Nematophagous fungus Arthrobotrys oligospora mimics olfactory cues of sex and food to lure its nematode prey.

To study the molecular basis for predator-prey coevolution, we investigated how Caenorhabditis elegans responds to the predatory fungus Arthrobotrys oligospora. C. elegans and other nematodes were attracted to volatile compounds produced by A. oligospora. Gas-chromatographic mass-spectral analyses of A. oligospora-derived volatile metabolites identified several odors mimicking food cues attractive to nematodes. One compound, methyl 3-methyl-2-butenoate (MMB) additionally triggered strong sex- and stage-specific attraction in several Caenorhabditis species. Furthermore, when MMB is present, it interferes with nematode mating, suggesting that MMB might mimic sex pheromone in Caenorhabditis species. Forward genetic screening suggests that multiple receptors are involved in sensing MMB. Response to fungal odors involves the olfactory neuron AWCs. Single-cell RNA-seq revealed the GPCRs expressed in AWC. We propose that A. oligospora likely evolved the means to use olfactory mimicry to attract its nematode prey through the olfactory neurons in C. elegans and related species.

NMR-spectroscopic screening of spider venom reveals sulfated nucleosides as major components for the brown recluse and related species.

Extensive chemical analyses of spider venoms from many species have revealed complex mixtures of biologically active compounds, of which several have provided important leads for drug development. We have recently shown that NMR spectroscopy can be used advantageously for a direct structural characterization of the small-molecule content of such complex mixtures. Here, we report the application of this strategy to a larger-scale analysis of a collection of spider venoms representing >70 species, which, in combination with mass spectrometric analyses, allowed the identification of a wide range of known, and several previously undescribed, small molecules. These include polyamines, common neurotransmitters, and amino acid derivatives as well as two additional members of a recently discovered family of natural products, the sulfated nucleosides. In the case of the well studied brown recluse spider, Loxosceles reclusa, sulfated guanosine derivatives were found to comprise the major small-molecule components of the venom.

Effects of AiiA-mediated quorum quenching in Sinorhizobium meliloti on quorum-sensing signals, proteome patterns, and symbiotic interactions.

Many behaviors in bacteria, including behaviors important to pathogenic and symbiotic interactions with eukaryotic hosts, are regulated by a mechanism called quorum sensing (QS). A "quorum-quenching" approach was used here to identify QS-regulated behaviors in the N-fixing bacterial symbiont Sinorhizobium meliloti. The AiiA lactonase from Bacillus produced in S. meliloti was shown to enzymatically inactivate S. meliloti's N-acyl homoserine lactone (AHL) QS signals, thereby disrupting normal QS regulation. Sixty proteins were differentially accumulated in the AiiA-producing strain versus the control in early log or early stationary phase cultures. Fifty-two of these QS-regulated proteins, with putative functions that include cell division, protein processing and translation, metabolite transport, oxidative stress, and amino acid metabolism, were identified by peptide mass fingerprinting. Transcription of representative genes was reduced significantly in the AiiA-producing strain, although the effects of AiiA on protein accumulation did not always correspond to effects on transcription. The QS signal-deficient strain was reduced significantly in nodule initiation during the first 12 h after inoculation onto Medicago truncatula host plants. The AiiA lactonase also was found to substantially inactivate two of the AHL mimic compounds secreted by M. truncatula. This suggests some structural similarity between bacterial AHLs and these mimic compounds. It also indicates that quorum quenching could be useful in identifying Sinorhizobium genes that are affected by such host QS mimics in planta.

Extending the scope of NMR spectroscopy with microcoil probes.

Capillary NMR (CapNMR) spectroscopy has emerged as a major breakthrough for increasing the mass-sensitivity of NMR spectroscopic analysis and enabling the combination of NMR spectroscopy with other analytical techniques. Not only is the acquisition of high-sensitivity spectra getting easier but the quality of CapNMR spectra obtained in many small-molecule applications exceeds what can be accomplished with conventional designs. This Minireview discusses current CapNMR technology and its applications for the characterization of mass-limited, small-molecule and protein samples, the rapid screening of small-molecule or protein libraries, as well as hyphenated techniques that combine CapNMR with other analytical methods.

sinI- and expR-dependent quorum sensing in Sinorhizobium meliloti.

Quorum sensing (QS) in Sinorhizobium meliloti, the N-fixing bacterial symbiont of Medicago host plants, involves at least half a dozen different N-acyl homoserine lactone (AHL) signals and perhaps an equal number of AHL receptors. The accumulation of 55 proteins was found to be dependent on SinI, the AHL synthase, and/or on ExpR, one of the AHL receptors. Gas chromatography-mass spectrometry and electrospray ionization tandem mass spectrometry identified 3-oxo-C(14)-homoserine lactone (3-oxo-C(14)-HSL), C(16)-HSL, 3-oxo-C(16)-HSL, C(16:1)-HSL, and 3-oxo-C(16:1)-HSL as the sinI-dependent AHL QS signals accumulated by the 8530 expR(+) strain under the conditions used for proteome analysis. The 8530 expR(+) strain secretes additional, unidentified QS-active compounds. Addition of 200 nM C(14)-HSL or C(16:1)-HSL, two of the known SinI AHLs, affected the levels of 75% of the proteins, confirming that their accumulation is QS regulated. A number of the QS-regulated proteins have functions plausibly related to symbiotic interactions with the host, including ExpE6, IdhA, MocB, Gor, PckA, LeuC, and AglE. Seven of 10 single-crossover beta-glucuronidase (GUS) transcriptional reporters in genes corresponding to QS-regulated proteins showed significantly different activities in the sinI and expR mutant backgrounds and in response to added SinI AHLs. The sinI mutant and several of the single-crossover strains were significantly delayed in the ability to initiate nodules on the primary root of the host plant, Medicago truncatula, indicating that sinI-dependent QS regulation and QS-regulated proteins contribute importantly to the rate or efficiency of nodule initiation. The sinI and expR mutants were also defective in surface swarming motility. The sinI mutant was restored to normal swarming by 5 nM C(16:1)-HSL.

Exploring uncharted terrain in nature's structure space using capillary NMR spectroscopy: 13 steroids from 50 fireflies.

Capillary NMR spectroscopy (CapNMR) was used to characterize 13 new cardenolides and related steroids from a severely mass-limited natural products sample derived from a rare firefly, Lucidota atra. These analyses were carried out on only partially purified samples, each containing 20-100 mug of up to three steroids. Compared to other NMR spectroscopic techniques, CapNMR provided an up to 3-fold gain in sensitivity while maintaining very high spectral quality, which was essential for the identification of the L. atra steroids. We show that CapNMR allows for routine 1H and 13C NMR spectroscopic characterization of small molecule samples containing as little as 40 nmol of material.

Direct binding of the quorum sensing regulator CepR of Burkholderia cenocepacia to two target promoters in vitro.

Burkholderia cenocepacia is an opportunistic human pathogen that can aggressively colonize the cystic fibrosis lung. This organism has a LuxR/LuxI-type quorum sensing system that enables cell-cell communication via exchange of acyl homoserine lactones (AHLs). The CepR and CepI proteins constitute a global regulatory system, controlling expression of at least 40 genes, including those controlling swarming motility and biofilm formation. In this study, we isolated seven lacZ fusions in a clinical isolate of B. cenocepacia that are inducible by octanoyl-HSL. Induction of all of these genes requires CepR. The cepI promoter was tested for induction by a set of 33 synthetic autoinducers and analogues, and was most strongly induced by long-chain AHLs lacking 3-oxo substitutions. Expression of this promoter was inhibited by high concentrations of three different autoinducers, each having six-carbon acyl chains. When CepR protein was overproduced in Escherichia coli, it accumulated in a soluble form in the presence of octanoyl-HSL, but accumulated only as insoluble inclusion bodies in its absence. Purified CepR-OHL complexes bound to specific DNA sequences at the cepI and aidA promoters with high specificity. These binding sites included a 16-nucleotide imperfect dyad symmetry. Both CepR binding sites are centred approximately 44 nucleotides upstream of the respective transcription start sites.

Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide-producing species of Halomonas.

Some members of the moderately halophilic genus Halomonas, such as H. eurihalina, H. maura, H. ventosae and H. anticariensis, produce exopolysaccharides with applications in many industrial fields. We report here that these four species also produce autoinducer molecules that are involved in the cell-to-cell signaling process known as quorum sensing. By using the N-acyl homoserine lactone (AHL) indicator strains Agrobacterium tumefaciens NTL4 (pZRL4) and Chromobacterium violaceum CV026, we discovered that all the Halomonas strains examined synthesize detectable AHL signal molecules. The synthesis of these compounds was growth-phase dependent and maximal activity was reached during the late exponential to stationary phases. One of these AHLs seems to be synthesized only in the stationary phase. Some of the AHLs produced by H. anticariens FP35(T) were identified by gas chromatography/mass spectrometry and electrospray ionization tandem mass spectrometry as N-butanoyl homoserine lactone (C(4)-HL), N-hexanoyl homoserine lactone (C(6)-HL), N-octanoyl homoserine lactone (C(8)-HL) and N-dodecanoyl homoserine lactone (C(12)-HL). This study suggests that quorum sensing may also play an important role in extreme environments.

Nutritional requirements of Allisonella histaminiformans, a ruminal bacterium that decarboxylates histidine and produces histamine.

Histamine is an inflammatory agent that contributes to bovine laminitis. Cattle fed silage-containing rations often have large populations of Allisonella histaminiformans, but this obligate histidine-decarboxylating bacterium could not be isolated from cattle fed timothy hay. The growth of A. histaminiformans was stimulated by yeast extract, protein hydrolysates, and water-soluble extracts of alfalfa or corn silage. Extracts of alfalfa were more potent than corn silage. Because growth and histamine production were not stimulated by Casamino Acids or a mixture of purified amino acids, it appeared that A. histaminiformans requires peptides. The idea that A. histaminiformans requires peptides is consistent with the observation that alfalfa silages often have a large amount of peptide nitrogen.

Chemical identification of N-acyl homoserine lactone quorum-sensing signals produced by Sinorhizobium meliloti strains in defined medium.

The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Both strains synthesized several long-chain AHLs. Defined medium cultures of strain AK631 synthesized a complex mixture of AHLs with short acyl side chains. Strain 1021 produced no short-chain AHLs when grown on defined medium and made a somewhat different set of long-chain AHLs than previously reported for cultures in rich medium. While the two strains produced several AHLs in common, the differences in AHLs produced suggest that there may be significant differences in their patterns of quorum-sensing regulation.

Characterization of the Sinorhizobium meliloti sinR/sinI locus and the production of novel N-acyl homoserine lactones.

Sinorhizobium meliloti is a soil bacterium which can establish a nitrogen-fixing symbiosis with the legume Medicago sativa. Recent work has identified a pair of genes, sinR and sinI, which represent a potential quorum-sensing system and are responsible for the production of N-acyl homoserine lactones (AHLs) in two S. meliloti strains, Rm1021 and Rm41. In this work, we characterize the sinRI locus and show that these genes are responsible for the synthesis of several long-chain AHLs ranging from 12 to 18 carbons in length. Four of these, 3-oxotetradecanoyl HL, 3-oxohexadecenoyl HL, hexadecenoyl HL, and octadecanoyl HL, have novel structures. This is the first report of AHLs having acyl chains longer than 14 carbons. We show that a disruption in sinI eliminates these AHLs and that a sinR disruption results in only basal levels of the AHLs. Moreover, the same sinI and sinR mutations also lead to a decrease in the number of pink nodules during nodulation assays, as well as a slight delay in the appearance of pink nodules, indicating a role for quorum sensing in symbiosis. We also show that sinI and sinR mutants are still capable of producing several short-chain AHLs, one of which was identified as octanoyl HL. We believe that these short-chain AHLs are evidence of a second quorum-sensing system in Rm1021, which we refer to here as the mel system, for "S. meliloti."