Anatol Eberhard

Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates.

Many bacteria, including several pathogens of plants and humans, use a pheromone called an autoinducer to regulate gene expression in a cell density-dependent manner. Agrobacterium autoinducer [AAI, N-(3-oxo-octanoyl)-L-homoserine lactone] of A. tumefaciens is synthesized by the TraI protein, which is encoded by the tumor-inducing plasmid. Purified hexahistidinyl-TraI (H6-TraI) used S-adenosylmethionine to make the homoserine lactone moiety of AAI, but did not use related compounds. H6-TraI used 3-oxo-octanoyl-acyl carrier protein to make the 3-oxo-octanoyl moiety of AAI, but did not use 3-oxo-octanoyl-coenzyme A. These results demonstrate the enzymatic synthesis of an autoinducer through the use of purified substrates.

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.

Analogs of the autoinducer of bioluminescence inVibrio fischer

The enzymes for luminescence inVibrio fischeri are induced only when a sufficient concentration of a metabolic product (autoinducer) specifically produced by this species accumulates. It has previously been shown that the autoinducer is 3-oxohexanoyl homoserine lactone and that it enters the cells by simple diffusion. To further study the mechanism of induction, we have synthesized several analogs of the autoinducer. The analogs were tested withV. fischeri for their inducing activity and for their ability to inhibit the action of the natural autoinducer. The compounds were found to display various combinations of inducing and inhibiting abilities. None of the compounds tested appeared to have any effect on cells ofV. harveyi strain MAV orPhotobacterium leiognathi strain 721, but several of the compounds decreased light output byP. phosphoreum strain 8265. These studies show that 1) the site of action of the autoinducer is not highly sterically constrained 2) the autoinducers of other species of luminous bacteria are likely to be quite different from that ofV. fischeri and 3) a simple mode in which one autoinducer molecule binds to a single receptor protein site and thus initiates luciferase synthesis is inadequate. The analogs should prove useful in the study of the binding site and mode of action of the autoinducer.

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.

Synthesis of the lux gene autoinducer in Vibrio fischeri is positively autoregulated

The enzymes for luminescence in Vibrio fischeri are induced only after the accumulation of a sufficient concentration of a metabolic product (the autoinducer) generated by the bacteria themselves. Genetic analyses by others have previously suggested that biosynthesis of the autoinducer is catalyzed by a single gene product (autoinducer synthetase) presumably from precursors typically present in the bacterial cell. Also, the biosynthesis was predicted to be autocatalytic such that in the presence of autoinducer, more autoinducer synthetase should be produced. We have directly tested these predictions and found that autoinducer synthesis is indeed positively autoregulated. In addition, we have demonstrated autoinducer synthesis in vitro and have tentatively identified the substrates of autoinducer synthetase as S-adenosylmethionine and 3-oxohexanoyl coenzyme A.

The phenolic vir gene inducer ferulic acid is O-demethylated by the VirH2 protein of an Agrobacterium tumefaciens Ti plasmid

Some or possibly all Ti plasmids of Agrobacterium tumefaciens encode a bicistronic operon designated virH, which encodes two proteins, VirH1 and VirH2, that resemble a family of cytochrome P450‐type monooxygenases. Expression of this operon is induced by a family of phenolic compounds that induce all other operons within the vir regulon. We hypothesized that either or both of these proteins might metabolize some or all of these phenolic compounds. We therefore tested induction of a vir promoter by a variety of phenolic compounds in isogenic strains that express or lack virH1 and virH2. Although some compounds were equally effective inducers regardless of the virH status, other compounds induced vir expression far more effectively in the virH mutant than in the virH‐proficient host. For all tested compounds, VirH2 appeared to be solely responsible for this effect. One such compound, ferulic acid, was chosen for biochemical analysis. Ferulic acid was degraded by a VirH‐proficient host but not by a VirH mutant. The wild‐type strain released large amounts of a more hydrophilic compound into the cell supernatant. This compound was tested by mass spectroscopy, nuclear magnetic resonance and UV spectroscopy and found to consist of caffeic acid. This indicates that wild‐type strains convert virtually all added ferulic acid to caffeic acid, and that VirH2 is essential for this O‐demethylation reaction. Ferulic acid was far more toxic than caffeic acid to the wild‐type strain, although the wild‐type strain was more resistant to ferulic acid than was the virH mutant. Caffeic acid was slowly removed from the broth, suggesting further metabolic reactions.

Decyl nitrite: An aldehyde analog in the bacterial bioluminescence reaction

Decyl nitrite was found to be an effective substitute for aliphatic aldehydes in the light producing reactions of bacterial luciferase. Two other aldehyde analogs, decyl formate and decyl formamide, were inactive. A kinetic isotope effect of 1.7 was found for 1- 2 H-dodecanal in comparison to dodecanal. These results support a Baeyer-Villiger type mechanism for bioluminescence proposed previously. Alkyl nitrites may be useful in the study of other enzymic reactions involving aldehyde substrates.

Luminous bacteria synthesize luciferase anaerobically

Four species of luminous bacteria, Photobacterium phosphoreum, P. leiognathi, P. fischeri and Beneckea harveyi (two strains of each), were shown to synthesize luciferase anaerobically. One of these, P. phosphoreum, produced as much luciferase anaerobically as it did aerobically, and all four species were found to grow almost equally rapidly under the two sets of conditions. Previous work with B. harveyi and P. fischeri had shown that aerobic luciferase synthesis can proceed only after an inhibitor in the complex medium has been removed and a species-specific autoinducer secreted. All strains tested also removed the inhibitor and secreted an autoinducer anaerobically. The small amount of luciferase produced anaerobically by some strains is thus apparently not due either to lack of removal of inhibitor or to insufficient production of autoinducer but may involve an oxygen-dependent control mechanism.