Phylogenetic characterization of the energy taxis receptor Aer in Pseudomonas and phenotypic characterization in Pseudomonas pseudoalcaligenes KF707.

Chemotaxis allows bacteria to sense gradients in their environment and respond by directing their swimming. Aer is a receptor that, instead of responding to a specific chemoattractant, allows bacteria to sense cellular energy levels and move towards favourable environments. In Pseudomonas, the number of apparent Aer homologues differs between the only two species it has been characterized in, Pseudomonas aeruginosa and Pseudomonas putida. Here we combined bioinformatic approaches with deletional mutagenesis in Pseudomonas pseudoalcaligenes KF707 to further characterize Aer. It was determined that the number of Aer homologues varies between zero and four throughout the genus Pseudomonas, and they were phylogenetically classified into five subgroups. We also used sequence analysis to show that these homologous receptors differ in their HAMP signal transduction domains. Genetic analysis also indicated that some Aer homologues have likely been subject to horizontal transfer. P. pseudoalcaligenes KF707 was unique among strains for having three Aer homologues as well as the receptors CttP and McpB. Phenotypic characterization in this strain showed that the most prevalent homologue of Aer was key, but not essential, for energy taxis. This study demonstrates that energy taxis in Pseudomonas varies between species and provides a new naming convention and associated phylogenetic details for Aer chemoreceptors.

Response of the Biocontrol Agent Pseudomonas pseudoalcaligenes AVO110 to Rosellinia necatrix Exudate.

The rhizobacterium Pseudomonas pseudoalcaligenes AVO110, isolated by the enrichment of competitive avocado root tip colonizers, controls avocado white root rot disease caused by Rosellinia necatrix Here, we applied signature-tagged mutagenesis (STM) during the growth and survival of AVO110 in fungal exudate-containing medium with the goal of identifying the molecular mechanisms linked to the interaction of this bacterium with R. necatrix A total of 26 STM mutants outcompeted by the parental strain in fungal exudate, but not in rich medium, were selected and named growth-attenuated mutants (GAMs). Twenty-one genes were identified as being required for this bacterial-fungal interaction, including membrane transporters, transcriptional regulators, and genes related to the metabolism of hydrocarbons, amino acids, fatty acids, and aromatic compounds. The bacterial traits identified here that are involved in the colonization of fungal hyphae include proteins involved in membrane maintenance (a dynamin-like protein and ColS) or cyclic-di-GMP signaling and chemotaxis. In addition, genes encoding a DNA helicase (recB) and a regulator of alginate production (algQ) were identified as being required for efficient colonization of the avocado rhizosphere.IMPORTANCE Diseases associated with fungal root invasion cause a significant loss of fruit tree production worldwide. The bacterium Pseudomonas pseudoalcaligenes AVO110 controls avocado white root rot disease caused by Rosellinia necatrix by using mechanisms involving competition for nutrients and niches. Here, a functional genomics approach was conducted to identify the bacterial traits involved in the interaction with this fungal pathogen. Our results contribute to a better understanding of the multitrophic interactions established among bacterial biocontrol agents, the plant rhizosphere, and the mycelia of soilborne pathogens.

Interaction of Pb(II) and biofilm associated extracellular polymeric substances of a marine bacterium Pseudomonas pseudoalcaligenes NP103.

Three-dimensional excitation-emission matrix (3D EEM) fluorescence spectroscopy and attenuated total reflectance fourier-transformed infrared spectroscopy (ATR-FTIR) was used to evaluate the interaction of biofilm associated extracellular polymeric substances (EPS) of a marine bacterium Pseudomonas pseudoalcaligenes NP103 with lead [Pb(II)]. EEM fluorescence spectroscopic analysis revealed the presence of one protein-like fluorophore in the EPS of P. pseudoalcaligenes NP103. Stern-Volmer equation indicated the existence of only one binding site (n=0.789) in the EPS of P. pseudoalcaligenes NP103. The interaction of Pb(II) with EPS was spontaneous at room temperature (∆G=-2.78kJ/K/mol) having binding constant (Kb) of 2.59M-1. ATR-FTIR analysis asserted the involvement of various functional groups such as sulphydryl, phosphate and hydroxyl and amide groups of protein in Pb(II) binding. Scanning electron microscopy (SEM) and fluorescence microscopy analysis displayed reduced growth of biofilm with altered surface topology in Pb(II) supplemented medium. Energy dispersive X-ray spectroscopy (EDX) analysis revealed the entrapment of Pb in the EPS. Uronic acid, a characteristic functional group of biofilm, was observed in 1H NMR spectroscopy. The findings suggest that biofilm associated EPS are perfect organic ligands for Pb(II) complexation and may significantly augment the bioavailability of Pb(II) in the metal contaminated environment for subsequent sequestration.

The Role of cheA Genes in Swarming and Swimming Motility of Pseudomonas pseudoalcaligenes KF707.

A genome analysis of Pseudomonas pseudoalcaligenes KF707, a PCBs degrader and metal-resistant soil microorganism, revealed the presence of two novel gene clusters named che2 and che3, which were predicted to be involved in chemotaxis-like pathways, in addition to a che1 gene cluster. We herein report that the histidine kinase coding genes, cheA2 and cheA3, have no role in swimming or chemotaxis in P. pseudoalcaligenes KF707, in contrast to cheA1. However, the cheA1 and cheA2 genes were both necessary for cell swarming, whereas the cheA3 gene product had a negative effect on the optimal swarming phenotype of KF707 cells.

DNA microarray analysis of the cyanotroph Pseudomonas pseudoalcaligenes CECT5344 in response to nitrogen starvation, cyanide and a jewelry wastewater.

Pseudomonas pseudoalcaligenes CECT5344 is an alkaliphilic bacterium that can use cyanide as nitrogen source for growth, becoming a suitable candidate to be applied in biological treatment of cyanide-containing wastewaters. The assessment of the whole genome sequence of the strain CECT5344 has allowed the generation of DNA microarrays to analyze the response to different nitrogen sources. The mRNA of P. pseudoalcaligenes CECT5344 cells grown under nitrogen limiting conditions showed considerable changes when compared against the transcripts from cells grown with ammonium; up-regulated genes were, among others, the glnK gene encoding the nitrogen regulatory protein PII, the two-component ntrBC system involved in global nitrogen regulation, and the ammonium transporter-encoding amtB gene. The protein coding transcripts of P. pseudoalcaligenes CECT5344 cells grown with sodium cyanide or an industrial jewelry wastewater that contains high concentration of cyanide and metals like iron, copper and zinc, were also compared against the transcripts of cells grown with ammonium as nitrogen source. This analysis revealed the induction by cyanide and the cyanide-rich wastewater of four nitrilase-encoding genes, including the nitC gene that is essential for cyanide assimilation, the cyanase cynS gene involved in cyanate assimilation, the cioAB genes required for the cyanide-insensitive respiration, and the ahpC gene coding for an alkyl-hydroperoxide reductase that could be related with iron homeostasis and oxidative stress. The nitC and cynS genes were also induced in cells grown under nitrogen starvation conditions. In cells grown with the jewelry wastewater, a malate quinone:oxidoreductase mqoB gene and several genes coding for metal extrusion systems were specifically induced.

Microcystis aeruginosa/Pseudomonas pseudoalcaligenes interaction effects on off-flavors in algae/bacteria co-culture system under different temperatures.

We conducted an experiment to study the interaction effects of Microcystis aeruginosa and Pseudomonas pseudoalcaligenes on off-flavors in an algae/bacteria co-culture system at three temperatures (24, 28 and 32°C). Gas chromatography-mass spectrometry was applied to measure off-flavor compounds dimethyl sulfide (DMS), dimethyl trisulfide (DMTS), 2-methylisoborneol, geosmin (GEO) and ß-cyclocitral. During the lag phase of co-cultured M. aeruginosa (first 15days), P. pseudoalcaligenes significantly increased the production of DMS, DMTS and ß-cyclocitral at all three temperatures. In the exponential phase of co-cultured M. aeruginosa (after 15days), M. aeruginosa became the main factor on off-flavors in the co-culture system, and ß-cyclocitral turned to the highest off-flavor compound. These results also indicated that DMS, DMTS and ß-cyclocitral were the main off-flavor compounds in our M. aeruginosa/P. pseudoalcaligenes co-culture system. Univariate analysis was applied to investigate the effects of M. aeruginosa and P. pseudoalcaligenes on the production of off-flavors. The results demonstrated that both M. aeruginosa and P. pseudoalcaligenes could increase the production of DMS and DMTS, while ß-cyclocitral was mainly determined by M. aeruginosa. Our results also provide some insights into understanding the relationship between cyanobacteria and heterotrophic bacteria.

A hydroponic system for growing gnotobiotic vs. sterile plants to study phytoremediation processes.

In some phytoremediation studies it is desirable to separate and define the specific contribution of plants and root-colonizing bacteria towards contaminant removal. Separating the influence of plants and associated bacteria is a difficult task for soil root environments. Growing plants hydroponically provides more control over the biological factors in contaminant removal. In this study, a hydroponic system was designed to evaluate the role of sterile plant roots, rhizodeposition, and root-associated bacteria in the removal of a model contaminant, phenol. A strain of Pseudomonas pseudoalcaligenes that grows on phenol was inoculated onto plant roots. The introduced biofilm persisted in the root zone and promoted phenol removal over non-augmented controls. These findings indicate that this hydroponic system can be a valuable tool for phytoremediation studies that investigate the effects of biotic and abiotic factors on pollution remediation.

Potential application of algicidal bacteria for improved lipid recovery with specific algae.

The utility of specific strains of natural algicidal bacteria isolated from shallow wetland sediments was evaluated against several strains of algae with potential immediate or future commercial value. Two strains of bacteria, Pseudomonas pseudoalcaligenes AD6 and Aeromonas hydrophila AD9, were identified and demonstrated to have algicidal activity against the microalgae Neochloris oleoabundans and Dunaliella tertiolecta. These bacteria were further evaluated for the potential to improve lipid extraction using a mild solvent extraction approach. Aeromonas hydrophila AD9 showed a nearly 12-fold increase in lipid extraction with D. tertiolecta, while both bacteria showed a sixfold improvement in lipid extraction with N. oleoabundans.

[Monosaccharide and fatty acid composition of exopolymer complex of bacteria-destructors of the protective coating of gas pipeline].

Monosaccharide and fatty acid composition of the exopolymer complex (EPC) of heterotrophic bacteria Pseudomonas pseudoalkaligenes 109, Pseudomonas sp. T/2, Rhodococcus erythropolis 102--destructors of the protective coating Polyken 980-25 has been studied. It is shown that qualitative and quantitative composition of EPC components changes depending on the model of bacteria growth. Arabinose, mannose, galactose and glucose are dominating saccharides. Xylose has been revealed only under conditions of the biofilm form of growth of all the studied bacteria; ribose only in the biofilm of Pseudomonas sp. T/2. The fatty acid composition of EPC contains saturated and unsaturated acids with 12-19 carbon atoms. Hexadecanoic acid (C 16:0) acid which content in the biofilm and plankton conditions is from 24.9 to 32.4% prevailed in the spectrum of fatty acids of EPC bacteria. Unsaturated fatty acids: hexadecanoic (C 16:1) and octadecenoic (C 18:1) ones have been revealed only in the biofilm of bacteria-destructors of the coating.

Two similar enhanced root-colonizing Pseudomonas strains differ largely in their colonization strategies of avocado roots and Rosellinia necatrix hyphae.

Pseudomonas alcaligenes AVO73 and Pseudomonas pseudoalcaligenes AVO110 were selected previously as efficient avocado root tip colonizers, displaying in vitro antagonism towards Rosellinia necatrix, causal agent of avocado white root rot. Despite the higher number of antagonistic properties shown in vitro by AVO73, only AVO110 demonstrated significant protection against avocado white root rot. As both strains are enhanced root colonizers, and as colonization is crucial for the most likely biocontrol mechanisms used by these strains, namely production of non-antibiotic antifungal compounds and competition for nutrients and niches, we decided to compare the interactions of the bacterial strains with avocado roots as well as with R. necatrix hyphae. The results indicate that strain AVO110 is superior in biocontrol trait swimming motility and establishes on the root tip of avocado plants faster than AVO73. Visualization studies, using Gfp-labelled derivatives of these strains, showed that AVO110, in contrast to AVO73, colonizes intercellular crevices between neighbouring plant root epidermal cells, a microhabitat of enhanced exudation. Moreover, AVO110, but not AVO73, also colonizes root wounds, described to be preferential penetration sites for R. necatrix infection. This result strongly suggests that AVO110 meets, and can attack, the pathogen on the root. Finally, when co-inoculated with the pathogen, AVO110 utilizes hyphal exudates more efficiently for proliferation than AVO73 does, and colonizes the hyphae more abundantly than AVO73. We conclude that the differences between the strains in colonization levels and strategies are likely to contribute to, and even can explain, the difference in disease-controlling abilities between the strains. This is the first report that shows that two similar bacterial strains, selected by their ability to colonize avocado root, use strongly different root colonization strategies and suggests that in addition to the total bacterial root colonization level, the sites occupied on the root are important for biocontrol.

The cyanotrophic bacterium Pseudomonas pseudoalcaligenes CECT5344 responds to cyanide by defence mechanisms against iron deprivation, oxidative damage and nitrogen stress.

Two-dimensional (2-D) electrophoresis approach has been used to test protein expression changes in response to cyanide in the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344. This is a cyanide-assimilating strain which also grows in media containing cyanide-enriched effluent from the jewellery industry. The bacterium efficiently uses this residue as the sole nitrogen source for aerobic growth under alkaline pH with negligible nitrogen losses as HCN. Cell-free extracts isolated from P. pseudoalcaligenes grown with a jewellery residue, free cyanide or ammonium chloride as nitrogen source were subjected to 2-D electrophoresis and the spot patterns were examined to determine differential protein expression. Electrophoretic plates exhibiting an average of 1000 spots showed significant differences in the expression of about 44 proteins depending on the nitrogen source. Some of these protein spots were analysed by Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Characterization of five of these proteins reveals that cyanide shock induces proteins related to iron acquisition, regulation of nitrogen assimilation pathways and oxidative stress repairing and protection.

Increased mesquite gum formation in nodal explants cultures after treatment with a microbial biomass preparation.

Prosopis laevigata nodal explants cultures were established in Murashige and Skoog medium. Simultaneously these cultures were subjected to stress with biotic elicitors and an environmental factor (temperature increase to promote heat stress) in order to promote and increase exuded mesquite gum production. The biotic elicitors were: Aspergillus nidulans and Pseudomonas pseudoalcaligenes both used in concentrations of 10, 20 and 30 mg, whereas the environmental condition was different incubation temperatures (25, 35 and 40 degrees C). The greatest gum production (approximately 13 mg of pooled gum from 100 explants after 14 days incubation) took place when the culture medium was added 10, 20 and 30 mg of autoclaved fungal mycelium of A. nidulans or 30 mg of autoclaved bacterial biomass of P. pseudoalcaligenes in combination with an incubation temperature of 35 degrees C. These treatments were non-significantly different among themselves (P < 0.05), but were significantly different to the rest of the treatments (P > 0.05).