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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 -
Fungal Biology Sep 2019The fungus causing target spot disease, Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei, poses an increasing threat to watermelon (Citrullus lanatus), muskmelon...
The fungus causing target spot disease, Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei, poses an increasing threat to watermelon (Citrullus lanatus), muskmelon (Cucumis melo), and cucumber (Cucumis sativus); the most economically important cucurbit crops grown in China. An understanding of the molecular mechanisms underlying the pathogenicity of C. cassiicola is essential for the development of new strategies to control this disease-causing fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) might be useful to obtain transformants of C. cassiicola, for the ultimate identification of genes involved in pathogenicity. In the present work, we established and optimized an ATMT protocol using A. tumefaciens strain AGL-1 carrying the vector pATMT1 for C. cassiicola. Efficiency of ATMT was 102-148 transformants per 10 conidia and successive subculturing of transformants on non-selective and selective media demonstrated that the integrated transfer (T)-DNA was stably inherited in C. cassiicola transformants. The integration of the hygromycin B phosphotransferase (hph) gene into C. cassiicola was validated by PCR and Southern blot analyses, which revealed that nearly 90 % of the transformants contained single-copy T-DNA. The transformants with altered phenotypes were characterized. Three of these transformants completely lost pathogenicity and other three showed strongly impaired pathogenicity relative to the Cc-GX strain on muskmelon leaves. These results strongly suggest that ATMT may be used as a molecular tool for identifying genes relevant to pathogenicity in the fungus C. cassiicola, an emerging threat to several agronomically important plants in China.
Topics: Agrobacterium tumefaciens; Ascomycota; China; Cucumis melo; DNA, Bacterial; Fungal Proteins; Mutagenesis, Insertional; Plant Diseases; Transformation, Genetic; Virulence
PubMed: 31416586
DOI: 10.1016/j.funbio.2019.05.011 -
Microbiological Research Oct 2022TriR serves as a repressor for a resistance-nodulation-cell division (RND) efflux pump TriABC involved in triclosan (TCS) resistance in Agrobacterium tumefaciens. The...
TriR serves as a repressor for a resistance-nodulation-cell division (RND) efflux pump TriABC involved in triclosan (TCS) resistance in Agrobacterium tumefaciens. The triR gene is transcribed divergently from the triABC operon. TriR specifically bound to the triR-triA intergenic region, at an imperfect 10 bp inverted repeat, 5'-TTGACTAttC-GgtTAGTCAA-3' (TriR box), that was revealed by DNase I footprinting and electrophoretic mobility shift assay. TCS treatment appeared to up-regulate triR and triABC expression, via preventing TriR binding to the triR-triA intergenic region. Promoter-lacZ fusions and β-galactosidase activity assay further demonstrated TriR-mediated repression of triABC and triR autoregulation. Site-directed mutagenesis confirmed the identified TriR box is essential for TriR repression. A. tumefaciens mutant strains disrupting either triR or triA were constructed to determine their biological functions. The triA mutant showed hypersensitivity to TCS and sodium dodecyl sulfate (SDS), whereas the triR mutant was hyper-resistant, compared to wild-type. In addition to TCS and SDS, overproduction of TriABC from a multi-copy plasmid conferred enhanced resistance to a quaternary ammonium compound, benzalkonium chloride. Molecular modelling was able to predict the model of TriR and docking simulations were able to anticipate plausible binding interactions between TriR and TCS ligand.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; DNA, Intergenic; Gene Expression Regulation, Bacterial; Operon; Promoter Regions, Genetic; Triclosan
PubMed: 35810656
DOI: 10.1016/j.micres.2022.127112 -
Microbiology (Reading, England) Feb 2019A core regulatory pathway that directs developmental transitions and cellular asymmetries in Agrobacterium tumefaciens involves two overlapping, integrated...
A core regulatory pathway that directs developmental transitions and cellular asymmetries in Agrobacterium tumefaciens involves two overlapping, integrated phosphorelays. One of these phosphorelays putatively includes four histidine sensor kinase homologues, DivJ, PleC, PdhS1 and PdhS2, and two response regulators, DivK and PleD. In several different alphaproteobacteria, this pathway influences a conserved downstream phosphorelay that ultimately controls the phosphorylation state of the CtrA master response regulator. The PdhS2 sensor kinase reciprocally regulates biofilm formation and swimming motility. In the current study, the mechanisms by which the A. tumefaciens sensor kinase PdhS2 directs this regulation are delineated. PdhS2 lacking a key residue implicated in phosphatase activity is markedly deficient in proper control of attachment and motility phenotypes, whereas a kinase-deficient PdhS2 mutant is only modestly affected. A genetic interaction between DivK and PdhS2 is revealed, unmasking one of several connections between PdhS2-dependent phenotypes and transcriptional control by CtrA. Epistasis experiments suggest that PdhS2 may function independently of the CckA sensor kinase, the cognate sensor kinase for CtrA, which is inhibited by DivK. Global expression analysis of the pdhS2 mutant reveals a restricted regulon, most likely functioning through CtrA to separately control motility and regulate the levels of the intracellular signal cyclic diguanylate monophosphate (cdGMP), thereby affecting the production of adhesive polysaccharides and attachment. We hypothesize that in A. tumefaciens the CtrA regulatory circuit has expanded to include additional inputs through the addition of PdhS-type sensor kinases, likely fine-tuning the response of this organism to the soil microenvironment.
Topics: Agrobacterium tumefaciens; Bacterial Adhesion; Bacterial Proteins; Biofilms; Cyclic GMP; Epistasis, Genetic; Gene Expression Regulation, Bacterial; Histidine Kinase; Locomotion; Mutation; Phosphorylation; Polysaccharides, Bacterial; Signal Transduction; Transcription Factors
PubMed: 30620265
DOI: 10.1099/mic.0.000758 -
The New Phytologist Jul 2018Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T-DNA from its Ti plasmid into the host genome and by diverting the host...
Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T-DNA from its Ti plasmid into the host genome and by diverting the host metabolism. We combined transcriptomics and genetics for understanding the A. tumefaciens lifestyle when it colonizes Arabidopsis thaliana tumors. Transcriptomics highlighted: a transition from a motile to sessile behavior that mobilizes some master regulators (Hfq, CtrA, DivK and PleD); a remodeling of some cell surface components (O-antigen, succinoglucan, curdlan, att genes, putative fasciclin) and functions associated with plant defense (Ef-Tu and flagellin pathogen-associated molecular pattern-response and glycerol-3-phosphate and nitric oxide signaling); and an exploitation of a wide variety of host resources, including opines, amino acids, sugars, organic acids, phosphate, phosphorylated compounds, and iron. In addition, construction of transgenic A. thaliana lines expressing a lactonase enzyme showed that Ti plasmid transfer could escape host-mediated quorum-quenching. Finally, construction of knock-out mutants in A. tumefaciens showed that expression of some At plasmid genes seemed more costly than the selective advantage they would have conferred in tumor colonization. We provide the first overview of A. tumefaciens lifestyle in a plant tumor and reveal novel signaling and trophic interplays for investigating host-pathogen interactions.
Topics: Agrobacterium tumefaciens; Arabidopsis; Arginine; Bacterial Proteins; Carbon; Cell Wall; Chemotaxis; Ecosystem; Gene Expression Regulation, Bacterial; Genome, Bacterial; Host-Pathogen Interactions; Iron; Mutation; Nitrogen; Plant Tumors; Plants, Genetically Modified; Sugar Phosphates
PubMed: 29701262
DOI: 10.1111/nph.15164 -
Journal of Bacteriology Jan 2016Succinic semialdehyde (SSA), an important metabolite of γ-aminobutyric acid (GABA), is a ligand of the repressor AttJ regulating the expression of the attJ-attKLM gene...
UNLABELLED
Succinic semialdehyde (SSA), an important metabolite of γ-aminobutyric acid (GABA), is a ligand of the repressor AttJ regulating the expression of the attJ-attKLM gene cluster in the plant pathogen Agrobacterium tumefaciens. While the response of A. tumefaciens to GABA and the function of attKLM have been extensively studied, genetic and physiological responses of A. tumefaciens to SSA remain unknown. In combination with microarray and genetic approaches, this study sets out to explore new roles of the SSA-AttJKLM regulatory mechanism during bacterial infection. The results showed that SSA plays a key role in regulation of several bacterial activities, including C4-dicarboxylate utilization, nitrate assimilation, and resistance to oxidative stress. Interestingly, while the SSA relies heavily on the functional AttKLM in mediating nitrate assimilation and oxidative stress resistance, the compound could regulate utilization of C4-dicarboxylates independent of AttJKLM. We further provide evidence that SSA controls C4-dicarboxylate utilization through induction of an SSA importer and that disruption of attKLM attenuates the tumorigenicity of A. tumefaciens. Taken together, these findings indicate that SSA could be a potent plant signal which, together with AttKLM, plays a vital role in promoting the bacterial prosurvival abilities during infection.
IMPORTANCE
Agrobacterium tumefaciens is a plant pathogen causing crown gall diseases and has been well known as a powerful tool for plant genetic engineering. During the long history of microbe-host interaction, A. tumefaciens has evolved the capabilities of recognition and response to plant-derived chemical metabolites. Succinic semialdehyde (SSA) is one such metabolite. Previous results have demonstrated that SSA functions to activate a quorum-quenching mechanism and thus to decrease the level of quorum-sensing signals, thereby avoiding the elicitation of a plant defense. Here, we studied the effect of SSA on gene expression at a genome-wide level and reported that SSA also promotes bacterial survival during infection. These findings provide a new insight on the biological significance of chemical signaling between agrobacteria and plant hosts.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Metabolic Networks and Pathways; Microarray Analysis; Microbial Viability; Nitrates; Operon; Oxidative Stress; gamma-Aminobutyric Acid
PubMed: 26755630
DOI: 10.1128/JB.00373-15 -
Molecular Microbiology May 2022Agrobacterium tumefaciens is a member of the Alphaproteobacteria that pathogenises plants and associates with biotic and abiotic surfaces via a single cellular pole. A....
Agrobacterium tumefaciens is a member of the Alphaproteobacteria that pathogenises plants and associates with biotic and abiotic surfaces via a single cellular pole. A. tumefaciens produces the unipolar polysaccharide (UPP) at the site of surface contact. UPP production is normally surface-contact inducible, but elevated levels of the second messenger cyclic diguanylate monophosphate (cdGMP) bypass this requirement. Multiple lines of evidence suggest that the UPP has a central polysaccharide component. Using an A. tumefaciens derivative with elevated cdGMP and mutationally disabled for other dispensable polysaccharides, a series of related genetic screens have identified a large number of genes involved in UPP biosynthesis, most of which are Wzx-Wzy-type polysaccharide biosynthetic components. Extensive analyses of UPP production in these mutants have revealed that the UPP is composed of two genetically, chemically, and spatially discrete forms of polysaccharide, and that each requires a specific Wzy-type polymerase. Other important biosynthetic, processing, and regulatory functions for UPP production are also revealed, some of which are common to both polysaccharides, and a subset of which are specific to each type. Many of the UPP genes identified are conserved among diverse rhizobia, whereas others are more lineage specific.
Topics: Adhesives; Agrobacterium tumefaciens; Bacterial Proteins; Biosynthetic Pathways; Gene Expression Regulation, Bacterial; Polysaccharides, Bacterial
PubMed: 35191101
DOI: 10.1111/mmi.14887 -
PloS One 2013The α-Proteobacterium Agrobacterium tumefaciens has proteins homologous to known regulators that govern cell division and development in Caulobacter crescentus, many of...
The α-Proteobacterium Agrobacterium tumefaciens has proteins homologous to known regulators that govern cell division and development in Caulobacter crescentus, many of which are also conserved among diverse α-Proteobacteria. In light of recent work demonstrating similarity between the division cycle of C. crescentus and that of A. tumefaciens, the functional conservation for this presumptive control pathway was examined. In C. crescentus the CtrA response regulator serves as the master regulator of cell cycle progression and cell division. CtrA activity is controlled by an integrated pair of multi-component phosphorelays: PleC/DivJ-DivK and CckA-ChpT-CtrA. Although several of the conserved orthologues appear to be essential in A. tumefaciens, deletions in pleC or divK were isolated and resulted in cell division defects, diminished swimming motility, and a decrease in biofilm formation. A. tumefaciens also has two additional pleC/divJhomologue sensor kinases called pdhS1 and pdhS2, absent in C. crescentus. Deletion of pdhS1 phenocopied the ΔpleC and ΔdivK mutants. Cells lacking pdhS2 morphologically resembled wild-type bacteria, but were decreased in swimming motility and elevated for biofilm formation, suggesting that pdhS2 may serve to regulate the motile to non-motile switch in A. tumefaciens. Genetic analysis suggests that the PleC/DivJ-DivK and CckA-ChpT-CtrA phosphorelays in A. tumefaciens are vertically-integrated, as in C. crescentus. A gain-of-function mutation in CckA (Y674D) was identified as a spontaneous suppressor of the ΔpleC motility phenotype. Thus, although the core architecture of the A. tumefaciens pathway resembles that of C. crescentus there are specific differences including additional regulators, divergent pathway architecture, and distinct target functions.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Caulobacter crescentus; Cell Cycle; Cell Division; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Metabolic Networks and Pathways; Phosphorylation; Protein Kinases; Transcription Factors
PubMed: 23437210
DOI: 10.1371/journal.pone.0056682 -
Philosophical Transactions of the Royal... Jul 2007The plant pathogen Agrobacterium tumefaciens induces the formation of crown gall tumours at wound sites on host plants by directly transforming plant cells. This disease... (Review)
Review
The plant pathogen Agrobacterium tumefaciens induces the formation of crown gall tumours at wound sites on host plants by directly transforming plant cells. This disease strategy benefits the bacteria as the infected plant tissue produces novel nutrients, called opines, that the colonizing bacteria can use as nutrients. Almost all of the genes that are required for virulence, and all of the opine uptake and utilization genes, are carried on large tumour-inducing (Ti) plasmids. The observation more than 25 years ago that specific opines are required for Ti plasmid conjugal transfer led to the discovery of a cell-cell signalling system on these plasmids that is similar to the LuxR-LuxI system first described in Vibrio fischeri. All Ti plasmids that have been described to date carry a functional LuxI-type N-acylhomoserine lactone synthase (TraI), and a LuxR-type signal receptor and transcriptional regulator called TraR. The traR genes are expressed only in the presence of specific opines called conjugal opines. The TraR-TraI system provides an important model for LuxR-LuxI-type systems, especially those found in the agriculturally important Rhizobiaceae family. In this review, we discuss current advances in the biochemistry and structural biology of the TraR-TraI system.
Topics: Agrobacterium tumefaciens; Conjugation, Genetic; Models, Molecular; Plant Proteins; Plant Tumors; Plants; Quorum Sensing
PubMed: 17360279
DOI: 10.1098/rstb.2007.2040 -
The New Phytologist Apr 2019Agrobacterium tumefaciens is a niche-constructing biotroph that exploits host plant metabolites. We combined metabolomics, transposon-sequencing (Tn-seq),...
Agrobacterium tumefaciens is a niche-constructing biotroph that exploits host plant metabolites. We combined metabolomics, transposon-sequencing (Tn-seq), transcriptomics, and reverse genetics to characterize A. tumefaciens pathways involved in the exploitation of resources from the Solanum lycopersicum host plant. Metabolomics of healthy stems and plant tumors revealed the common (e.g. sucrose, glutamate) and enriched (e.g. opines, γ-aminobutyric acid (GABA), γ-hydroxybutyric acid (GHB), pyruvate) metabolites that A. tumefaciens could use as nutrients. Tn-seq and transcriptomics pinpointed the genes that are crucial and/or upregulated when the pathogen grew on either sucrose (pgi, kdgA, pycA, cisY) or GHB (blcAB, pckA, eno, gpsA) as a carbon source. While sucrose assimilation involved the Entner-Doudoroff and tricarboxylic acid (TCA) pathways, GHB degradation required the blc genes, TCA cycle, and gluconeogenesis. The tumor-enriched metabolite pyruvate is at the node connecting these pathways. Using reverse genetics, we showed that the blc, pckA, and pycA loci were important for aggressiveness (tumor weight), proliferation (bacterial charge), and/or fitness (competition between the constructed mutants and wild-type) of A. tumefaciens in plant tumors. This work highlighted how a biotroph mobilizes its central metabolism for exploiting a wide diversity of resources in a plant host. It further shows the complementarity of functional genome-wide scans by transcriptomics and Tn-seq to decipher the lifestyle of a plant pathogen.
Topics: Agrobacterium tumefaciens; Carbon; DNA Transposable Elements; Gene Library; Genes, Bacterial; Host-Pathogen Interactions; Hydroxybutyrates; Solanum lycopersicum; Metabolome; Mutation; Nitrogen; Plant Stems; Plant Tumors; Sucrose; Transcriptome; gamma-Aminobutyric Acid
PubMed: 30447163
DOI: 10.1111/nph.15598