Potato plants genetically modified to produce N-acylhomoserine lactones increase susceptibility to soft rot erwiniae.

Many gram-negative bacteria employ N-acylhomoserine lactones (AHL) to regulate diverse physiological processes in concert with cell population density (quorum sensing [QS]). In the plant pathogen Erwinia carotovora, the AHL synthesized via the carI/expI genes are responsible for regulating the production of secreted plant cell wall-degrading exoenzymes and the antibiotic carbapen-3-em carboxylic acid. We have previously shown that targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in planta of the cognate AHL signaling molecules N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL), which in turn, were able to complement a carI-QS mutant. In the present study, we demonstrate that transgenic potato plants containing the yenI gene are also able to express AHL and that the presence and level of these AHL in the plant increases susceptibility to infection by E. carotovora. Susceptibility is further affected by both the bacterial level and the plant tissue under investigation.

Preliminary findings of quorum signal molecules in clinically stable lung allograft recipients.

BACKGROUND: Infection with bacteria such as Pseudomonas is common in lung allograft recipients, particularly during chronic rejection. Analysis of sputum samples from patients with cystic fibrosis infected with Pseudomonas aeruginosa or Burkholderia cepacia has indicated the presence of bacterial N-acylhomoserine lactones (AHLs) quorum sensing signalling molecules. AHLs not only control the expression of bacterial virulence genes but are also involved in stimulating the maturation of antibiotic resistant biofilms and host chemokine release. It was hypothesised that AHLs may be detected even in clinically stable lung transplant recipients free of clinical infection or rejection. METHODS: Three 60 ml samples of bronchoalveolar lavage (BAL) fluid were taken from nine stable lung transplant recipients 3-12 months after transplantation. Detection of AHLs was carried out on dichloromethane extracted supernatants using the bioluminescence based AHL reporter plasmid pSB1075. This responds to the presence of AHLs with long acyl chains (C10-C14), generating light. Synthetic AHLs were included as positive controls. RESULTS: Five of the nine BAL fluid supernatants exhibited AHL activity, suggesting the presence of AHLs with long N-acyl chains. There was no correlation between the levels of AHLs detected or their absence and BAL fluid microbiology or diagnosis before transplantation. CONCLUSIONS: This is the first evidence for the presence of AHL quorum sensing signals in human lung allograft recipients, even in subjects with no rejection or apparent infection. Further longitudinal follow up of these preliminary findings is required to elucidate potential links with infection, rejection, and allograft deterioration.

Plants genetically modified to produce N-acylhomoserine lactones communicate with bacteria.

N-acylhomoserine lactones (AHLs) play a critical role in plant/microbe interactions. The AHL, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), induces exoenzymes that degrade the plant cell wall by the pathogenic bacterium Erwinia carotovora. Conversely, the antifungal activity of the biocontrol bacterium Pseudomonas aureofaciens 30-84 is due (at least in part) to phenazine antibiotics whose synthesis is regulated by N-hexanoylhomoserine lactone (HHL). Targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in plants of the cognate AHL signaling molecules (OHHL and HHL). The AHLs produced by the transgenic plants were sufficient to induce target gene expression in several recombinant bacterial AHL biosensors and to restore biocontrol activity to an HHL-deficient P. aureofaciens strain. In addition, pathogenicity was restored to an E. carotovora strain rendered avirulent as a consequence of a mutation in the OHHL synthase gene, carI. The ability to generate bacterial quorum-sensing signaling molecules in the plant offers novel opportunities for disease control and for manipulating plant/microbe interactions.

N-acyl-homoserine lactone-mediated regulation of phenazine gene expression by Pseudomonas aureofaciens 30-84 in the wheat rhizosphere.

Pseudomonas aureofaciens 30-84 is a soilborne bacterium that colonizes the wheat rhizosphere. This strain produces three phenazine antibiotics which suppress take-all disease of wheat by inhibition of the causative agent Gaeumannomyces graminis var. tritici. Phenazines also enhance survival of 30-84 within the wheat rhizosphere in competition with other organisms. Expression of the phenazine biosynthetic operon is controlled by the phzR/phzI N-acyl-homoserine lactone (AHL) response system (L. S. Pierson III et al., J. Bacterial 176:3966-3974, 1994; D. W. Wood and L. S. Pierson III, Gene 168:49-53, 1996). By using high-pressure liquid chromatography coupled with high-resolution mass spectrometry, the AHL produced by PhzI has now been identified as N-hexanoyl-homoserine lactone (HHL). In addition, the ability of HHL to serve as an interpopulation signal molecule in the wheat rhizosphere has been examined by using isogenic reporter strains. Disruption of phzI reduced expression of the phenazine biosynthetic operon 1,000-fold in the wheat rhizosphere. Coinoculation of an isogenic strain which produced the endogenous HHL signal restored phenazine gene expression in the phzI mutant to wild-type levels in situ. These results demonstrate that HHL is required for phenazine expression in situ and is an effective interpopulation signal molecule in the wheat rhizosphere.

Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a spectrum of exoproducts many of which have been implicated in the pathogenesis of human infection. Expression of some of these factors requires cell-cell communication involving the interaction of a small diffusible molecule, an "autoinducer," with a positive transcriptional activator. In P. aeruginosa PAO1, LasI directs the synthesis of the autoinducer N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), which activates the positive transcriptional activator, LasR. Recently, we have discovered a second signaling molecule-based modulon in PAO1, termed vsm, which contains the genes vsmR and vsmI. Using HPLC, mass spectrometry, and NMR spectroscopy we now establish that in Escherichia coli, VsmI directs the synthesis of N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL). These compounds are present in the spent culture supernatants of P. aeruginosa in a molar ratio of approximately 15:1 and their structures were unequivocally confirmed by chemical synthesis. Addition of either BHL or HHL to PAN067, a pleiotropic P. aeruginosa mutant unable to synthesize either of these autoinducers, restored elastase, chitinase, and cyanide production. In E. coli carrying a vsmR/vsmI'::lux transcriptional fusion, BHL and HHL activated VsmR to a similar extent. Analogues of these N-acyl-L-homoserine lactones in which the N-acyl side chain has been extended and/or oxidized at the C-3 position exhibit substantially lower activity (e.g., OdDHL) or no activity (e.g., dDHL) in this lux reporter assay. These data indicate that multiple families of quorum sensing modulons interactively regulate gene expression in P. aeruginosa.

High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man.

High-performance liquid chromatographic assay procedures have been developed for naproxen, ibuprofen and diclofenac in human plasma and synovial fluid samples. A single liquid-liquid extraction procedure was used to isolate each compound from acidified biological matrix prior to the quantitative analysis. A Spherisorb ODS column (12.5 cm x 4.6 mm I.D.) was used for all the chromatography. Naproxen was eluted with a mobile phase of methanol-Sörensen's buffer at pH 7 (37:63, v/v). Ibuprofen and diclofenac were eluted using mobile phases of methanol-water at pH 3.3 (65:35, v/v and 63:37, v/v, respectively). Diphenylacetic acid was used as the internal standard for the assay of naproxen and flurbiprofen was used in the analysis of ibuprofen and diclofenac. Inter- and intra-day coefficients of variation were less than 7%. The assays were used in clinical studies of the three drugs in osteo- and rheumatoid arthritis patients.

Histology of the Interactions of Paecilomyces lilacinus with Meloidogyne incognita on Tomato.

Excised tomato roots were examined histologically for interactions of the fungus Paecilomyces lilacinus and Meloidogyne incognita race 1. Root galling and giant-cell formation were absent in tomato roots inoculated with nematode eggs infected with P. lilacinus. Few to no galls and no giant-cell formation were found in roots dipped in a spore suspension of P. lilacinus and inoculated with M. incognita. Numerous large galls and giant cells were present in roots inoculated only with M. incognita. P. lilacinus colonized the surface of epidermal cells as well as the internal cells of epidermis and cortex. The possibility of biological protection of plant surfaces with P. lilacinus against root-knot nematodes is discussed.

Histopathology of Selected cultivars of Tobacco infected with Meloidogyne species.

Rates of nematode penetration and the histopathology of root infections in fluecured tobacco cultivars 'McNair-944,' 'Speight G-28,' and 'NC-89' with either Meloidogyne arenaria, M. incognita, M. hapla, or M. javanica were investigated. Penetration of root tips by juveniles of all species into the M. incognita-resistant NC-89 and G-28 was much less than that on the susceptible McNair-944. Few juveniles of M. incognita were detected in resistant cultivars 7 and 14 days after inoculation. Infection sites exhibited some cavities and extensive necrotic tissue at 14 days; less necrotic tissue and no intact nematodes were observed 35 days after inoculation. Although some females of M. arenaria reached maturity and produced eggs, considerable necrosis was induced in the resistant cultivars. Meloidogyne hapla and M. javanica developed on all cultivars, but there was necrotic tissue at some infection sites in the resistant cultivars. The occurrence of single multistructured nuclei in the syncytia of most M. hapla infections differed from the numerous small nuclei found in syncytia caused by the other three species.