Quantitative headspace analysis of selected odorants from latrines in Africa and India.

This analytical investigation focuses on the quantification of odorant molecules in the headspace of latrines. Hydrogen sulfide and methyl mercaptan were derivatized under a more stable N-ethyl maleimide conjugate. Since the amount of odorant molecules is very low in the gas phase, we developed a method that had two steps of concentration. The first step consisted of the accumulation of volatiles in buffered water by bubbling 350 L of air in a bottle. The second step consisted of loading the water on a 1 g solid-phase extraction cartridge, shipping it to our laboratories, and desorbing with Et2O, which achieved a total concentration factor of 3.5 × 10(6). The acidification of the water phase gave us access to the acids, and an additional bottle containing an acidic ion-exchange resin gave us access to trimethyl amine. The limits of quantification in the gas phase were 8.7 × 10(-4) µg/L air for hydrogen sulfide, 1 × 10(-4) µg/L air for methyl mercaptan, 1 × 10(-3) µg/L air for butyric acid, 1 × 10(-4) µg/L air for p-cresol, 1 × 10(-5) µg/L air for indole, and 1 × 10(-5) µg/L air for skatole. The system was calibrated by using olfactometers, which can deliver a precisely known quantity of volatiles into the air. We were able to quantify all compounds near their odor detection thresholds (ODTs). All ODTs were measured in our laboratory with the same olfactometry method. This allowed accurate and comparable ODT values for malodorant compounds from toilets.

Cinnamic acid, an autoinducer of its own biosynthesis, is processed via Hca enzymes in Photorhabdus luminescens.

Photorhabdus luminescens, an entomopathogenic bacterium and nematode symbiont, has homologues of the Hca and Mhp enzymes. In Escherichia coli, these enzymes catalyze the degradation of the aromatic compounds 3-phenylpropionate (3PP) and cinnamic acid (CA) and allow the use of 3PP as sole carbon source. P. luminescens is not able to use 3PP and CA as sole carbon sources but can degrade them. Hca dioxygenase is involved in this degradation pathway. P. luminescens synthesizes CA from phenylalanine via a phenylalanine ammonia-lyase (PAL) and degrades it via the not-yet-characterized biosynthetic pathway of 3,5-dihydroxy-4-isopropylstilbene (ST) antibiotic. CA induces its own synthesis by enhancing the expression of the stlA gene that codes for PAL. P. luminescens bacteria release endogenous CA into the medium at the end of exponential growth and then consume it. Hca dioxygenase is involved in the consumption of endogenous CA but is not required for ST production. This suggests that CA is consumed via at least two separate pathways in P. luminescens: the biosynthesis of ST and a pathway involving the Hca and Mhp enzymes.

Identification and characterization of the receptor for the Bacillus sphaericus binary toxin in the malaria vector mosquito, Anopheles gambiae.

The binary toxin (Bin) from Bacillus sphaericus exhibits a highly insecticidal activity against Culex and Anopheles mosquitoes. The cytotoxicity of Bin requires an interaction with a specific receptor present on the membrane of midgut epithelial cells in larvae. A direct correlation exists between binding affinity and toxicity. The toxin binds with high affinity to its receptor in its primary target, Culex pipiens, and displays a lower affinity to the receptor in Anopheles gambiae, which is less sensitive to Bin. Although the Bin receptor has previously been identified and named Cpm1 in C. pipiens, its structure in Anopheles remains unknown. In this study, we hypothesize that the Anopheles Bin receptor is an ortholog of Cpm1. By screening the Anopheles genomic database, we identified a candidate gene (Agm3) which is expressed primarily on the surface of midgut cells in larvae and which functions as a receptor for Bin. A Cpm1-like gene is also present in the Bin-refractory species Aedes aegypti. Overall, our results indicate that the three mosquito genes examined share a very similar organization and are strongly conserved at the amino acid level, in particular in the NH(2)-terminus, a region believed to contain the ligand binding site, suggesting that relatively few amino acids residues are critical for high affinity binding of the toxin.

Simulating latrine conditions to assess perfume performance against malodour.

To evaluate perfume performance in toilets, we built model toilets in which critical factors such as background malodour, climate, and airflow were controlled. The models were equipped with an odour generator that injected hydrogen sulfide, methyl mercaptan, butyric acid, para-cresol, and indole, allowing us to accurately and reliably reconstitute toilet malodour headspace. The malodorant concentrations matched the quantitative headspace analysis performed in African and Indian toilets. The toilet malodour headspace performance was validated by chemical and sensory analysis. Olfactory stimuli were presented to participants in different simulated climates to assess the effect of climate on the perception of odours. The sensory data show that increasing temperature and humidity decreased the intensity ratings of odours without altering their quality. Perfume can be delivered in these toilets by forced evaporation to control the headspace concentration, or by delivery systems such as cellulosic pads, liquids, and powders. Our experimental set-up allowed us to establish dose-response curves to assess the performance of a perfume in reducing toilet malodour and increasing perceived pleasantness.

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens.

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.

Identification of Hydrogen Disulfanes and Hydrogen Trisulfanes in H2S Bottle, in Flint, and in Dry Mineral White Wine.

Through the accidental contamination of a gas cylinder of H2S, the importance of polysulfanes for flint, gun powder, and match odors was discovered. The hydrogen disulfane was prepared from disulfanediylbis[methyl(diphenyl)silane], and its odor descriptor was evaluated in the gas phase from a gas chromatograph coupled to an olfaction port. The occurrence of this compound in flint and pebbles was confirmed by analyses after derivatization with pentafluorobromobenzene. The occurrence of this sulfane was also confirmed in two dry white Swiss Chasselas wines, sorted by a large-scale sensory analysis from 80 bottles and evaluated by 62 wine professionals. The occurrence of disulfane was confirmed for the two wines described as the most mineral. Polysulfane comprises a class of compounds contributing to the flint odor and that may contribute to the wine mineral odor descriptor. Due to the high volatility and instability pure HSSH was not isolated but kept in solution and its odor profile was described by gas chromatography coupled to an olfaction port as flint, matches, and fireworks with a higher odor intensity compared to H2S.

Crystal protein synthesis is dependent on early sporulation gene expression in Bacillus sphaericus.

Insertional mutations in the spo0A and spoIIAC genes of Bacillus sphaericus 2362 were prepared by conjugation with Escherichia coli using a suicide plasmid containing cloned portions of the target genes. The mutants resembled their Bacillus subtilis counterparts phenotypically and were devoid of crystal proteins as determined by electron microscopy, SDS-PAGE and Western blots. The mutants had greatly reduced toxicity to anopheline mosquito larvae compared to the parental strain. We conclude that crystal protein synthesis in this bacterium is dependent on expression of early sporulation genes.

High resistance to Bacillus sphaericus binary toxin in culex pipiens (Diptera: Culicidae): the complex situation of west Mediterranean countries.

This study was aimed at clarifying the nature of the resistance to Bacillus sphaericus Neide (Bs) that Culex pipiens L. has developed in west Mediterranean countries, France, and Tunisia. Two recessive and sex-linked mutants, sp-1R and sp-2R, were previously detected in southern France. Here, the Tunisian resistance was also shown to involve a single recessive and sex-linked gene that was temporarily named sp-T(R). In addition, sp-1R, sp-2R, and sp-T(R) were shown to separately confer a similar high resistance level (> 5,000-fold) in the homozygous state. Knowing that sp-1R resistance does not alter the binding of Bs binary toxin to its specific receptor, we investigated this character in sp-2RR and sp-T(RR) homozygotes. This was performed by in vitro experiments in which larval brush border membrane fractions (BBMF) were exposed to the 125I-Bin2 toxin of B. sphaericus strain 1593. The toxin-receptor binding was found disrupted by sp-2R but not by sp-T(R). Comparing the binding kinetics among nine Culex pipiens strains of diverse origins revealed that the Bs receptors of sp-1RR and Sp-T(RR) homozygous larvae were displaying the highest affinity toward Bs binary toxins. These results are discussed with regard to alternative assumptions on the dynamics of high Bs-resistance and on the emerging possibilities to test them in a near future.

Transposon-mediated resistance to Bacillus sphaericus in a field-evolved population of Culex pipiens (Diptera: Culicidae).

The binary toxin is the major active component of Bacillus sphaericus, a microbial larvicide used for controlling some vector mosquito-borne diseases. B. sphaericus resistance has been reported in many part of the world, leading to a growing concern for the usefulness of this environmental friendly insecticide. Here we characterize a novel mechanism of resistance to the binary toxin in a natural population of the West Nile virus vector, Culex pipiens. We show that the insertion of a transposable element-like DNA into the coding sequence of the midgut toxin receptor induces a new mRNA splicing event, unmasking cryptic donor and acceptor sites located in the host gene. The creation of the new intron causes the expression of an altered membrane protein, which is incapable of interacting with the toxin, thus providing the host mosquito with an advantageous phenotype. As a large portion of insect genomes is composed of transposable elements or transposable elements-related sequences, this new mechanism may be of general importance to appreciate their significance as potent agents for insect resistance to the microbial insecticides.

The HcaR regulatory protein of Photorhabdus luminescens affects the production of proteins involved in oxidative stress and toxemia.

Comparison of the proteomes of wild-type Photorhabdus luminescens and its hcaR derivative, grown in insect hemolymph, showed that hcaR disruption decreased the production of toxins (tcdA1, mcf, and pirAB) and proteins involved in oxidative stress response (SodA, AhpC, Gor). The disruption of hcaR did not affect growth rate in insects, but did delay the virulence of P. luminescens in Bombyx mori and Spodoptera littoralis larvae. This delayed virulence was associated with a lower toxemia rather than delay in bacteremia. The disruption of hcaR also increased bacterial sensitivity to hydrogen peroxide. A sodA mutant and an hcaR mutant had similar phenotypes in terms of sensitivity to hydrogen peroxide, virulence, toxin gene expression, and growth rate in insects. Thus, the two processes affected by hcaR disruption - toxemia and oxidative stress response - appear to be related. Besides, expression of toxin genes tcdA1, mcf, and pirAB was decreased by paraquat challenge. We provide here the first demonstration of the importance of toxemia for P. luminescens virulence. Our results also highlight the power of proteomic analysis for detecting unexpected links between different, concomitant processes in bacteria.

Effects of a mosquitocidal toxin on a mammalian epithelial cell line expressing its target receptor.

The spread of diseases transmitted by Anopheles and Culex mosquitoes, such as malaria and West Nile fever, is a growing concern for human health. Bacillus sphaericus binary toxin (Bin) is one of the few available bioinsecticides able to control populations of these mosquitoes efficiently. We previously showed that Bin binds to Cpm1, an alpha-glucosidase located on the apical side of Culex larval midgut epithelium. We analysed the effects of Bin by expressing a construct encoding Cpm1 in the mammalian epithelial MDCK cell line. Cpm1 is targeted to the apical side of polarized MDCK, where it is anchored by glycosylphosphatidylinositol (GPI) and displays alpha-glucosidase activity. Bin bound to transfected cells and induced a non-specific current presumably related to the opening of pores. The formation of these pores may be related to the location of the toxin/receptor complex in lipid raft microdomains. Finally, Bin promoted the time-dependent appearance of intracytoplasmic vacuoles but did not drive cell lysis. Thus, the dual functionality (enzyme/toxin receptor) of Cpm1 is fully conserved in MDCK cells and Cpm1 is an essential target protein for Bin cytotoxicity in Culex mosquitoes.

Characterization of Bacillus thuringiensis ser. jordanica (serotype H71), a novel serovariety isolated in Jordan.

The novel strain of Bacillus thuringiensis J112 isolated from a soil sample in Jordan was classified and characterized in terms of toxicity against dipteran and nematode larvae, crystal protein pattern, plasmid profile, and cry gene content. A new name, Bacillus thuringiensis serovariety jordanica (H serotype 71), is proposed for the reference strain J112. The parasporal crystal proteins were toxic to 3(rd) instar larvae of Drosophila melanogaster and to 2(nd) stage juveniles of root knot nematodes Meloidogyne javanica and M. incognita, but showed poor mosquitocidal activity towards Culex pipiens molestus and Culiseta longiareolata larvae. Solubilized and trypsin-digested crystal proteins possessed moderate hemolytic activity against sheep erythrocytes. SDS-polyacrylamide gel electrophoresis revealed that crystals are composed of several polypeptides ranging from 24 to 170 kDa, of which the 20-, 42-, 140-, and 170-kDa proteins were the major components. Analysis of the plasmid pattern of J112 revealed the presence of two large plasmidic bands of about 160 and 205 kbp. PCR with total DNA from strain J112 and specific primers for cry1, cry2, cry3, cry4, and cyt2A genes revealed that cry1, cry3A, cry4, cry5 and cyt2a genes are present.

Loss of the membrane anchor of the target receptor is a mechanism of bioinsecticide resistance.

The mosquitocidal activity of Bacillus sphaericus is because of a binary toxin (Bin), which binds to Culex pipiens maltase 1 (Cpm1), an alpha-glucosidase present in the midgut of Culex pipiens larvae. In this work, we studied the molecular basis of the resistance to Bin developed by a strain (GEO) of C. pipiens. Immunohistochemical and in situ hybridization experiments showed that Cpm1 was undetectable in the midgut of GEO larvae, although the gene was correctly transcribed. The sequence of the cpm1(GEO) cDNA differs from the sequence we previously reported for a susceptible strain (cpm1(IP)) by seven mutations: six missense mutations and a mutation leading to the premature termination of translation. When produced in insect cells, Cpm1(IP) was attached to the membrane by a glycosylphosphatidylinositol (GPI). In contrast, the premature termination of translation of Cpm1(GEO) resulted in the targeting of the protein to the extracellular compartment because of truncation of the GPI-anchoring site. The interaction between Bin and Cpm1(GEO) and the enzyme activity of the receptor were not affected. Thus, Bin is not toxic to GEO larvae because it cannot interact with the midgut cell membrane, even though its receptor site is unaffected. This mechanism contrasts with other known resistance mechanisms in which point mutations decrease the affinity of binding between the receptor and the toxin.

AstR-AstS, a new two-component signal transduction system, mediates swarming, adaptation to stationary phase and phenotypic variation in Photorhabdus luminescens.

Photorhabdus luminescens is an insect-pathogenic bacterium that forms a symbiosis with specific entomopathogenic nematodes. In this bacterium, a symbiosis-'deficient' phenotypic variant (known as the secondary variant or form II) arises at a low frequency during prolonged incubation. A knock-out mutant was generated of the regulator of a newly identified two-component regulatory system, designated AstR-AstS. Interestingly, this mutation altered the timing of phenotypic switching. Variant cells arose in the mutant strain several days before they did in the wild-type population, suggesting that AstRS is directly or indirectly involved in the genetic mechanism underlying variant cell formation. This mutation also affected motility and antibiotic synthesis. To identify AstRS-regulated genes, a comparative analysis using two-dimensional gel electrophoresis was performed. Seventeen proteins with modified synthesis in stationary phase were identified by mass spectrometry and shown to be involved in electron-transport systems, energy metabolism, iron acquisition and stress responses. The results imply that AstRS is involved in the adaptation of cells to the stationary phase, whilst negatively affecting the competitive advantage of form I cells. The link between AstRS-dependent stationary-phase adaptation and phenotypic variation is discussed.