Agrobacterium tumefaciens can obtain sulphur from an opine that is synthesized by octopine synthase using S-methylmethionine as a substrate.

Agrobacterium tumefaciens incites plant tumours that produce nutrients called opines, which are utilized by the bacteria during host colonization. Various opines provide sources of carbon, nitrogen and phosphorous, but virtually nothing was previously known about how A. tumefaciens acquires sulphur during colonization. Some strains encode an operon required for the catabolism of the opine octopine. This operon contains a gene, msh, that is predicted to direct the conversion of S-methylmethionine (SMM) and homocysteine (HCys) to two equivalents of methionine. Purified Msh carried out this reaction, suggesting that SMM could be an intermediate in opine catabolism. Purified octopine synthase (Ocs, normally expressed in plant tumours) utilized SMM and pyruvate to produce a novel opine, designated sulfonopine, whose catabolism by the bacteria would regenerate SMM. Sulfonopine was produced by tobacco and Arabidopsis when colonized by A. tumefaciens and was utilized as sole source of sulphur by A. tumefaciens. Purified Ocs also used 13 other proteogenic and non-proteogenic amino acids as substrates, including three that contain sulphur. Sulfonopine and 11 other opines were tested for induction of octopine catabolic operon and all were able to do so. This is the first study of the acquisition of sulphur, an essential element, by this pathogen.

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.

Simple electrospray mass spectrometry detection of acylhomoserine lactones.

Simple (non-tandem) electrospray mass spectrometry (ESMS) can detect acyl homoserine lactones (AHL) in bacteriological media in picomole amounts. The chemical reactivity of AHLs and their extraction behaviour into ethyl acetate, coupled with detection in the ESMS, has shown that these lactones can be detected as the protonated pseudomolecular ions themselves as well as solvent and ammonium adducts, and as dimers. ESMS detects and identifies these molecules, utilizing simple chemical properties of AHLs.

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.

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.

Signal quenching, detoxification and mineralization of vir gene-inducing phenolics by the VirH2 protein of Agrobacterium tumefaciens.

Plant tumorigenesis by Agrobacterium tumefaciens requires approximately 20 Vir proteins, transcription of which is induced by a family of phenolic compounds released from plant wound sites. One Vir protein, VirH2, plays a role in the metabolism of at least one phenolic inducer inasmuch as it converts ferulic acid, a potent vir gene inducer, to the non-inducer caffeate by O-demethylation of a methoxyl group. Here, we tested VirH2-dependent O-demethylation of 16 other vir-inducing phenolics, and detected this activity for each compound. However, O-demethylation rates differed enormously, with the strongest vir gene inducers such as acetosyringone being demethylated extremely slowly. Compounds containing two methoxyl groups were demethylated at both positions. In general, phenolic inducers were more toxic than their demethylated counterparts. A virH2 mutant was more sensitive than the wild type to growth inhibition by virtually all phenolic inducers tested, indicating that VirH2 detoxifies these compounds. VirH2 also played a role in mineralization of some phenolics. It converted vanillate to protocatechuate, which was then mineralized via the beta-ketoadipate pathway. Vanillyl alcohol and vanillin were also mineralized after being oxidized to vanillate. All three compounds served as sole sources of carbon, whereas the remaining 13 compounds did not.

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.

Quorum sensing controls exopolysaccharide production in Sinorhizobium meliloti.

Sinorhizobium meliloti is a soil bacterium capable of invading and establishing a symbiotic relationship with alfalfa plants. This invasion process requires the synthesis, by S. meliloti, of at least one of the two symbiotically important exopolysaccharides, succinoglycan and EPS II. We have previously shown that the sinRI locus of S. meliloti encodes a quorum-sensing system that plays a role in the symbiotic process. Here we show that the sinRI locus exerts one level of control through regulation of EPS II synthesis. Disruption of the autoinducer synthase gene, sinI, abolished EPS II production as well as the expression of several genes in the exp operon that are responsible for EPS II synthesis. This phenotype was complemented by the addition of acyl homoserine lactone (AHL) extracts from the wild-type strain but not from a sinI mutant, indicating that the sinRI-specified AHLs are required for exp gene expression. This was further confirmed by the observation that synthetic palmitoleyl homoserine lactone (C(16:1)-HL), one of the previously identified sinRI-specified AHLs, specifically restored exp gene expression. Most importantly, the absence of symbiotically active EPS II in a sinI mutant was confirmed in plant nodulation assays, emphasizing the role of quorum sensing in symbiosis.

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."