Giuliano Degrassi

Plant Growth-Promoting Pseudomonas putida WCS358 Produces and Secretes Four Cyclic Dipeptides: Cross-Talk with Quorum Sensing Bacterial Sensors

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.

An overview of general features of risk assessments of genetically modified crops

The intentional introduction into the environment or market of genetically modified organisms (GMOs) is nearly always governed by a framework of science-based risk assessment and risk management measures. This is usually implemented through the integration of hazard identification and characterisation of all of the elements of risk associated with a new GM crop or derived product. Typical categories of hazards arising from the introduction of transgenic crops include: possible unintended negative health effects in a susceptible subgroup of the consumer (target) population; the evolution of resistance in the targeted pest/pathogen populations when the transgene confers resistance to a pest or pathogen; non-target hazards associated directly or indirectly with the transgenic plant or transgene product outside the plant; and those associated with the integration and subsequent expression of the transgene in a different organism or species following gene flow. The consequences of likely exposure to these and other hazards are considered in this introduction to the main issues raised when evaluating the possible risks arising from the importation or cultivation of genetically modified crops.

Genetics of ferulic acid bioconversion to protocatechuic acid in plant-growth-promoting Pseudomonas putida WCS358.

Transposon Tn5 genomic mutants of plant-growth-promoting Pseudomonas putida strain WCS358 have been isolated which no longer utilize ferulic 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 at 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 (34.3 kDa), encoded by two genes, vanA and vanB, 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.

Involvement of a quorum-sensing-regulated lipase secreted by a clinical isolate of Burkholderia glumae in severe disease symptoms in rice.

ABSTRACT 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.

The acetyl xylan esterase of Bacillus pumilus belongs to a family of esterases with broad substrate specificity.

The Bacillus pumilus gene encoding acetyl xylan esterase (axe) was identified and characterized. The axe gene was expressed and the recombinant enzyme produced in Escherichia coli was purified and characterized. The recombinant enzyme displayed similar properties to the acetyl xylan esterase (AXE) purified from B. pumilus. The AXE primary structure was 76% identical to the cephalosporin C deacetylase of B. subtilis, and 40% to two recently identified AXEs from Thermoanaerobacterium and Thermotoga maritima. These four proteins are of similar size and represent a new family of esterases having a broad substrate specificity. The recombinant AXE was demonstrated to have activity on several acetylated substrates, including on cephalosporin C.

DEGRADATION OF TRANS-FERULIC AND P-COUMARIC ACID BY ACINETOBACTER CALCOACETICUS DSM 586

Cell suspensions of Acinetobacter calcoaceticus strain DSM 586 and DSM 590 were able to grow on benzoic, p-hydroxybenzoic and vanillic acid as sole carbon source. Testing the utilization of trans-ferulic and p-coumaric acid, we found that the sole A. calcoaceticus DSM 586 efficiently degraded the lignocellulose related monomers. Cells induced with trans-ferulic acid were able to oxidize trans-ferulic, p-coumaric, vanillic, p-hydroxybenzoic and protocatechuic acid at rates higher than the uninduced culture. The same activity was found in the p-coumaric acid induced culture. Two aromatic compounds, vanillic and p-hydroxybenzoic acid, were isolated from culture filtrates of trans-ferulic and p-coumaric acid grown cells, respectively, and further characterized by high performance liquid chromatography. 1H- and 13C-nuclear magnetic resonance and ultraviolet spectrophotometry. Cell extracts of trans-ferulic or p-coumaric acid induced cultures were shown to rapidly convert protocatechuic acid to beta-carboxymuconic acid. Moreover, A. calcoaceticus DSM 586 produced high levels of protocatechuic 3,4-dioxygenase compared to cathecol 1,2-dioxygenase and gentisate 1,2-dioxygenase in the degradation of trans-ferulic or p-coumaric acid. Based upon these results, a reaction sequence for the complete degradation of trans-ferulic and p-coumaric acid in A. calcoaceticus DSM 586 is proposed.

Cloning and characterisation of the rpoS gene from plant growth-promoting Pseudomonas putida WCS358: RpoS is not involved in siderophore and homoserine lactone production

The rpoS gene which encodes a stationary phase sigma factor has been identified and characterised from the rhizosphere-colonising plant growth-promoting Pseudomonas putida strain WCS358. The predicted protein sequence has extensive homologies with the RpoS proteins form other bacteria, in particular with the RpoS sigma factors of the fluorescent pseudomonads. A genomic transposon insertion in the rpoS gene was constructed, these mutants were analysed for their ability to produce siderophore (iron-transport agent) and the autoinducer quorum-sensing molecules called homoserine lactones (AHL). It was determined that RpoS was not involved in the regulation of siderophore and AHL production, synthesis of these molecules is important for gene expression at stationary phase. P. putida WCS358 produces at least three different AHL molecules.

A thermostable α-arabinofuranosidase from xylanolytic Bacillus pumilus: purification and characterisation

Bacillus pumilus PS213 secretes an alpha-L-arabinofuranosidase (AF) when grown in the presence of arabinogalactan or oat meal. The enzyme has been purified to homogeneity and characterised. Its molecular mass, as determined by gel filtration, is 220 kDa, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band of approximately 60 kDa. According to the result of the mass spectrometry analysis showing a molecular mass of 56 kDa, the enzyme should be a homotetramer. The isoelectric point was found to be 5.2, the enzyme activity was optimal at 55 degrees C and pH 7.0. The enzyme retained 80% of its activity after 2 h at 65 degrees C and lost 50% of activity at 75 degrees C after 135 min. The Michaelis constant K(m) and V(max) for p-nitrophenylarabinofuranoside at 37 degrees C were 1.7 mM and 52.9 U mg(-1), respectively. N-terminal sequence analysis and internal peptide fragments showed homology with glycosyl hydrolases of family 51.

A proteomic study of Xanthomonas oryzae pv. oryzae in rice xylem sap

Xanthomonas oryzae pv. oryzae (Xoo) is the second most important rice pathogen, causing a disease called bacterial leaf blight. Xoo colonizes and infects the vascular tissue resulting in tissue necrosis and wilting causing significant yield losses worldwide. In this study Xoo infected vascular fluid (xylem sap) was recovered and analyzed for secreted Xoo proteins. Three independent experiments resulted in the identification of 324 different proteins, 64 proteins were found in all three samples which included many of the known virulence-associated factors. In addition, 10 genes encoding for the identified proteins were inactivated and one mutant displayed statistically a significant loss in virulence when compared to the wild type Xoo, suggesting that a new virulence-associated factor has been revealed. The usefulness of this approach in understanding the lifestyle and unraveling the virulence-associated factors of phytopathogenic vascular bacteria is discussed.

Bacterial LuxR solos have evolved to respond to different molecules including signals from plants.

A future challenge will be understanding the extensive communication that most likely takes place in bacterial interspecies and interkingdom signaling between plants and bacteria. A major bacterial inter-cellular signaling system in Gram-negative bacteria is LuxI/R quorum sensing (QS) based on the production (via the LuxI-family proteins) and detection (via the LuxR-family proteins) of N-acyl homoserine lactones (AHLs) signaling molecules. LuxR proteins which have the same modular structure of QS LuxRs but are devoid of a cognate LuxI AHL synthase are called solos. LuxR solos have been shown to be responsible to respond to exogenous AHLs produced by neighboring cells as well endogenously produced AHLs. It is now also evident that some LuxR proteins have evolved from the ability to binding AHLs and respond to other molecules/signals. For example, recent research has shown that a sub-family of LuxR solos responds to small molecules produced by plants. This indicates the presence of a uni-directional interkingdom signaling system occurring from plants to bacteria. In addition LuxR solos have now been also implicated to respond to endogenously produced signals which are not AHLs. In this Mini Review article we will discuss current trends and implications of the role of LuxR solos in bacterial responses to other signals using proteins related to AHL QS systems.