Biophysical control of the growth of Agrobacterium tumefaciens using extremely low frequency electromagnetic waves at resonance frequency.

Isolated Agrobacterium tumefaciens was exposed to different extremely low frequencies of square amplitude modulated waves (QAMW) from two generators to determine the resonance frequency that causes growth inhibition. The carrier was 10 MHz sine wave with amplitude ±10 Vpp which was modulated by a second wave generator with a modulation depth of ± 2Vpp and constant field strength of 200 V/m at 28 °C. The exposure of A. tumefaciens to 1.0 Hz QAMW for 90 min inhibited the bacterial growth by 49.2%. In addition, the tested antibiotics became more effective against A. tumefaciens after the exposure. Furthermore, results of DNA, dielectric relaxation and TEM showed highly significant molecular and morphological changes due to the exposure to 1.0 Hz QAMW for 90 min. An in-vivo study has been carried out on healthy tomato plants to test the pathogenicity of A. tumefaciens before and after the exposure to QAMW at the inhibiting frequency. Symptoms of crown gall and all pathological symptoms were more aggressive in tomato plants treated with non-exposed bacteria, comparing with those treated with exposed bacteria. We concluded that, the exposure of A. tumefaciens to 1.0 Hz QAMW for 90 min modified its cellular activity and DNA structure, which inhibited the growth and affected the microbe pathogenicity.

Rhizobium radiobacter en absceso pulmonar asociado a neumonía necrotizante postgripal / Rhizobium radiobacter in pulmonary abscess associated with postgripal necrotizing pneumonia

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Temperature effects on bacterial phytochrome.

Bacteriophytochromes (BphPs) are light-sensing regulatory proteins encoded in photosynthetic and non-photosynthetic bacteria. This protein class incorporate bilin as their chromophore, with majority of them bearing a light- regulated His kinase or His kinase related module in the C-terminal. We studied the His kinase actives in the temperature range of 5°C to 40°C on two BphPs, Agp1 from Agrobacterium tumefaciens and Cph1 from cyanobacterium Synechocystis PCC 6803. As reported, the phosphorylation activities of the far red (FR) irradiated form of the holoprotein is stronger than that of the red (R) irradiated form in both phytochromes. We observed for the apoprotein and FR irradiated holoprotein of Agp1 an increase in the phosphorylation activities from 5°C to 25°C and a decrease from 25°C to 40°C. At 5°C the activities of the apoprotein were significantly lower than those of the FR irradiated holoprotein, which was opposite at 40°C. A similar temperature pattern was observed for Cph1, but the maximum of the apoprotein was at 20°C while the maximum of the FR irradiated holoprotein was at 10°C. At 40°C, prolonged R irradiation leads to an irreversible bleaching of Cph1, an effect which depends on the C-terminal His kinase module. A more prominent and reversible temperature effect on spectral properties of Agp1, mediated by the His kinase, has been reported before. His kinases in phytochromes could therefore share similar temperature characteristics. We also found that phytochrome B mutants of Arabidopsis have reduced hypocotyl growth at 37°C in darkness, suggesting that this phytochrome senses the temperature or mediates signal transduction of temperature effects.

Shape-dependent bactericidal activity of TiO2 for the killing of Gram-negative bacteria Agrobacterium tumefaciens under UV torch irradiation.

This paper demonstrated the relative bactericidal activity of photoirradiated (6W-UV Torch, λ > 340 nm and intensity = 0.64 mW/cm(2)) P25-TiO2 nanoparticles, nanorods, and nanotubes for the killing of Gram-negative bacterium Agrobacterium tumefaciens LBA4404 for the first time. TiO2 nanorod (anatase) with length of 70-100 nm and diameter of 10-12 nm, and TiO2 nanotube with length of 90-110 nm and diameter of 9-11 nm were prepared from P-25 Degussa TiO2 (size, 30-50 nm) by hydrothermal method and compared their biocidal activity both in aqueous slurry and thin films. The mode of bacterial cell decomposition was analyzed through transmission electron microscopy (TEM), Fourier transform-infrared (FT-IR), and K(+) ion leakage. The antimicrobial activity of photoirradiated TiO2 of different shapes was found to be in the order P25-TiO2 > nanorod > nanotube which is reverse to their specific surface area as 54 < 79 < 176 m(2) g(-1), evidencing that the highest activity of P25-TiO2 nanoparticles is not due to surface area as their crystal structure and surface morphology are entirely different. TiO2 thin films always exhibited less photoactivity as compared to its aqueous suspension under similar conditions of cell viability test. The changes in the bacterial surface morphology by UV-irradiated P25-TiO2 nanoparticles was examined by TEM, oxidative degradation of cell components such as proteins, carbohydrates, phospholipids, nucleic acids by FT-IR spectral analysis, and K(+) ion leakage (2.5 ppm as compared to 0.4 ppm for control culture) as a measure of loss in cell membrane permeability.

The tomato UV-damaged DNA-binding protein-1 (DDB1) is implicated in pathogenesis-related (PR) gene expression and resistance to Agrobacterium tumefaciens.

Plants defend themselves against potential pathogens via the recognition of pathogen-associated molecular patterns (PAMPs). However, the molecular mechanisms underlying this PAMP-triggered immunity (PTI) are largely unknown. In this study, we show that tomato HP1/DDB1, coding for a key component of the CUL4-based ubiquitin E3 ligase complex, is required for resistance to Agrobacterium tumefaciens. We found that the DDB1-deficient mutant (high pigment-1, hp1) is susceptible to nontumorigenic A. tumefaciens. The efficiency of callus generation from the hp1 cotyledons was extremely low as a result of the necrosis caused by Agrobacterium infection. On infiltration of nontumorigenic A. tumefaciens into leaves, the hp1 mutant moderately supported Agrobacterium growth and developed disease symptoms, but the expression of the pathogenesis-related gene SlPR1a1 and several PTI marker genes was compromised at different levels. Moreover, exogenous application of salicylic acid (SA) triggered SlPR1a1 gene expression and enhanced resistance to A. tumefaciens in wild-type tomato plants, whereas these SA-regulated defence responses were abolished in hp1 mutant plants. Thus, HP1/DDB1 may function through interaction with the SA-regulated PTI pathway in resistance against Agrobacterium infection.

A photolyase-like protein from Agrobacterium tumefaciens with an iron-sulfur cluster.

Photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. While photolyases can repair UV-induced DNA lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. Here we report about two photolyase-related proteins, named PhrA and PhrB, found in the phytopathogen Agrobacterium tumefaciens. PhrA belongs to the class III cyclobutane pyrimidine dimer (CPD) photolyases, the sister class of plant cryptochromes, while PhrB belongs to a new class represented in at least 350 bacterial organisms. Both proteins contain flavin adenine dinucleotide (FAD) as a primary catalytic cofactor, which is photoreduceable by blue light. Spectral analysis of PhrA confirmed the presence of 5,10-methenyltetrahydrofolate (MTHF) as antenna cofactor. PhrB comprises also an additional chromophore, absorbing in the short wavelength region but its spectrum is distinct from known antenna cofactors in other photolyases. Homology modeling suggests that PhrB contains an Fe-S cluster as cofactor which was confirmed by elemental analysis and EPR spectroscopy. According to protein sequence alignments the classical tryptophan photoreduction pathway is present in PhrA but absent in PhrB. Although PhrB is clearly distinguished from other photolyases including PhrA it is, like PhrA, required for in vivo photoreactivation. Moreover, PhrA can repair UV-induced DNA lesions in vitro. Thus, A. tumefaciens contains two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF) that contains an iron-sulfur cluster.

Spectral properties of phytochrome Agp2 from Agrobacterium tumefaciens are specifically modified by a compound of the cell extract.

Phytochromes are widely distributed photoreceptors that are converted by light between the red absorbing Pr and the far-red absorbing Pfr form. The soil bacterium Agrobacterium tumefaciens contains two phytochromes, Agp1 and Agp2, which act as light-regulated histidine kinases. Whereas most phytochromes are stable in the Pr form, Agp2 and few other phytochromes convert into Pfr in darkness. We have shown in a previous publication that the spectral properties of recombinant Agp2 are modified by compounds of the cell extract from an Agrobacterium agp1(-)/agp2(-) double knockout mutant. In the present work we performed concentration series which show that the interaction is specific and that the modifying factor has a concentration of ca. 0.2 microM. We have also performed a series of mixing experiments with the truncated protein Agp2-M2, which consists of the N-terminal chromophore module (501 amino acids). The cell extract inhibited the photoconversion of Agp2-M2 in an unspecific way. In concentration series, this negative effect was less pronounced when lower concentrations of Agp2-M2 were used. In the presence of excess Agp2-M2 apoprotein, the cell extract did no longer modify the spectral properties of Agp2. The data suggest that the factor of the cell extract interacts specifically with the N-terminal moiety of Agp2.

A novel approach for improving the productivity of ubiquinone-10 producing strain by low-energy ion beam irradiation.

Agrobacterium tumefaciens ATCC4452 cells were irradiated by nitrogen ion beam, a new mutagen, with energy of 10 keV and fluence ranging from 2.6x10(14) ions/cm2 to 6.5x10(15) ions/cm2. A similar "saddle shape" survival curve due to ion beam irradiation appeared again in this study. Some mutants with high yield of ubiquinone-10 were induced by ion implantation. High mutation rate and wide mutation spectrum were also observed in the experiment. These results suggested that the mutagenic effect of such low-energy ion influx into bacterium cells could result from multiple processes involving direct collision of particles with cytoplasm, nucleolus, and cascade atomic and molecular reactions due to plentiful primary and secondary particles.

Light strongly promotes gene transfer from Agrobacterium tumefaciens to plant cells.

Light conditions during Agrobacterium-based plant transformation, the most routinely used method in plant genetic engineering, differ widely and, to our knowledge, have not been studied systematically in relation to transformation efficiency. Here, light effects were examined in two already optimized transformation procedures: coculture of Agrobacterium tumefaciens with callus from two genotypes of the crop plant Phaseolus acutifolius (tepary bean) and coculture of root segments from two ecotypes of Arabidopsis thaliana. Except for the light conditions during coculture, all steps followed established procedures. Coculture was done either under continuous darkness, under a commonly used photoperiod of 16 h light/8 h darkness or under continuous light. beta-glucuronidase (GUS) production due to the transient expression of an intron-containing uidA gene in the binary vector was used to evaluate T-DNA transfer. In all situations, uidA expression correlated highly and positively with the light period used during coculture; it was inhibited severely by darkness and enhanced more under continuous light than under a 16 h light/8 h dark photoperiod. The promotive effect of light was observed with Agrobacterium strains harboring either a nopaline-, an octopine- or an agropine/succinamopine-type non-oncogenic helper Ti plasmid. The observed positive effect of light has obvious implications for developing and improving transient and stable transformation protocols, specifically those involving dark coculture conditions.

Characterization of a virG mutation that confers constitutive virulence gene expression in Agrobacterium.

Transformation of plants by Agrobacterium tumefaciens is mediated by a set of virulence (vir) genes that are specifically induced by plant signal molecules through the VirA/VirG two-component regulatory system. The plant signal is transmitted from VirA to VirG by a cascade of phosphorylation reactions followed by the sequence-specific DNA binding of the VirG protein to the vir gene promoters which then activates their transcription. In this report, we describe a VirG mutant which is able to activate vir gene expression independently of the VirA molecule and the two plant signal molecules, acetosyringone and monosaccharides. A strain of Agrobacterium containing this virG gene but lacking a functional virA gene was able to induce tumours on all three plants that were tested. A single amino acid change of asparagine (N) to aspartate (D) at position 54, adjacent to the site of VirG phosphorylation, aspartate 52, resulted in this constitutive phenotype. In vitro phosphorylation experiments showed that the mutant protein cannot be phosphorylated by VirA, suggesting that the negative charge resulting from the N to D switch mimics the phosphorylated conformation of the VirG molecule. The same amino acid change in the virG gene of the supervirulent strain A281 also resulted in a constitutive phenotype. However, the vir genes were not induced to high levels when compared with the levels of the constitutive virG of strain A348.