Acute and subacute toxicities of biogenic tellurium nanorods in mice.

The current study was performed to evaluate the acute and subacute toxicities of biogenic tellurium nanorods (Te NRs). The Te NRs were prepared using Pseudomonas pseudoalcaligenes strain Te in a culture medium containing K2TeO3 (1 mM) and their physiochemical properties were investigated using TEM, EDX and XRD. The median lethal dose (LD50) of Te NRs and potassium tellurite (K2TeO3) were determined in mice and the subacute toxicity was also evaluated. The experimental design involved certain general toxicological, haematological, serum and histopathological investigations. The TEM and XRD analyses showed that the biogenic nanoparticles were rod-shaped and hexagonal. The toxicological evaluation showed that the LD50 values of Te NRs and K2TeO3 were 60 and 12.5 mg/kg, respectively. Higher doses of Te NRs (6 mg/kg) and K2TeO3 (1.25 mg/kg) were accompanied by signs of toxicity, including lower body weight, elevation in MDA and depletion in GSH content, SOD and CAT activity, and changes in biochemistry parameters. No obvious histopathological changes were observed in the treatment with Te NRs. In conclusion, the biogenic Te NRs were less toxic as compared to K2TeO3, and the no-observed-adverse-effect level (NOAEL) dose of Te NRs in 14 days subacute toxicity study was lower than 1.2 mg/kg.

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.

Metabolomic investigation of the bacterial response to a metal challenge.

Pseudomonas pseudoalcaligenes KF707 is naturally resistant to the toxic metalloid tellurite, but the mechanisms of resistance are not known. In this study we report the isolation of a KF707 mutant (T5) with hyperresistance to tellurite. In order to characterize the bacterial response and the pathways leading to tolerance, we utilized Phenotype MicroArray technology (Biolog) and a metabolomic technique based on nuclear magnetic resonance spectroscopy. The physiological states of KF707 wild-type and T5 cells exposed to tellurite were also compared in terms of viability and reduced thiol content. Our analyses showed an extensive change in metabolism upon the addition of tellurite to KF707 cultures as well as different responses when the wild-type and T5 strains were compared. Even in the absence of tellurite, T5 cells displayed a "poised" physiological status, primed for tellurite exposure and characterized by altered intracellular levels of glutathione, branched-chain amino acids, and betaine, along with increased resistance to other toxic metals and metabolic inhibitors. We conclude that hyperresistance to tellurite in P. pseudoalcaligenes KF707 is correlated with the induction of the oxidative stress response, resistance to membrane perturbation, and reconfiguration of cellular metabolism.

[Transformation of core Pseudomonas pseudoalcaligene insecticidal protein gene and its insecticidal expression in tobacco].

OBJECTIVE: We studied the effect of the signal peptide sequence (SPS) on the expression of Pseudomonas pseudoalcaligenes insecticidal protein gene (ppip). METHODS: We obtained the core pseudomonas pseudoalcaligenes insecticidal protein gene (cppip, ppip without the UTR and SPS) by PCR and ligated it into pCAMBIA2301 to generate plant express vector pCPPIP, which was then transformed into tobacco to investigate the insecticidal activity of cppip expression products by locust bioassays. The Kanamycin resistance segregation ratio was determined by the germination rate of T0-generation seeds of the transgenic tobacco. Integration of ppip into genomic DNA was detected by PCR and confirmed by Southern blotting. RESULTS: The bioassay with the 2nd and 3rd instar larvae of Locusta orthoptera showed that the crude proteins extracted from cppip transformed plants caused an average mortality of 83.37%. In contrast, the protein extracts from ppip transformed plants caused a much lower mortality (15.65%). The growth of locust was highly inhibited by the expression products of cppip when compared with the locusts fed with the protein extracts from wild type tobacco or tobacco transformed with intact ppip gene. CONCLUSION: The results indicated that the SPS might affect the insecticidal activity of ppip expressed in plants. The data of this study are helpful for cost-effective genetic engineering of plants with ppip gene.

Cyanide metabolism of Pseudomonas pseudoalcaligenes CECT5344: role of siderophores.

Cyanide is one of the most potent and toxic chemicals produced by industry. The jewelry industry of Córdoba (Spain) generates a wastewater (residue) that contains free cyanide, as well as large amounts of cyano-metal complexes. Cyanide is highly toxic to living systems because it forms very stable complexes with transition metals that are essential for protein function. In spite of its extreme toxicity, some organisms have acquired mechanisms to avoid cyanide poisoning. The biological assimilation of cyanide needs the concurrence of three separate processes: (i) a cyanide-insensitive respiratory chain, (ii) a system for iron acquisition (siderophores) and (iii) a cyanide assimilation pathway. Siderophores are low-molecular-mass compounds (600-1500 Da) that scavenge iron (Fe(3+)) ions (usually with extremely high affinity) from the environment under iron-limiting conditions. There are two main classes of siderophores: catechol and hydroxamate types. The catechol-type siderophores chelate ferric ion via a hydroxy group, whereas the hydroxamate-type siderophores bind iron via a carbonyl group with the adjacent nitrogen. In the presence of cyanide, bacterial proliferation requires this specific metal uptake system because siderophores are able to break down cyano-metal complexes. Pseudomonas pseudoalcaligenes CECT5344 is able to use free cyanide or cyano-metal complexes as nitrogen source. A proteomic approach was used for the isolation and identification, in this strain, of a protein that was induced in the presence of cyanide, namely CN0, that is involved in siderophore biosynthesis in response to cyanide. An overview of bacterial cyanide degradation pathways and the involvement of siderophores in this process are presented.

[N-terminal analysis and antibody preparation of insecticidal protein form Pseudomonas pseudoalcaligenes].

The insecticidal protein from Pseudomonas pseudoaligenes was and exotoxin which had toxicity on locusts. In order to elucidate its molecular properties an amino acid sequence, the insecticidal protein was purified from the culture supernatant by ultrafiltration, ion-exchange chromatography and gel filtration, and showed a single band on SDS-PAGE. Analysis of the purified insecticidal protein dentified N-terminal sequence of ten amino acid residues. Its polyclonal antibody was also obtained by immunizing rabbit with the insecticidal protein recovered form SDS-PAGE gel. The antibody titer determined by ELISA method was 1:12,800, indicating that it has high reactivity. Western blot analysis revealed that the antibody was spectific to 26 kD insecticidal protein, and did not cross-react with other proteins produced by the bacterium, suggesting that a specific antibody with high titer was obtained and could be used for further investigations of the gene cloning and expression of insecticidal protein.