The most universal cell-cell signaling mechanism in Gram-negative bacteria occurs via the production and response to a class of small diffusible molecules called N-acylhomoserine lactones (AHLs). This communication is called quorum sensing and is responsible for the regulation of several physiological processes and many virulence factors in pathogenic bacteria. The detection of these molecules has been rendered possible by the utilization of genetically engineered bacterial biosensors which respond to the presence of exogenously supplied AHLs. In this study, using diverse bacterial biosensors, several biosensor activating fractions were purified by organic extraction, HPLC and TLC of cell-free culture supernatants of plant growth-promoting Pseudomonas putida WCS358. Surprisingly, it was observed that the most abundant compounds in these fractions were cyclic dipeptides (diketopiperazines, DKPs), a rather novel finding in Gram-negative bacteria. The purification, characterization, chemical synthesis of four DKPs are reported and their possible role in cell-cell signaling is discussed.

Burkholderia glumae is an emerging rice pathogen in several areas around the world. Closely related Burkholderia species are important opportunistic human pathogens for specific groups of patients, such as patients with cystic fibrosis and patients with chronic granulomatous disease. Here we report that the first clinical isolate of B. glumae, strain AU6208, has retained its capability to be very pathogenic to rice. As previously reported for rice isolate B. glumae BGR1 (and also for the clinical isolate AU6208), TofI or TofR acyl homoserine lactone (AHL) quorum sensing played a pivotal role in rice virulence. We report that AHL quorum sensing in B. glumae AU6208 regulates secreted LipA lipase and toxoflavin, the phytotoxin produced by B. glumae. B. glumae AU6208 lipA mutants were no longer pathogenic to rice, indicating that the lipase is an important virulence factor. It was also established that type strain B. glumae ATCC 33617 did not produce toxoflavin and lipase and was nonpathogenic to rice. It was determined that in strain ATCC 33617 the LuxR family quorum-sensing sensor/regulator TofR was inactive. Introducing the tofR gene of B. glumae AU6208 in strain ATCC 33617 restored its ability to produce toxoflavin and the LipA lipase. This study extends the role of AHL quorum sensing in rice pathogenicity through the regulation of a lipase which was demonstrated to be a virulence factor. It is the first report of a clinical B. glumae isolate retaining strong rice pathogenicity and finally determined that B. glumae can undergo phenotypic conversion through a spontaneous mutation in the tofR regulator.

Transposon Tn5 genomic mutants of plant-growth-promoting Pseudomonas putida strain WCS358 have been isolated which no longer utilize ferufic and coumaric acids as sole sources of carbon and energy. Genetic studies confirmed previous biochemical data showing that ferulic acid is degraded via vanillic acid, and coumaric acid via hydroxybenzoic acid. The genes involved in these enzymic steps were cloned and characterized. Two proteins designated Fca (26-5 kDa) and Vdh (50.3 kDa) were identified as responsible for the conversion of ferulic acid to vanillic acid; the proteins are encoded by the fca and vdh genes which are organized in an operon structure in the chromosome. The Vdh protein is 69% identical a t the amino acid level to the Vdh protein recently identified in Pseudomonas sp. strain HR199 and converts vanillin to vanillic acid. Homology studies revealed that the Vdh proteins exhibited significant identity to aldehyde dehydrogenases from different organisms whereas Fca belonged to the enoyl-CoA hydratase family of proteins. Two proteins, designated VanA (39.9 kDa) and VanB (343 kDa), encoded by two genes, vanA and van& are organized in an operon in the chromosome. They were found to be responsible for the demethylation of vanillic acid to protocatechuic acid. The VanA proteins showed no homology to any other known protein, while VanB belonged to the ferredoxin family of proteins. This two-component enzyme system demethylated another phenolic monomer, veratric acid, thus indicating broad specificity. Studies of the regulation of the vanAB operon demonstrated that the genes were induced by the substrate, vanillic acid; however, the strongest induction was observed when cells were grown in the presence of the product of the reaction, protocatechuic acid.

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Bacterial Panicle Blight caused by Burkholderia glumae is a major disease of rice, which has dramatically affected rice production around the world in the last years. In this study we describe the assessment of three Streptomyces isolates as biocontrol agents for B. glumae . Additionally, the presence of other plant-growth promoting abilities and their possible beneficial effects upon their inoculation on rice plants was evaluated as an ecological analysis for their future inoculation in rice crops. Two isolates (A20 and 5.1) inhibited growth of virulent B. glumae strains, as well as a wide range of bacterial and fungal species, while a third strain (7.1) showed only antifungal activity. In vitro tests demonstrated the ability of these strains to produce siderophores, Indoleacetic acid (IAA), extracellular enzymes and solubilizing phosphate. Greenhouse experiments with two rice cultivars indicated that Streptomyces A20 is able to colonize rice plants and promote plant growth in both cultivars. Furthermore, an egfp tagged mutant was generated and colonization experiments were performed, indicating that Streptomyces A20 –GFP was strongly associated with root hairs, which may be related to the plant growth promotion observed in the gnotobiotic experiments. In order to characterize the antimicrobial compounds produced by strain A20 bacteria, mass spectrometry analyses were performed. This technique indicated that A20 produced several antimicrobial compounds with sizes below 3 kDa and three of these molecules were identified as Streptotricins D, E and F. These findings indicate the potential of Streptomyces A20 as a biocontrol inoculant to protect rice plants against bacterial diseases.

This study presents a computational analysis of the structures of lipase B from Candida antarctica (CalB) and two penicillin G acylases (PGAs), from eukaryotic and prokaryotic sources, respectively. Molecular simulations were used to point out the regions of the enzymes that are prone to interact with immobilisation supports. In order to evaluate the accessibility of the active site, the location of the amino acid residues involved in the formation of covalent bonds with the polymers was visualised. The mapping of the distribution of hydrophobic and hydrophilic regions on the enzyme surface provided a view of the areas of the protein that can establish either hydrophobic or hydrophilic interactions with the carriers. Experimental data obtained from the immobilisation of the enzymes on supports bearing different chemical functionalities suggest the involvement of the glycan moiety in enzyme-polymer interactions. In the case of PGA the glycan moiety can constitute an extra site for the covalent linkage of the enzyme on the polymer.

Sphaeropsidin A, the main phytotoxin produced by Diplodia cupressi, as well as the two natural analogues sphaeropsidins B and C and 14 derivatives obtained by chemical modifications were assayed for antibacterial activity against Xanthomonas oryzae pv. oryzae, Pseudomonas fuscovaginae, and Burkholderia glumae, the causal agents of severe bacterial rice diseases. The results showed a strong and specific activity of sphaeropsidin A against X. oryzae pv. oryzae, while no activity was observed against the other two pathogens. The results of structure-activity relationship studies showed that structural features important to impart this antibacterial activity are the presence of the C-7 carbonyl group and the hemiketalic lactone functionality. The C-13 vinyl group, the double bond of ring C, and/or the tertiary C-9 hydroxy group, as well as the pimarane arrangement of the tricylic carbon skeleton, were also important for the antibacterial activity. These findings may be useful in designing novel compounds for practical applications in agriculture.