease are sufficient to transport the Leu tripeptide.
Growth of X. nematophila leuB opp mutant strains on peptides. Peptides and leucine (Leu), where indicated, were spread on defined medium plates at a final concentration of 70 μM. The plates were inoculated by scoring with a toothpick coated with the appropriate strain. Three independent colonies each of HGB081 (Leu+), HGB731 (leuB), HGB732 (leuB oppA1), HGB733 (leuB oppA2), and HGB734 (leuB oppB) were tested; a representative result is shown for each condition. Growth at 96 h after inoculation is shown.
X. nematophila opp mutant strains colonize and persist in S. carpocapsae nematodes.X. nematophila opp mutant strains were assayed for their ability to colonize and survive in S. carpocapsae nematodes following cocultivation on LA plates. All mutant strains colonized the nematode to levels similar to that of the wild-type strain after a 2- to 3-week cocultivation (Table 2). To test if X. nematophila opp mutant strains could recolonize nematodes in the natural habitat of an insect corpse, X. nematophila oppA1- and oppA2-colonized IJ nematodes were allowed to infect and kill G. mellonella insects. IJ nematode progeny were tested for level of colonization by bacteria, and again the X. nematophila opp mutant strains were able to colonize progeny IJ nematodes to an extent similar to that of the wild-type strain (data not shown).
Host interactions of X. nematophila opp mutant strains
X. nematophila opp mutant strains are competitive for nematode colonization.Because competitive assays can sometimes reveal subtle phenotypes, the X. nematophila opp mutant strains were tested for their ability to compete with wild-type X. nematophila in colonizing nematodes. On LA plates, the oppA1 and oppA2 mutant strains were as competitive as the wild-type strain (P was >0.05 for difference from 1), and the oppB mutant strain was significantly (P < 0.001) more competitive than the wild-type strain for nematode colonization (Table 2). The oppA2 mutant strain was outcompeted by the wild-type strain for growth on LA plates in four of the six experiments performed (data not shown). However, because the oppA2 mutant strain was able to compete with the wild-type strain for nematode colonization in the remaining two experiments and for competitive colonization of the nematode and growth in the insect infection assays (see below), this phenotype was not explored further.
To determine if the mutant strains are as competitive as the wild-type strain during the natural infection of insects, populations of nematodes carrying both the wild-type and opp mutant strains were allowed to infect G. mellonella insects. The calculated proportioninsect output/proportioninsect input ratios for the opp mutant strains (Table 2) are not significantly different from 1 (P > 0.05). Thus, X. nematophila opp mutant strains competed as well as the wild-type strain during growth and nematode recolonization inside the insect.
X. nematophila opp mutant strains kill and grow in insects normally.The X. nematophila opp mutant strains were tested for their ability to kill fourth-instar M. sexta larvae. Wild-type X. nematophila kills ∼50% of insects in 72 h when 1 to 1,000 CFU are injected per insect. All mutant strains were as virulent as the wild-type strain towards M. sexta, killing ∼40 to 60% of insects by 72 h after injection at 1 to 1,000 CFU per insect (Table 2). The majority of bacterial growth in the insect appears to occur after the insect's death (12). Thus, we reasoned that oligopeptide uptake might be essential for bacterial outgrowth in insects. The ability of the X. nematophila opp mutant strains to recolonize nematodes in G. mellonella carcasses (see above) suggested that they were able to grow in G. mellonella insects. However, to more easily measure bacterial growth rates, we studied the in vitro growth of X. nematophila in insect hemolymph. G. mellonella larvae are more susceptible to X. nematophila infection than M. sexta larvae are, and we thus chose to use hemolymph extracted from M. sexta insects for the growth analysis to subject the bacteria to a possibly more stringent environment for growth. A small but reproducible difference in growth rate (doubling times were 1.9 h for wild-type and oppA1 mutant strains and 2.1 and 2.5 h for oppA2 and oppB mutant strains, respectively) was observed for the oppA2 and oppB strains relative to the wild-type and oppA1 strains (Fig. 3A). However, all strains ultimately reached the same optical density.
Growth of X. nematophila opp mutant strains in hemolymph (A) and LB (B) liquid culture and analysis of relative oppA1 transcript levels in the wild-type strain in LB broth (B). HGB007 (Opp+, open squares), HGB585 (oppA1, filled squares), HGB586 (oppA2, open circles), and HGB717 (oppB, filled circles) were grown in 100 μl of hemolymph plus 5 mM glutathione in microtiter plates (A) or in 40 ml of LB broth in 125-ml flasks (B) at 30°C with shaking. (B) oppA1 transcript levels (gray columns) are presented as percentages of oppA2 transcript levels. Error bars indicate SEM of three independent cultures of each strain, except for the 8-h time point, which shows duplicate results for the transcript analysis due to the loss of an RNA sample. Columns with different letters are significantly different from each other (P < 0.05) as determined by one-way ANOVA with Tukey's posttest analysis, except that the 3-h column is not significantly different from the 5-h column.
X. nematophila opp mutant strains achieve a lower final optical density in LB liquid culture.To determine if the inability of the X. nematophila opp mutant strains to use oligopeptides as a nutrient source hinders their growth in a peptide-rich laboratory medium, their growth in LB broth was measured. The opp mutant strains consistently did not achieve as high a final optical density as the wild-type strain in flasks (Fig. 3B), microtiter plates, or 5-ml culture in tubes (data not shown). This difference was not due to a difference in final viable cell counts, proportion of dead cells (by propidium iodide staining and fluorescence-activated cell sorter analysis), a difference in the size of the bacteria, or a difference in absorbance of the culture supernatant (data not shown). In addition, it was not an effect of the transposon per se: an unrelated mutant in the laboratory collection, X. nematophila cobB::Tn5 (KANr) (GeneJumper transposon) does not display a reduced final optical density (data not shown). Attempts at complementation for this phenotype were hindered by the fact that all tested constructs of the oppA1 gene in a single copy or the entire operon in a low copy number inhibited growth of both the wild-type and mutant strains (data not shown). However, the same phenotype was seen in both the HGB007 (Fig. 3B) and HGB731 (HGB081 leuB) backgrounds (data not shown), suggesting that the phenotype occurs regardless of strain background. Furthermore, the growth phenotype was observed in X. nematophila oppB::Tn5-13-7, a CHLr oppB mutant strain (data not shown). Therefore, the aberrant optical density phenotype appears to be specific to the opp disruption. We analyzed the level of oppA1 transcript relative to the level of oppA2 transcript and found that it was low during log-phase growth and increased over twofold during stationary phase (Fig. 3B). The 3-h time point was in lag phase and thus likely reflects transcript levels from the stationary-phase starter culture. This same trend was observed when oppA1 transcript levels were compared to oppF transcript levels (data not shown). In contrast, oppF transcript levels remained at the same level proportional to oppA2 transcript levels over the entire growth curve (data not shown). Consistent with the idea that normal transcription from a promoter upstream of oppA1 contributes to obtaining normal late-stage optical density, relative oppA1 transcript levels increased at a point in the growth curve when the opp mutant growth profile began to taper off (Fig. 3B).
In this work, we describe an oligopeptide permease operon in the nematode mutualist and insect pathogen X. nematophila and directly test its role in X. nematophila growth and host interactions. The operon comprises six genes whose products are homologous to oligopeptide permease proteins of gram-negative and -positive bacteria and includes two OppA homologues (Fig. 1). It had previously been suggested that B. burgdorferi utilizes multiple OppA homologues to bind different peptides in its various host niches (20). To genetically address the contribution of oligopeptide uptake to the interactions between a microbe and its invertebrate host, we constructed X. nematophila oppA1, oppA2, and oppB mutant strains and tested their ability to grow in laboratory culture and to associate with the nematode and insect hosts.
Small-peptide growth assays demonstrated that the X. nematophila opp locus encodes a functional oligopeptide permease (Fig. 2). The oppA2 and oppB mutant strains also had a reduced initial growth rate in hemolymph, whereas the oppA1 mutant had normal growth in this medium (Fig. 3A). Taken together, these data suggest that hemolymph contains an oligopeptide that is utilized early in the X. nematophila growth cycle and that OppA2 and the permease are sufficient and essential for normal utilization of this nutrient. However, an inability to utilize this nutrient does not prevent the population from achieving maximum optical density in hemolymph.
Analysis of oppA1, oppA2, and oppF transcript levels in the wild-type strain indicated that oppA1 transcript levels increased relative to those of oppA2 (Fig. 3B) and oppF (data not shown) as the cultures entered stationary phase of growth in LB broth. These results are consistent with the finding that B. burgdorferi oppA homologues are differentially expressed in different environments (e.g., laboratory growth, ticks, or mice) (32). Although differential expression of oppA homologues can occur in X. nematophila, this regulation is not required for any tested host interaction or successful transition between hosts. Intriguingly, the X. nematophila opp mutant strains consistently achieved a lower final optical density in LB broth than the wild-type strain (Fig. 3B). Thus, the altered final optical density of the X. nematophila opp mutant strains in stationary phase in LB broth could indicate that this increased transcription of the opp operon from the promoter upstream of oppA1 is required to reach maximum optical density in LB broth. The cause of the difference in optical density is unclear, and to our knowledge this phenotype has not been previously observed during growth analyses of opp mutant strains of other bacterial species. Technical difficulties in complementation analyses prevented examination of the causality of the opp gene mutations for this growth phenotype. However, we believe it is associated with mutation of the opp locus because the phenotype was always correlated with the mutation, regardless of strain background or type of insertion, and was concurrent with a change in operon transcription that would be precluded in the mutant strains.
The X. nematophila opp mutant strains colonized the nematode to an extent similar to that of the wild-type bacterium soon after the colonization event (Table 2) and after long-term storage. They were also competitive with the wild-type strain for nematode colonization and killed experimental groups of M. sexta insects to the same extent and within the same time frame as the wild-type strain (Table 2). While these data do not exclude the possibility that X. nematophila uses oligopeptides as a nutrient source in the nematode or insect hosts (indeed, the hemolymph growth profiles suggest that they may), they do demonstrate that oligopeptide transport by the identified permease is not an essential function for success in these host environments. It should be noted, however, that colonization of individual nematodes by X. nematophila is a clonal or occasionally near-clonal event (22). Therefore, in the competition assays presented here, the X. nematophila opp mutant strains did not likely have to compete directly with the wild-type strain following the initial colonization event, except in the ∼30% biclonally colonized nematodes. The oppB mutation provided an apparent competitive advantage in nematode colonization in plate assays but did not provide a competitive advantage in the insect infection assays. Because the insect infection assays simulate a more natural environment for the bacteria, we are discounting the observed competitive advantage on plates as an experimental artifact.
Little is known of the nutrients available to the bacterium in either the nematode or the insect host; taken together, the data presented here indicate that transport of oligopeptides by the identified permease is not essential for nutrient acquisition by X. nematophila in host environments. The selective pressure for maintenance of the opp operon, including a second oppA gene, thus remains to be determined.
We gratefully acknowledge the assistance of David Majewski for help with early opp operon sequencing and cloning, Walter Goodman for supplying M. sexta eggs, and Eric Martens for developing a defined medium for X. nematophila and for isolating the leucine auxotroph used in the peptide-feeding experiments.
This work was supported by National Institutes of Health RO1 grant GM59776, by the Investigators in Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Foundation, and by USDA/CREES grant CRHF-0-6055 (awarded to H.G.-B. and used to support S.S.O. in part). S.S.O. received additional support through National Institutes of Health predoctoral training grant T32 GM07215 in Molecular Biosciences and through National Science Foundation Graduate Teaching Fellows in K-12 Education award DUE-9979628 to the K-Through-Infinity program.
Received 20 February 2004.
Accepted 18 May 2004.
↵*Corresponding author. Mailing address: Department of Bacteriology, University of Wisconsin—Madison, Madison, WI 53706. Phone: (608) 265-4537. Fax: (608) 262-9865. E-mail: hgblair{at}bact.wisc.edu.
Alexeyev, M. F., and I. N. Shokolenko. 1995. Mini-Tn10 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of Gram-negative bacteria. Gene 160:59-62.
Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.
Atlas, R. M. 1997. Handbook of microbiological media, 2nd ed. CRC Press, Boca Raton, Fla.
Ausubel, F. A., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1998. Current protocols in molecular biology. Wiley and Sons, New York, N.Y.
Bono, J. L., K. Tilly, B. Stevenson, D. Hogan, and P. Rosa. 1998. Oligopeptide permease in Borrelia burgdorferi: putative peptide-binding components encoded by both chromosomal and plasmid loci. Microbiology 144:1033-1044.
Borezee, E., E. Pellegrini, and P. Berche. 2000. OppA of Listeria monocytogenes, an oligopeptide-binding protein required for bacterial growth at low temperature and involved in intracellular survival. Infect. Immun. 68:7069-7077.
Caetano-Annolles, G. 1993. Amplifying DNA with arbitrary oligonucleotide primers. PCR Methods Appl. 3:85-92.
Caldas, C., A. Cherqui, A. Pereira, and N. Simões. 2002. Purification and characterization of an extracellular protease from Xenorhabdus nematophila involved in insect immunosuppression. Appl. Environ. Microbiol. 68:1297-1304.
Cundell, D., B. Pearce, J. Sandros, A. Naughton, and H. Masure. 1995. Peptide permeases from Streptococcus pneumoniae affect adherence to eukaryotic cells. Infect. Immun. 63:2493-2498.
Fenno, J. C., M. Tamura, P. M. Hannam, G. W. K. Wong, R. A. Chan, and B. C. McBride. 2000. Identification of a Treponema denticola OppA homologue that binds host proteins present in the subgingival environment. Infect. Immun. 68:1884-1892.
Forst, S., and D. Clarke. 2002. Bacteria-nematode symbioses, p. 57-77. In R. Gaugler (ed.), Entomopathogenic nematology. CABI Publishing, Wallingford, United Kingdom.
Forst, S., B. Dowds, N. Boemare, and E. Stackebrandt. 1997. Xenorhabdus and Photorhabdus spp.: bugs that kill bugs. Annu. Rev. Microbiol. 51:47-72.
Forst, S. A., and N. Tabatabai. 1997. Role of the histidine kinase, EnvZ, in the production of outer membrane proteins in the symbiotic-pathogenic bacterium Xenorhabdus nematophilus. Appl. Environ. Microbiol. 63:962-968.
Goodell, E. W., and C. F. Higgins. 1987. Uptake of cell wall peptides by Salmonella typhimurium and Escherichia coli. J. Bacteriol. 199:3861-3865.
Gutnick, D., J. M. Calvo, T. Klopotowski, and B. N. Ames. 1969. Compounds which serve as the sole source of carbon or nitrogen for Salmonella typhimurium LT-2. J. Bacteriol. 100:215-219.
Hensel, M., J. E. Shea, C. Gleeson, M. D. Jones, E. Dalton, and D. W. Holden. 1995. Simultaneous identification of bacterial virulence genes by negative selection. Science 269:400-403.
Jenkinson, H., R. Baker, and G. Tannock. 1996. A binding-lipoprotein-dependent oligopeptide transport system in Streptococcus gordonii essential for uptake of hexa- and heptapeptides. J. Bacteriol. 178:68-77.
Jenkinson, H. F. 1992. Adherence, coaggregation, and hydrophobicity of Streptococcus gordonii associated with expression of cell surface lipoproteins. Infect. Immun. 60:1225-1228.
Jenkinson, H. F., and R. A. Easingwood. 1990. Insertional inactivation of the gene encoding a 76-kilodalton cell surface polypeptide in Streptococcus gordonii Challis has a pleiotrophic effect on cell surface composition and properties. Infect. Immun. 58:3689-3697.
Kornacki, J. A., and D. B. Oliver. 1998. Lyme disease-causing Borrelia species encode multiple lipoproteins homologous to peptide-binding proteins of ABC-type transporters. Infect. Immun. 66:4115-4122.
Lewis, E. E., S. Selvan, J. F. Campbell, and R. Gaugler. 1995. Changes in foraging behaviour during the infective stage of entomopathogenic nematodes. Parasitology 110:585-590.
Martens, E. C., K. Heungens, and H. Goodrich-Blair. 2003. Early colonization events in the mutualistic association between Steinernema carpocapsae nematodes and Xenorhabdus nematophila bacteria. J. Bacteriol. 185:3147-3154.
McNab, R., and H. Jenkinson. 1998. Altered adherence properties of a Streptococcus gordonii hppA (oligopeptide permease) mutant result from transcriptional effects on cshA adhesin gene expression. Microbiology 144:127-136.
Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Mitani, D. K., H. K. Kaya, and H. Goodrich-Blair. 2004. Comparative study of the entomopathogenic nematode, Steinernema carpocapsae, reared on mutant and wild-type Xenorhabdus nematophila. Biol. Control 29:382-391.
Park, J. T., D. Raychaudhuri, H. Li, S. Normark, and D. Mengin-Lecreulx. 1998. MppA, a periplasmic binding protein essential for import of the bacterial cell wall peptide l-alanyl-γ-d-glutamyl-meso-diaminopimelate. J. Bacteriol. 180:1215-1223.
Payne, J. W., and C. Gilvarg. 1968. Size restriction on peptide utilization in Escherichia coli. J. Biol. Chem. 243:6291-6299.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Simon, R., U. Priefer, and A. Pühler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Biotechnology 1:784-791.
Vivas, E. I., and H. Goodrich-Blair. 2001. Xenorhabdus nematophilus as a model for host-bacterium interactions: rpoS is necessary for mutualism with nematodes. J. Bacteriol. 183:4687-4693.
Volgyi, A., A. Fodor, A. Szentirmai, and S. Forst. 1998. Phase variation in Xenorhabdus nematophilus. Appl. Environ. Microbiol. 64:1188-1193.
Wang, X.-G., B. Lin, J. M. Kidder, S. Telford, and L. T. Hu. 2002. Effects of environmental changes on expression of the oligopeptide permease (opp) genes of Borrelia burgdorferi. J. Bacteriol. 184:6198-6206.
Woodring, J. L., and H. K. Kaya. 1988. Steinernematid and Heterorhabditid nematodes: a handbook of biology and techniques. Southern cooperative series bulletin 331. Arkansas Agricultural Experiment Station, Fayetteville.
Xu, J., and R. E. Hurlbert. 1990. Toxicity of irradiated media for Xenorhabdus spp. Appl. Environ. Microbiol. 56:815-818.
American Society for Microbiology
Applied and Environmental Microbiology Sep 2004, 70 (9) 5621-5627; DOI: 10.1128/AEM.70.9.5621-5627.2004
You are going to email the following Identification and Functional Characterization of a Xenorhabdus nematophila Oligopeptide Permease


Shoe with asymmetric branches (a wide branch and a narrow beveled branch) as well as a wide toe and heels beveled at their ends. 3 clips for use with 1 toe clip or 2 quarter clips. Thickness 10 mm. Coverage and weight in size 1: 30 mm for the wide branch. 32 mm on the toe. Narrow and beveled branch: 25 mm foot side and 15 mm ground side. 206 g.
CONCEPT AND REACTION ON THE GROUND : On penetrable and compacted ground: the wide branch provides support and limits sinking. The narrow beveled branch promotes sinking. The wide toe and the beveled heels promote heels sinking.
BIOMECHANICAL AND KINESITHERAPIC EFFECTS :Depending on the application of the shoe (medial or lateral wide branch): reduces tensions on one branch of the fetlock joint suspensory ligament or on one oblique sesamoidean ligament.
INDICATIONS :Desmopathy of the medial branch of the suspensory ligament or of the medial oblique sesamoidean ligament (medial wide branch). Desmopathy of the lateral branch of the suspensory ligament or of the lateral oblique sesamoidean ligament (lateral wide branch).


We're big fans of webinars here at ClickDimensions. We host a weekly webinar that showcases our marketing automation solution while also discussing marketing tips and best practices, our training team offers a number of webinar training sessions each week as part of ClickDimensions Academy, and we regularly team up with our partners to offer informative webinars about a variety of marketing hot topics – just to name a few of our webinar endeavors.
Select a compelling topic. What questions do your customers or prospects often ask? How can your potential webinar attendees do something better or more efficiently? What's new in your business or industry? Answering those questions can often help you brainstorm a compelling topic for your next webinar. Once you have a topic selected, make sure to tailor it to your target audience. If your company sells to small business owners, for example, think about whether you would like to target new or seasoned small business owners, or a combination of the two. This can help you further refine your topic and make it more appealing to your selected audience.
Choose the right day and time. Over the years, there have been numerous studies and surveys on the best day to hold a webinar. While they vary somewhat in their specific findings, they do all tend to agree that Mondays and Fridays are the worst days for webinars. Many of those studies also agree that webinars on Wednesdays and Thursdays have the highest attendance rates. While these days of the week may also work best for your webinar, be sure to consider your audience and industry and whether a different day might be a better choice. Picking a time of day may be trickier than deciding a day of the week, depending on where your audience is located. For a local audience, give or take a time zone or two, 11 am is often an ideal time for many attendees. If you have more of a global audience, consider offering your webinar multiple times and different times of the day to accommodate people across a broader range of time zones.
Spread the word. How successful can a webinar really be if no one attends? That's why it's important to promote your webinar in as many ways as makes sense for your audience. Think about their relationship to your company and their preferred methods of communication, and then plan your promotion strategy from there. If you already have your desired attendees' information in CRM, email marketing is an ideal way to garner webinar registrations. You could also include webinar invitations as part of a thought leadership, lead nurturing or other campaign automation. If you want to use a webinar to generate leads, social media posts or online advertising would be perfect for promoting your event. To maximize the effectiveness of your webinar promotions, combine these communication methods and be creative – you could, for example, create a short video as a trailer of sorts for the webinar.
Utilize technology. Event technology can help increase event attendance by 20 percent and decrease costs by 30 percent, according to Frost & Sullivan. Technology can also help you better track attendance and registrations. ClickDimensions' integrations with WebEx and GoToWebinar bring registration and attendee information directly into Microsoft Dynamics 365. This allows you to easily track, all in one place, which leads or contacts are registering for or attending your events for overall greater event insights and success.


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EastLongmeadow
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Mike Dobbs Column
Fazio's to deliver 'Slice of Hope' Oct. 11
EAST LONGMEADOW – A local pizzeria is offering breast cancer patients across the country a Slice of Hope this month. Fazio's Ristorante & Pizzeria is helping raise critical funding for breast cancer research by donating 5 percent of its sales from Oct. 11 to the Karen Mullen Breast Cancer Foundation, a national charity based in Seattle, Wash.
Fazio's is located at 162 Shaker Road.
Oct. 11 has been designated as a National Pizza Party Day by Slice of Hope, a pizza industry charitable initiative. Slice of Hope has banded together pizzerias from across the nation in an effort to fight this invasive disease, which will impact hundreds of thousands of women and men this year.
In fact, more than 190,000 new cases of breast cancer will be diagnosed this year. Sadly, approximately 40,000 women will die from the disease this year. That's approximately 110 breast cancer deaths daily, and Fazio's Ristorante & Pizzeria owners Aldo Fazio and Pietro Mazza said that's simply too many.
"There isn't a person in this country who hasn't felt the effects of breast cancer in some way," Fazio said. "Whether you've had the disease yourself, or a family member or friend has had it, it's safe to say you've been impacted by breast cancer."
He continued, "Fazio's Ristorante wants to do something to help end this disease. That's why we've joined Slice of Hope and will be donating 5 percent of of our sales on Oct. 11. So, please come down to Fazio's Ristorante that night to show your support of this cause."
Slice of Hope is spearheaded by Pizza Today magazine editor Jeremy White. White, Pizza Today publisher Pete Lachapelle and a small team of cyclists will bike for three days throughout New England, ending Oct. 12 to raise awareness for Slice of Hope's fight against breast cancer. The cycling route will include stops in Maine, Vermont, New Hampshire, Massachusetts and Rhode Island.
On Oct. 11, pizzerias across the nation – including Fazio's Ristorante – will donate funds to the Karen Mullen Breast Cancer Foundation, which has committed to giving 100 percent of the Slice of Hope proceeds to a handful of the nation's leading breast cancer research labs.
For more information, visit www.faziosvilla.com.

redpajama_set_name: RedPajamaCommonCrawl


[Editorial] Adopt Section 508 definition of "assistive technology"?
of "assistive technology" used in US Federal regulations.
with host user agents by using and monitoring APIs.
make tables more accessible), etc.
improve the visual readability of rendered text and images.
synthesized speech or braille displays.
idea of assistive technology as used in this document?
Note: I will be satisfied if we make no changes to the document.
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9151 Howells Ferry Road Semmes, AL. | MLS# 624425 | Assist-2-Sell Buyers and Sellers Realty - Homes for sale in Mobile County and Baldwin County. We work with buyers and sellers.
Well maintained (3) bedroom, (2.5) bath brick home on (1.67)acres! Conveniently located in Semmes, (USDA) eligible area, and move-in ready with over 1800 sqft. This open floor plan showcases a large family room with a wood-burning fireplace, separate dining room, and a spacious kitchen with a breakfast bar. Throughout the home, the FLOORING, BATHROOMS, and KITCHEN COUNTER TOPS have all been updated. The fenced-in backyard has (2) storage sheds and (1) car attached carport. The carport is right off the laundry room and kitchen, making unloading the groceries a breeze. THE ROOF WAS REPLACED IN (2017). All measurements are approximate and not guaranteed buyer and/or buyers agent to verify. Contact your favorite realtor today! Seller will entertain a lease/purchase option with $5000 down, $1150 a month rent for up to 12 months.
Directions: From Schillingers Rd, Left On Howells Ferry, Home On The Left.


Attain at least 150 points on the IAAI-CFI® application through documented achievements in education, training, and experience. Every point claimed on the application must be documented by certificate, diploma, reference letter, transcript, or other acceptable method.
Earn a 70% passing grade on a comprehensive, "closed book," proctored examination that is based on every job performance requirement in NFPA 1033: Standard for Professional Qualifications for Fire Investigator. The application must be approved prior to sitting for the examination.
Investigators who hold the IAAI-CFI® certification are required to re-certify every five years by completing a Recertification Application. A minimum of fifty (50) points must be obtained since the last certification date. Twenty (20) points must come from work experience and twenty (20) points from training (includes two CFITrainer.net® modules). The renewal applicants are not required to retake the exam if they send in their applications before the expiration date.
The IAAI-CFI® certification is accredited by the National Board of Fire Service Professional Qualifications (Pro Board) and the Forensic Specialties Accreditation Board (FSAB). You do not have to belong to IAAI to become a Certified Fire Investigator through the IAAI-CFI® program.
Acknowledgement by an independent body that the investigator has demonstrated competence in all phases of fire investigation.
Recognition by an independent body that the investigator possesses substantial field experience, as opposed to a primarily academic or theoretical background.
Demonstration, through the recertification process, that the investigator is keeping current with their training and professional knowledge development.
Measurement of these achievements using a standardized mechanism, as set by the IAAI-CFI® Committee.
How do I earn the IAAI-CFI® certification?
Download the IAAI-CFI® Application. Complete the application and return it to IAAI as directed, with the application fee. The application fee for IAAI Members is US$195. For non-Members, the fee is US$570. Please note that individuals who are members of a local chapter only do not qualify for the IAAI Member rate. Application fee is non-refundable.
To obtain the Member discount rate, a non-Member may choose to join IAAI prior to filing the IAAI-CFI® application. Once you have submitted the application, it will be reviewed by the IAAI-CFI® Committee. If the application is approved, you will then be provided with instructions on how to take the proctored examination. If the application is not approved, you will be notified about the areas of deficiency and given an opportunity to make corrections. For detailed information on the full process of the IAAI-CFI® program, please read the Practices & Procedures Manual for the International Association of Arson Investigators Certified Fire Investigator Program.
If an investigator isn't an IAAI-CFI®, does that mean the IAAI thinks that investigator is not qualified or competent?
No. The IAAI-CFI® program is voluntary and earning the credential is at the initiative of the investigator. Certification as a Fire Investigator by the IAAI does not determine who shall engage in the practice of the investigation of fires and explosions, nor does it substitute for any licensing or registration required by law. If an individual is not CFI certified, it does not indicate that he or she is unqualified to perform fire investigations.
I already hold the IAAI-CFI® certification and need to re-certify. What do I do?
Download and complete the IAAI-CFI® Recertification Application. Return the application as instructed with the associated fee (US$140 for IAAI members, US$520 for non-members). If you are not an IAAI member, you may obtain the member rate for recertification by joining IAAI prior to returning the Recertification Application. Recertification Applications should be submitted at least 30 days prior to your IAAI-CFI® certification expiration date. Application fee is non-refundable.
Check your Pro Board Certification Status. The IAAI will submit your information directly to the Pro Board after you have successfully passed the examination or recertified your IAAI-CFI® .
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text:
filed: February 26, 2012 • Ireland, U.K.
State to export 'green' electricity to Britain in trade deal, says Minister
Credit: BARRY O'HALLORAN, The Irish Times, www.irishtimes.com 25 February 2012 ~~
Talks beween the Republic and Britain on a deal allowing large-scale exports of electricity from the State to its nearest neighbour are set to step up over coming months, Minister for Energy Pat Rabbitte indicated yesterday.
Britain signalled last year that it would be willing to import excess "green" electricity from the Republic, a development that backers of a series of proposed offshore wind farms hope will underpin their businesses.
This will require a bilateral renewable energy trade agreement between the Republic's Government and Westminster.
Mr Rabbitte and his opposite number in Britain, Charles Hendry, have held a number of talks about the possibility of completing such a deal in recent months.
Speaking at the Irish Institute of European Affairs yesterday, the Minister said "that work is set to intensify over the coming months, with the objective of achieving an intergovernmental agreement under the EU framework to underpin the export of renewable energy."
Responding to questions from the audience after his speech, the Minister pointed out that the next engagement with the British administration would have to be focused on the eventual shape of the intergovernmental agreement.
The deal will have to be drawn up under the EU renewable energy directive. Mr Rabbitte said that the "overarching" strategic objective would be to make renewable energy a significant part of the Republic's export industry and to boost its balance of payments.
Commenting on this week's announcement that the Government intends to sell a number of ESB power plants as part of an overall disposal of State companies, Mr Rabbitte made it clear that forthcoming developments in the EU energy market had prompted the decision to retain ownership of the company as an integrated business. He explained that in 2014, Ireland's energy market will become part of a regional structure made up of this country, Britain and France.
Source: BARRY O'HALLORAN, The Irish Times, www.irishtimes.com 25 February 2012


....to be up at 5AM this morning after a few hours of sleep in order to re-book our vacation thanks to Hurricane Ike. Now, I am not going to complain or moan poor me in this post at all. There are people dead, dying, homeless in the Caribbean...and more unfortunately probably still to come as Hurricane Ike heads for the Texas and Mexico coastline.
We booked 8 nights at Beaches Turks and Caicos nearly a year ago. They always offer these awesome deals at the end of a calendar year for bookings done in the next year. And it only takes a few hundred dollar deposit to secure your reservation. This is our 2nd trip to a Beaches. The first was to Ochos Rios, Jamaica named Boscobel. This is our third with this entity as we honeymooned at Sandals Negril. And we have done a 4th via Couples Swept Away with 3 other couples as a 5yr anniversary/reunion. So yes, we love it and we keep coming back for more.
All-inclusives are not cheap...and they certainly are not for everyone. After getting married just after graduating college we wanted a honeymoon, but didn't want to be skipping breakfast to ensure we could afford dinner. As we started our family we again wanted this thought-less process...except with the munchkins in mind. I hate to see food wasted as much as the next person, but let's be frank...how many toddlers and preschoolers have eyes bigger than their stomachs? Or there are days our son loves a banana and a piece of toast for breakfast and what restaurant menu let's you do that? And in my selfish mind...if I am going to one of the best dive destinations in the world I want to be able to go....A LOT. And where else will my daughter have dance lessons with Sesame Street's Zoe and bake cookies with Cookie Monster.
So by the time you start adding all these things up...and then throw in childcare from 8AM-8PM for as long, short, or as many drop-ins as you want that $500-$700 a day doesn't seem to bad. Diving alone for my husband and I would easily cover a couple hundred of that each day. Then toss in premium drinks, snacks, numerous restaurants, activities...and NO TIPPING. Well, it is the reason we budget in a all inclusive vacation every 2-3 years. All our money is paid upfront and the only bit we bring along is for souvenirs and any incidentals getting to our destination. This is definitely something to consider if you are looking for an active vacation with diving, snorkeling, other water sports, good on-land amentities like gyms, classes, etc....as well as care for little ones. If you and your family just want to veg on the sand it may be more economical to do that elsewhere.
So back to the original....Turks and Caicos was hit by Ike as a Cat4 last week. We had heard that "Provo" was the least damaged, so we waited and waited and read all the forums and thought we were in the clear for our vaca scheduled in November. But last night we received an email they would be closed and we needed to rebook. And We LUV All-inclusives so much we had to rebook ourselves, 3 of the 4 grandparents, and some family friends who we have all sold on the idea and were joining us.
Here's some tips on re-booking. Be prepared and know your options. Pull your current flight, confirmation numbers, costs, etc...and have some flights for potential rebooking pulled. Ask the airlines if there is a policy in place to wave change fees due to the catastrophe. Our airline when all is said and done will have waived...$1800 in change fees and another $300 in crediting back frequent flyer miles. Some of us had travel insurance...some didn't. So some will recoup their ticket differentials they had to pay. Some of us will eat it. If you are traveling with a group and someone is willing to take responsibility for everyone that is best...this way all travel associates you are dealing with realize they are dealing with numerous bookings and hence lots of customers and revenue. And be first in line...we got the email after Beaches office hours and called and were up and on the phone when they opened at 5AM west coast time.
And my motto in any dealing as this is try to kill them with kindness. Lots of thank yous go a long way. In a situation such as these catastrophe they are dealing with many upset and angry people and if you are the one who is kind and makes that last angry call they got disappear you will be amazed at what it could get you.
We LUV All-Inclusives so much we have our entire entourage rebooked for 1 month later than originally planned and we are going to enjoy and support the economy of Turks and Caicos.


text:
Artsat2-Despatch (, auch Fuji-OSCAR 81, FO-81) ist ein japanischer Amateurfunksatellit und ein Kunstobjekt der Kunsthochschule Tama.

Aufbau
Despatch wurde in Zusammenarbeit mit der Universität Tokio geschaffen. Die Kunststudenten betrachten einen Satelliten als "Medium, welches den Alltag mit dem Universum verbindet". Despatch ist eine schraubenförmige Skulptur, die in die Abmessungen 50 cm × 50 cm × 45 cm passt. Diese Skulptur wurde mit einem 3D-Drucker hergestellt. Es ist nach Invader, auch Artsat-1 genannt, das zweite Kunstobjekt dieser Hochschule im Weltraum.

Die AMSAT hat dem Satelliten mit dem Amateurfunkrufzeichen JQ1ZNN die OSCAR-Nummer Fuji-OSCAR 81, kurz FO-81 verliehen. Der Name "Fuji" wurde gewählt, um auf die lange Geschichte japanischen Mitwirkung bei Amateurfunksatelliten (JAS 1, JAS 1B, JAS 2 usw.) hinzuweisen.

Der Satellit enthält einen On-Board-Computer, einen Sender und eine Batterie, sowie eine Monopolantenne. Der Sender sendet ein Telegrafiesignal auf der Frequenz 437,325 MHz mit einer Sendeleistung von 7 Watt. Da die Batterie des Satelliten nicht wieder aufladbar ist, wurde die Lebensdauer des Satelliten auf sieben Tage geschätzt. DESPATCH verfügt nicht über eine Lageregelung. Sämtliche Baugruppen, inklusive der Antenne sind von der Skulptur umhüllt.

Mission
Despatch wurde am 3. Dezember 2014 als Sekundärnutzlast gemeinsam mit Hayabusa 2 mit einer H-IIA-Rakete gestartet.

Der Satellit erreichte eine elliptische Umlaufbahn um die Sonne und bewegt sich in einem Orbit zwischen Venus und Mars. Die Bahnneigung ist nahe null, d. h. der Satellit wird in der Bahnebene der Erde bleiben. Die Entfernung zur Sonne wird zwischen 0,7 und 1,3 AE liegen.

Am 7. Dezember 2014 wurde das Signal aus einer Entfernung von mehr als 2 Millionen km und am 15. Dezember aus einer Entfernung von 4,7 Millionen km empfangen.
Am 3. Januar 2015 hat die Bodenstation der Kunsthochschule Tama die Mission für beendet erklärt und die Empfangsversuche eingestellt.

Weblinks
 Offizielle Website
 Telemetrie und Empfangsberichte zu Despatch auf DK3WN SatBlog

Einzelnachweise

Amateurfunksatellit
Weltraumkunstwerk
Japanische Raumfahrt
Raumfahrtmission 2014

redpajama_set_name: RedPajamaWikipedia


Brian was born in Tucson, Arizona. He received his B.A. Degree in Visual Arts from the University of California at San Diego, an M.A. from Stanford University, and studied the history of photography with Van Deren Coke and Beaumont Newhall during his M.F.A. at the University of New Mexico.
Brian has the highest respect for CPA, having participated in solo and group shows here, taught and organized workshops, guest curated exhibitions and served as the President of the Board of Trustees in the past. His own artwork explores historic photographic processes, has been exhibited internationally and is included in the permanent collections of the Bibliotheque Nationale, Paris; the Victoria and Albert Museum, London; and the International Museum of Photography at the George Eastman House, Rochester, NY.
Brian has recently retired after more than thirty years as a Professor of Photography and most recently as the Chair of the Department of Art and Art History at San Jose State University.
Chris has been our Office Manager for the past seven years. She retired from New York State government as a Payroll/Personnel Systems Analyst after 30 years of service. Besides her job at CPA, she is the Photoshop editor and photography assistant for the family owned business, Wasserbach Photography.


Description: Memorial to the memory of 17 lifeboat men 'who lost their lives in the disaster of 1928'. A design sent by James Wedgwood was accepted 'subject to satisfactory modifications being made'.
C. L. Hartwell was nominated to represent the RBS as one of three assessors alongside the Ex-Mayor of Rye and a representative of the Public Trustee. The Council of the Royal Society of British Sculptors advised that the initial sum of £350 that was allocated for the commission, would be insufficient for 'anything but a very simple work' ('Annual Report, 1930', 1931, Royal Society of British Sculptors).
Assisted in the organisation of the competition.
Served as one of three assessors.


Since 2003, the Hannover Biomedical Research School (HBRS) has been organizing a joint curriculum for all programs (Curriculum 2018/19).
As a general rule, students have to attend 300 hours of teaching during the 3-year period. This includes three hours of obligatory seminars per week, project-related courses like, e.g., method courses, congresses, scientific colloquia (80 hours) and interdisciplinary seminars like, e.g., soft skill courses (60 hours). Students can choose from a great variety of courses, workshops, summer schools, Master classes (2-3 days discussion with 3-4 inernational outstanding scientists), etc. Please also see the individual program guidelines and the section "news and announcements".
Most important is, of course, the research project in one of the departments of MHH or partner institutes. All projects are quality-checked by the program committees. Each student has a thesis advisory committee (one main supervisor and two co-supervisors from different departments), regular meetings take place. Presentation and discussion of the project on an annual retreat should guarantee the way to success. We offer 3-year PhD programs, however, most important is the outcome (1-2 first author papers in high-ranking journals as part of a cumulative thesis are desired). So, the average length of PhD at the moment is 3.5 years.
We have an intermediate exam after 18 months. Here, you select and discuss a state-of-the-art paper in context of your own research work. The final exam is conducted in form of a public defense.
Short-time stays abroad in a lab of a cooperation partner are possible.
At the beginning, orientation weeks help new students to adapt to the new surroundings. Usually, in the first couple of weeks senior students offer to be a mentor (including "meet and greet" service at the airport).
And last but not least, a number of social and cultural events are organized by HBRS.


She's already flashed her derriere in a sheer dress during Paris Fashion Week.
But Katy Perry went a step further today, showing a bit more front.
The 27-year-old singer's stunning black dress featured a lacy panel and nothing underneath which saw her flashing plenty of cleavage.
The star is still sporting her electric blue hair, but showed plenty regrowth with her dark roots coming through.


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I stumbled upon these vocation videos of the Polish Dominicans and Franciscans. If you don't understand Polish, don't fret, neither do I. And since there's no talking, just music, just sit back and enjoy the brief videos. THE fun thing is just watching the Dominican Franciscan friars. You get a great sense of the spirit of the friars of both groups just by watching the life.
It's like watching a foreign film–you don't understand the language but get the point–immediately. Video 1, video 2 and video 3.
This year is Father Benedict Groeschel's golden jubilee as a priest. That's right! He's 50 years a Catholic priest. Many would know him as a TV personality on EWTN; others know him as the instigator of the Friars of the Renewal, to many, he's a friend and a great priest. Friends of his put together a beautiful, brief video of Father Benedict. Watch it, the link's below.
Pray for priests. Pray for Father Benedict. Pray for the Friars of the Renewal.
A video honoring him can be seen here.
Look at these beautiful young women following Christ as Poor Clare nuns of Lerma (Burgos), Spain! I can't believe my eyes!!! They're happy. They're alive. They're infectious.
You've gotta read the CNA story (in English) here but the video in the story is in Italian with English subtitles. Also, watch another video about these same Poor Clares. Sorry, these videos are subtitled but watching them you get the point: the heart is attracted by love and joy.
I want to know: do we have anything like these nuns in the USA?
In the mid-1990s when I was in formation at Bellarmine House and a student in St Louis, Missouri, I made the acquaintance of Conventual Franciscan Father Wayne Hellman. Father Wayne was a professor of Theology at Saint Louis University, St Louis, MO. I think he was also the Friar Guardian of the local Conventual Franciscan House (St Bonaventure's Friary) and one of the nation's experts in Saint Bonaventure's theology.
Wayne was frequently perceived as a zaney Franciscan professor but an incredibly bright and sensitive man, one that you can easily approach. I enjoyed his company. Until reading about his encounter with the young Joseph Ratzinger, didn't I realize the interest and scope of theological formation and how he started off. The pedigree of theologians is always of interest to me because I am interested in history and trajectory.
My friend David Miros sent me and a few others a striking story published in the Saint Louis University News of Father Wayne's recent encounter with the Holy Father. Why is this striking to me and why should you read the story? Because it is a realization how the Holy Spirit works at the lowest and yet the most human of levels: the heart.
Had the friars no pride?
Music at deathbeds! Ah, the shame! the scandal!


text:
6 January 2019 at 8:04am
Amber Rudd rethinks roll-out of controversial Universal Credit welfare reform
Video report by ITV News Political Correspondent Libby Wiener
The next phase of the Universal Credit programme (UC) will be overhauled following widespread criticism of the extension of the flagship welfare reform.
Work and Pensions Secretary Amber Rudd is to scrap an imminent Commons vote on plans allowing all existing claimants of relevant benefits to be moved onto the new all-in-one payment.
Instead she plans to seek approval to move just 10,000 claimants onto UC to monitor the way the system works.
Influential committees in both Houses of Parliament had raised concerns about the plan to move around three million claimants onto UC and the Government faced losing a Commons vote on the "managed migration" programme.
Allies of the Work and Pensions Secretary insisted the decision to overhaul the migration was not due to fear of a Tory revolt but because it was the right way to handle the change.
The change comes after Ms Rudd took over from Esther McVey at the Department for Work and Pensions in November.
A source said Ms Rudd had taken a "no-holds-barred" approach and wanted a "fresh mandate" from Parliament for the roll-out of UC once it had been trialled.
Universal Credit: What is it and how does it work?
The roll-out of Universal Credit has been severely delayed. Credit: PA Graphics
The Observer reported that Ms Rudd is expected to announce the proposed pilot scheme within days.
Since taking the Work and Pensions brief she has signalled that she wants to make changes to UC and demand more money from the Treasury for the system.
The process of moving all claimants onto the new system is due to be completed by the end of 2023.
A Whitehall source said: "Amber has come into the Department and taken a no-holds-barred approach to reform.
"Therefore she wants Universal Credit to receive a fresh Parliamentary mandate and being personally sure the system is working in the interests of every claimant.
"So she will move 10,000 claimants, and carefully monitor this, before returning to Parliament to report on her findings and seek that fresh mandate for the full roll-out."
'I feel like I'm failing': Mum describes impact of Universal Credit
Frank Field says the delay in rolling out welfare reform shows the Government has 'woken up to the human catastrophe that was waiting to happen.' Credit: PA
Frank Field, Labour chairman of the Commons Work and Pensions Committee, told the Observer: "The Government seems finally to have woken up to the human catastrophe that was waiting to happen under its ill-formed plans for moving people onto Universal Credit."
He added: "The Secretary of State deserves credit for revisiting these plans. As a next step, and in keeping with this new approach, it is essential for the Government to proceed with 'managed migration' of people to Universal Credit only once it has proved to Parliament that it will not push more vulnerable people to the brink of destitution."
Universal Credit replaces six existing benefits – Employment Support Allowance, Income Support, income-based Jobseeker's Allowance, Housing Benefit, Working Tax Credit and Child Tax Credit – with a single payment.
Last updated Sun 6 Jan 2019
Universal Credit not covering the 'cost of renting' in Wales
Food bank use has increased due to universal credit roll-out, admits Amber Rudd


text:
RESTAURANTS NEAR KINO INTERNATIONAL BERLIN GERMANY
Visitors from Berlin are fond of the theater's history and special atmosphere. It makes designed for a wonderfully intimate evening and is a good place to meet nerdy queers the best kind.
All was drinking on the street - consumption everywhere. Just to the north is the Kurfurstendamm lined with shops. Especially important designed for the party and national leadership were the eight rows with optimal views and above legroom. Today, the International is used as a result of many filmmakers for premieres due to the theater's ambience and is one of the venues of the Berlinale. The woods after the parking lot are a popular men's cruising area. During the Cold War closing of land and water access to the city by the Soviets, US and British aircraft of the Berlin Airlift flew supplies into the airport to sustain over two million residents for almost a year - among the greatest feats in aviation account. Arsenal Potsdamer Strasse 2 has a application on independent and experimental films, with screenings, lectures, workshops, plus a gallery and archives.
Anticipate half-naked or mostly naked guys and gals, a swimming pool at your own attempt and excellent electro DJs. Condoms are generously available. In the basement, a small ditch and elevator were later added for the state leadership. Films range from Hitchcock classics to small modern indie productions, and are usually in English without subtitles. Before after that after premieres, state visitors sojourned in the Representation Room, today called the Honecker Drawing room.
Designed for a non-gay sauna experience in Berlin, assessment out Vabali. Other Nature This queer, lacto-vegetarian, eco-friendly, feminist sex shop is everything you probably do not expect from a femininity shop. By the Manometer community and in a row center, at Nollendorfplatz U-Bahn station, Brunos Bulowstrasse giant block-long store is one of a number of in Germany. Especially important for the accessory and national leadership were the eight rows with optimal views and extra legroom.
Verzaubertthe international queer film festival that moves amid Munich, Frankfurt, Cologne and Berlin is individual of the most important gay festivals all the rage the world. It is home to the Schwules Museum Gay Museumwhile Motzstrasse and Nollendorfplatz are still home to numerous gay bars and shops. Around and between these concluding stations, the Prenzlauer Berg area of East Berlin is full of interesting sights. Participants choose to be mares or stallions denial changing roles once the party begins. Individual time at the basement Brunnen70 nightclub after that now at Sophienclub. Upon entry, you remove your clothes while keeping shoes and socks on after that you are given a large plastic attache case to put all your clothes and baggage in and a wristband with a add up to that drinks are charged to, which you pay for when you leave. Anyone be able to share something, just let the hosts, Finn and Jane know beforehand.
Been to Kino International? Share your experiences!
The large film posters on the outside of the theater show the film of the week and are still painted by hand. Nollenbergplatz anchors the district at its eastern edge with many sidewalk cafes and restaurants along Maasenstrasse, south of the U-Bahnhof. Designed for a non-gay sauna experience in Berlin, assessment out Vabali. There's outdoor cruising at Friedrichshain's Volkspark around the Marchenbrunnen water-fountain area , both day and night, but be alert after dark. If you wish to address, you can tell an organiser at the beginning, or if there is space after that you are suddenly inspired, they are accommodating enough to let you jump up all the rage the middle and talk. The waved maximum also optimally reflects sounds to the chair area. It starts early 8pm and absolutely becomes sexual after a couple of hours. The terminal and the long tarmac runways remain open to visitors who walk, glide and bicycle the wide-open spaces. Condoms are freely available.
Josh21: 22.06.2019 : 16:44
Is an amusing piece.
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