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Current Protocols in Microbiology Feb 2012Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also...
Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens this is called the Ti (tumor-inducing) plasmid. During pathogenesis, virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool, and is also a model microorganism that has been well studied for host-microbe associations, horizontal gene transfer, cell-cell communication, and biofilm formation. This unit describes standard protocols for laboratory cultivation of A. tumefaciens.
Topics: Agrobacterium tumefaciens; Bacteriological Techniques; Maintenance; Plant Diseases; Plant Tumor-Inducing Plasmids; Plants; Virulence Factors
PubMed: 22307549
DOI: 10.1002/9780471729259.mc03d01s24 -
Phytopathology Oct 2015Agrobacterium species are soilborne gram-negative bacteria exhibiting predominantly a saprophytic lifestyle. Only a few of these species are capable of parasitic growth... (Review)
Review
Agrobacterium species are soilborne gram-negative bacteria exhibiting predominantly a saprophytic lifestyle. Only a few of these species are capable of parasitic growth on plants, causing either hairy root or crown gall diseases. The core of the infection strategy of pathogenic Agrobacteria is a genetic transformation of the host cell, via stable integration into the host genome of a DNA fragment called T-DNA. This genetic transformation results in oncogenic reprogramming of the host to the benefit of the pathogen. This unique ability of interkingdom DNA transfer was largely used as a tool for genetic engineering. Thus, the artificial host range of Agrobacterium is continuously expanding and includes plant and nonplant organisms. The increasing availability of genomic tools encouraged genome-wide surveys of T-DNA tagged libraries, and the pattern of T-DNA integration in eukaryotic genomes was studied. Therefore, data have been collected in numerous laboratories to attain a better understanding of T-DNA integration mechanisms and potential biases. This review focuses on the intranuclear mechanisms necessary for proper targeting and stable expression of Agrobacterium oncogenic T-DNA in the host cell. More specifically, the role of genome features and the putative involvement of host's transcriptional machinery in relation to the T-DNA integration and effects on gene expression are discussed. Also, the mechanisms underlying T-DNA integration into specific genome compartments is reviewed, and a theoretical model for T-DNA intranuclear targeting is presented.
Topics: Agrobacterium tumefaciens; Cell Nucleus; DNA, Bacterial; Gene Transfer Techniques; Host-Pathogen Interactions; Plant Diseases; Plants
PubMed: 26151736
DOI: 10.1094/PHYTO-12-14-0380-RVW -
Microbiology Spectrum Sep 2021Agrobacterium tumefaciens strain S33 can catabolize nicotine via a hybrid of the pyridine and pyrrolidine pathways. Most of the enzymes involved in this biochemical...
Agrobacterium tumefaciens strain S33 can catabolize nicotine via a hybrid of the pyridine and pyrrolidine pathways. Most of the enzymes involved in this biochemical pathway have been identified and characterized, except for the one catalyzing the oxidation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine. Based on a previous genomic and transcriptomic analysis, an open reading frame (ORF) annotated to encode aldehyde dehydrogenase (Ald) in the nicotine-degrading cluster was predicted to be responsible for this step. In this study, we heterologously expressed the enzyme and identified its function by biochemical assay and mass spectrum analysis. It was found that Ald catalyzes the NAD-specific dehydrogenation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine. With the nonhydroxylated analog 3-succinoyl-semialdehyde-pyridine (SAP) as a substrate, Ald had a specific activity of 10.05 U/mg at pH 9.0 and apparent values of around 58.68 μM and 0.41 mM for SAP and NAD, respectively. Induction at low temperature and purification and storage in low-salt buffers were helpful to prevent its aggregation and precipitation. Disruption of the gene caused a lower growth rate and biomass of strain S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine. Ald has a broad range of substrates, including benzaldehyde, furfural, and acetaldehyde. Recombinant Escherichia coli cells harboring the gene can efficiently convert furfural to 2-furoic acid at a specific rate of 0.032 mmol min g dry cells, extending the application of Ald in the catalysis of bio-based furan compounds. These findings provide new insights into the biochemical mechanism of the nicotine-degrading hybrid pathway and the possible application of Ald in industrial biocatalysis. Nicotine is one of the major toxic -heterocyclic aromatic alkaloids produced in tobacco plants. Manufacturing tobacco and smoking may lead to some environmental and public health problems. Microorganisms can degrade nicotine by various biochemical pathways, but the biochemical mechanism for nicotine degradation has not been fully elucidated. In this study, we identified an aldehyde dehydrogenase responsible for the oxidation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine; this was the only uncharacterized enzyme in the hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33. Similar to the known aldehyde dehydrogenase, the NAD-specific homodimeric enzyme presents a broad substrate range with high activity in alkaline and low-salt-containing buffers. It can catalyze not only the aldehyde from nicotine degradation but also those of benzaldehyde, furfural, and acetaldehyde. It was found that recombinant Escherichia coli cells harboring the gene could efficiently convert furfural to valuable 2-furoic acid, demonstrating its potential application for enzymatic catalysis.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Biodegradation, Environmental; Kinetics; NAD; Nicotine; Oxidoreductases; Pyridines; Substrate Specificity; Succinates
PubMed: 34378958
DOI: 10.1128/Spectrum.00924-21 -
International Journal of Molecular... Oct 2022The stable genetic transformation of soybean is time-consuming and inefficient. As a simple and practical alternative method, hairy root transformation mediated by is...
The stable genetic transformation of soybean is time-consuming and inefficient. As a simple and practical alternative method, hairy root transformation mediated by is widely applied in studying root-specific processes, nodulation, biochemical and molecular functions of genes of interest, gene editing efficiency of CRISPR/Cas9, and biological reactors and producers. Therefore, many laboratories have developed unique protocols to obtain hairy roots in composite plants composed of transgenic roots and wild-type shoots. However, these protocols still suffer from the shortcomings of low efficiency and time, space, and cost consumption. To address this issue, we developed a new protocol efficient regeneration and transformation of hairy roots (eR&T) in soybean, by integrating and optimizing the main current methods to achieve high efficiency in both hairy root regeneration and transformation within a shorter period and using less space. By this eR&T method, we obtained 100% regeneration of hairy roots for all explants, with an average 63.7% of transformation frequency, which promoted the simultaneous and comparative analysis of the function of several genes. The eR&T was experimentally verified reporters, protein subcellular localization, and CRISPR/Cas9 gene editing experiments. Employing this approach, we identified several novel potential regulators of nodulation, and nucleoporins of the Nup107-160 sub-complex, which showed development-dependent and tissue-dependent expression patterns, indicating their important roles in nodulation in soybean. Thus, the new eR&T method is an efficient and economical approach for investigating not only root and nodule biology, but also gene function.
Topics: Glycine max; Transformation, Genetic; Nuclear Pore Complex Proteins; Plants, Genetically Modified; Plant Roots; Agrobacterium; Biology
PubMed: 36293115
DOI: 10.3390/ijms232012261 -
PloS One 2017Curdlan is a linear polysaccharide considered a dietary fiber and with gelation properties. This study evaluated the structure, morphology and the physicochemical and...
Description of recovery method used for curdlan produced by Agrobacterium sp. IFO 13140 and its relation to the morphology and physicochemical and technological properties of the polysaccharide.
Curdlan is a linear polysaccharide considered a dietary fiber and with gelation properties. This study evaluated the structure, morphology and the physicochemical and technological properties of curdlan produced by Agrobacterium sp. IFO 13140 recovered by pre-gelation and precipitation methods. Commercial curdlan submitted or otherwise to the pre-gelation process was also evaluated. The data obtained from structural analysis revealed a similarity between the curdlan produced by Agrobacterium sp. IFO 13140 (recovered by both methods) and the commercial curdlans. The results showed that the curdlans evaluated differed significantly in terms of dispersibility and gelation, and only the pre-gelled ones had significant potential for food application, because this method influence on the size of the particles and in the presence of NaCl. In terms of technological properties, the curdlan produced by Agrobacterium sp. IFO 13140 (pre-gelation method) had a greater water and oil holding capacity (64% and 98% greater, respectively) and a greater thickening capacity than the pre-gelled commercial curdlan. The pre-gelled commercial curdlan displayed a greater gelling capacity at 95°C than the others. When applied to food, only the pre-gelled curdlans improved the texture parameters of yogurts and reduced syneresis. The curdlan gels, which are rigid and stable in structure, demonstrated potential for improving the texture of food products, with potential industrial use.
Topics: Agrobacterium; Culture Media; Dietary Carbohydrates; Food Technology; Gels; Polysaccharides, Bacterial; Rheology; Sodium; Solvents; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman; Temperature; Viscosity; Water; Yogurt; beta-Glucans
PubMed: 28245244
DOI: 10.1371/journal.pone.0171469 -
Applied and Environmental Microbiology Aug 2007In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur...
In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the fur mutant was more sensitive than the wild type to an iron chelator, 2,2'-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations. A. tumefaciens sitA, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (sitABCD), was strongly repressed by Mn(2+) and, to a lesser extent, by Fe(2+), and this regulation was Fur dependent. Moreover, the fur mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the fur mutant showed constitutive up-expression of the manganese uptake sit operon. Fur(At) showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The fur mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the fur mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4.
Topics: Agrobacterium tumefaciens; Amino Acid Sequence; Bacterial Proteins; Gene Expression Regulation, Bacterial; Heat-Shock Response; Homeostasis; Iron; Manganese; Molecular Sequence Data; Oxidative Stress; Plant Diseases; Repressor Proteins; Nicotiana; Virulence
PubMed: 17545320
DOI: 10.1128/AEM.00531-07 -
Applied and Environmental Microbiology Feb 2021GW4 is a heterotrophic arsenite-oxidizing bacterium with a high resistance to arsenic toxicity. It is now a model organism for studying the processes of arsenic...
GW4 is a heterotrophic arsenite-oxidizing bacterium with a high resistance to arsenic toxicity. It is now a model organism for studying the processes of arsenic detoxification and utilization. Previously, we demonstrated that under low-phosphate conditions, arsenate [As(V)] could enhance bacterial growth and be incorporated into biomolecules, including lipids. While the basic microbial As(V) resistance mechanisms have been characterized, global metabolic responses under low phosphate remain largely unknown. In the present work, the impacts of As(V) and low phosphate on intracellular metabolite and lipid profiles of GW4 were quantified using liquid chromatography-mass spectroscopy (LC-MS) in combination with transcriptional assays and the analysis of intracellular ATP and NADH levels. Metabolite profiling revealed that oxidative stress response pathways were altered and suggested an increase in DNA repair. Changes in metabolite levels in the tricarboxylic acid (TCA) cycle along with increased ATP are consistent with As(V)-enhanced growth of GW4. Lipidomics analysis revealed that most glycerophospholipids decreased in abundance when As(V) was available. However, several glycerolipid classes increased, an outcome that is consistent with maximizing growth via a phosphate-sparing phenotype. Differentially regulated lipids included phosphotidylcholine and lysophospholipids, which have not been previously reported in The metabolites and lipids identified in this study deepen our understanding of the interplay between phosphate and arsenate on chemical and metabolic levels. Arsenic is widespread in the environment and is one of the most ubiquitous environmental pollutants. Parodoxically, the growth of certain bacteria is enhanced by arsenic when phosphate is limited. Arsenate and phosphate are chemically similar, and this behavior is believed to represent a phosphate-sparing phenotype in which arsenate is used in place of phosphate in certain biomolecules. The research presented here uses a global approach to track metabolic changes in an environmentally relevant bacterium during exposure to arsenate when phosphate is low. Our findings are relevant for understanding the environmental fate of arsenic as well as how human-associated microbiomes respond to this common toxin.
Topics: Agrobacterium tumefaciens; Arsenates; Lipid Metabolism; Phosphates
PubMed: 33361371
DOI: 10.1128/AEM.02261-20 -
Phytopathology Sep 2017Agrobacterium is a genus of soilborne gram-negative bacteria. Members carrying oncogenic plasmids can cause crown gall disease, which has significant economic costs,...
Agrobacterium is a genus of soilborne gram-negative bacteria. Members carrying oncogenic plasmids can cause crown gall disease, which has significant economic costs, especially for the orchard and nursery industries. Early and rapid detection of pathogenic Agrobacterium spp. is key to the management of crown gall disease. To this end, we designed oligonucleotide primers and probes to target virD2 for use in a molecular diagnostic tool that relies on isothermal amplification and lateral-flow-based detection. The oligonucleotide tools were tested in the assay and evaluated for detection limit and specificity in detecting alleles of virD2. One set of primers that successfully amplified virD2 when used with an isothermal recombinase was selected. Both tested probes had detection limits in picogram amounts of DNA. Probe 1 could detect all tested pathogenic isolates that represented most of the diversity of virD2. Finally, the coupling of lateral-flow detection to the use of these oligonucleotide primers in isothermal amplification helped to reduce the onerousness of the process, and alleviated reliance on specialized tools necessary for molecular diagnostics. The assay is an advancement for the rapid molecular detection of pathogenic Agrobacterium spp.
Topics: Agrobacterium; DNA, Bacterial; Genome, Bacterial; Nucleic Acid Amplification Techniques; Phylogeny; Nicotiana
PubMed: 28569126
DOI: 10.1094/PHYTO-04-17-0144-R -
The Plant Cell Oct 2012Bacteria of the genus Agrobacterium are very useful and unusual plant pathogens. Through a rare inter-kingdom DNA transfer, the bacteria move some of their genes into...
Bacteria of the genus Agrobacterium are very useful and unusual plant pathogens. Through a rare inter-kingdom DNA transfer, the bacteria move some of their genes into their host's genome, thereby inducing the host cells to proliferate and produce opines, nutrients sources for the pathogen. Agrobacterium's ability to transfer DNA makes can be adapted to introduce other genes, such as those encoding useful traits, into plant genomes. The development of Agrobacterium as a tool to transform plants is a landmark event in modern plant biology. This lecture provides an introduction to Agrobacterium tumefaciens and related species, focusing on their modes of pathogenicity, their usefulness as tools for plant transformation, and their use as a model for the study of plant-pathogen interactions.
Topics: Agrobacterium tumefaciens; Botany; Genetic Engineering; Plant Diseases; Plants; Teaching Materials; Transformation, Genetic
PubMed: 23213133
DOI: 10.1105/tpc.112.tt1012 -
Molecular Membrane Biology 2005The type IV secretion systems (T4SS) are widely distributed among the gram-negative and -positive bacteria. These systems mediate the transfer of DNA and protein... (Review)
Review
The type IV secretion systems (T4SS) are widely distributed among the gram-negative and -positive bacteria. These systems mediate the transfer of DNA and protein substrates across the cell envelope to bacterial or eukaryotic cells generally through a process requiring direct cell-to-cell contact. Bacteria have evolved T4SS for survival during establishment of pathogenic or symbiotic relationships with eukaryotic hosts. The Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation machines serve as models for detailed mechanistic studies aimed at elucidating the nature of translocation signals, machine assembly pathways and architectures, and the dynamics of substrate translocation. The A. tumefaciens VirB/D4 T4SS are polar-localized organelles composed of a secretion channel and an extracellular T pilus. These T4SS are assembled from 11 or more subunits. whose membrane topologies, intersubunit contacts and, in some cases, 3-dimensional structures are known. Recently, powerful in vivo assays have identified C-terminal translocation signals, defined for the first time the translocation route for a DNA substrate through a type IV secretion channel, and supplied evidence that ATP energy consumption contributes to a late stage of machine morphogenesis. Together, these recent findings describe the mechanics of type IV secretion in unprecedented detail.
Topics: Adenosine Triphosphate; Agrobacterium tumefaciens; Bacterial Proteins; Biological Transport; Conjugation, Genetic; DNA, Bacterial; Gram-Negative Bacteria; Gram-Positive Bacteria; Membrane Proteins; Protein Transport; Virulence Factors
PubMed: 16092524
DOI: 10.1080/09687860500063316