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Molecular Microbiology Aug 2019The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers effector proteins into prokaryotic and eukaryotic preys. This secretion system has emerged...
The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers effector proteins into prokaryotic and eukaryotic preys. This secretion system has emerged as a key player in regulating the microbial diversity in a population. In the plant pathogen Agrobacterium tumefaciens, the signalling cascades regulating the activity of this secretion system are poorly understood. Here, we outline how the universal eubacterial second messenger cyclic di-GMP impacts the production of T6SS toxins and T6SS structural components. We demonstrate that this has a significant impact on the ability of the phytopathogen to compete with other bacterial species in vitro and in planta. Our results suggest that, as opposed to other bacteria, c-di-GMP turns down the T6SS in A. tumefaciens thus impacting its ability to compete with other bacterial species within the rhizosphere. We also demonstrate that elevated levels of c-di-GMP within the cell decrease the activity of the Type IV secretion system (T4SS) and subsequently the capacity of A. tumefaciens to transform plant cells. We propose that such peculiar control reflects on c-di-GMP being a key second messenger that silences energy-costing systems during early colonization phase and biofilm formation, while low c-di-GMP levels unleash T6SS and T4SS to advance plant colonization.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Type IV Secretion Systems; Type VI Secretion Systems
PubMed: 31102484
DOI: 10.1111/mmi.14279 -
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 -
Canadian Journal of Microbiology Jan 2021Agroinfiltration is used to treat plants with modified strains of for the purpose of transient in planta expression of genes transferred from the bacterium. These genes...
Agroinfiltration is used to treat plants with modified strains of for the purpose of transient in planta expression of genes transferred from the bacterium. These genes encode valuable recombinant proteins for therapeutic or industrial applications. Treatment of large quantities of plants for industrial-scale protein production exposes bacteria (harboring genes of interest) to agroinfiltration medium that is devoid of nutrients and carbon sources for prolonged periods of time (possibly upwards of 24 h). Such conditions may negatively influence bacterial viability, infectivity of plant cells, and target protein production. Here, we explored the role of timing in bacterial culture preparation for agroinfiltration using mass spectrometry-based proteomics to define changes in cellular processes. We observed distinct profiles associated with bacterial treatment conditions and exposure timing, including significant changes in proteins involved in pathogenesis, motility, and nutrient acquisition systems as the bacteria adapt to the new environment. These data suggest a progression towards increased cellular remodelling over time. In addition, we described changes in growth- and environment-specific processes over time, underscoring the interconnectivity of pathogenesis and chemotaxis-associated proteins with transport and metabolism. Overall, our results have important implications for the production of transiently expressed target protein products, as prolonged exposure to agroinfiltration medium suggests remodelling of the bacterial proteins towards enhanced infection of plant cells.
Topics: Adaptation, Physiological; Agricultural Inoculants; Agrobacterium tumefaciens; Bacterial Proteins; Culture Media; Molecular Farming; Plants, Genetically Modified; Proteomics; Recombinant Proteins
PubMed: 32721220
DOI: 10.1139/cjm-2020-0239 -
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 -
Microbiological Research Jan 2016The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate...
The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae.
Topics: Agrobacterium tumefaciens; Ascomycota; Gene Silencing; Genetic Vectors; Mutagenesis, Insertional; Oryza; Plant Diseases; Transformation, Genetic
PubMed: 26686612
DOI: 10.1016/j.micres.2015.09.008 -
Microbiological Research Sep 2019Microbial oxidation of antimonite [Sb(III)] to antimonate [Sb(V)] is a detoxification process which contributes to Sb(III) resistance. Antimonite oxidase AnoA is...
Microbial oxidation of antimonite [Sb(III)] to antimonate [Sb(V)] is a detoxification process which contributes to Sb(III) resistance. Antimonite oxidase AnoA is essential for Sb(III) oxidation, however, the regulation mechanism is still unknown. Recently, we found that the expressions of phosphate transporters were induced by Sb(III) using proteomics analysis in Agrobacterium tumefaciens GW4, thus, we predicted that the phosphate regulator PhoB may regulate bacterial Sb(III) oxidation and resistance. In this study, comprehensive analyses were performed and the results showed that (1) Genomic analysis revealed two phoB (named as phoB1 and phoB2) and one phoR gene in strain GW4; (2) Reporter gene assay showed that both phoB1 and phoB2 were induced in low phosphate condition (50 μM), but only phoB2 was induced by Sb(III); (3) Genes knock-out/complementation, Sb(III) oxidation and Sb(III) resistance tests showed that deletion of phoB2 significantly inhibited the expression of anoA and decreased bacterial Sb(III) oxidation efficiency and Sb(III) resistant. In contrast, deletion of phoB1 did not obviously affect anoA's expression level and Sb(III) oxidation/resistance; (4) A putative Pho motif was predicted in several A. tumefaciens strains and electrophoretic mobility shift assay (EMSA) showed that PhoB2 could bind with the promoter sequence of anoA; (5) Site-directed mutagenesis and short fragment EMSA revealed the exact DNA binding sequence for the protein-DNA interaction. These results showed that PhoB2 positively regulates Sb(III) oxidation and PhoB2 is also associated with Sb(III) resistance. Such regulation mechanism may provide a great contribution for bacterial survival in the environment with Sb and for bioremediation application.
Topics: Agrobacterium tumefaciens; Antimony; Arsenites; Bacterial Proteins; Drug Resistance, Bacterial; Electrophoretic Mobility Shift Assay; Gene Deletion; Gene Expression Regulation, Bacterial; Gene Knockout Techniques; Mutagenesis, Site-Directed; Oxidation-Reduction; Phosphate Transport Proteins; Phosphates; Proteomics
PubMed: 31284939
DOI: 10.1016/j.micres.2019.04.008 -
Functional Plant Biology : FPB Aug 2021Hypericum perforatum L. (St. John's wort) is a well-known medicinal plant that possesses secondary metabolites with beneficial pharmacological properties. However,...
Hypericum perforatum L. (St. John's wort) is a well-known medicinal plant that possesses secondary metabolites with beneficial pharmacological properties. However, improvement in the production of secondary metabolites via genetic manipulation is a challenging task as H. perforatum remains recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, the transcripts of key genes involved in several plant defence responses (secondary metabolites, RNA silencing, reactive oxygen species (ROS) and specific defence genes) were investigated in H. perforatum suspension cells inoculated with A. tumefaciens by quantitative real-time PCR. Results indicated that key genes from the xanthone, hypericin and melatonin biosynthesis pathways, the ROS-detoxification enzyme HpAOX, as well as the defence genes Hyp-1 and HpPGIP, were all upregulated to rapidly respond to A. tumefaciens elicitation in H. perforatum. By contrast, expression levels of genes involved in hyperforin and flavonoid biosynthesis pathways were markedly downregulated upon A. tumefaciens elicitation. In addition, we compared the expression patterns of key genes in H. perforatum leaf tissues with and without dark glands, a major site of secondary metabolite production. Overall, we provide evidence for the upregulation of several phenylpropanoid pathway genes in response to elicitation by Agrobacterium, suggesting that production of secondary metabolites could modulate H. perforatum recalcitrance to A. tumefaciens-mediated transformation.
Topics: Agrobacterium tumefaciens; Gene Expression; Hypericum; Plant Oils
PubMed: 34112313
DOI: 10.1071/FP20292 -
Journal of Bacteriology Jan 2021The type VI secretion system (T6SS) is a widespread antibacterial weapon capable of secreting multiple effectors for inhibition of competitor cells. Most of the...
The type VI secretion system (T6SS) is a widespread antibacterial weapon capable of secreting multiple effectors for inhibition of competitor cells. Most of the effectors in the system share the same purpose of target intoxication, but the rationale for maintaining various types of effectors in a species is not well studied. In this study, we showed that a peptidoglycan amidase effector in , Tae, cleaves d-Ala--diaminopimelic acid (mDAP) and d-Glu bonds in peptidoglycan and is able to suppress the growth of recipient cells. The growth suppression was effective only under the condition in which cells are actively growing. In contrast, the Tde DNase effectors in the strain possessed a dominant killing effect under carbon starvation. Microscopic analysis showed that Tde triggers cell elongation and DNA degradation, while Tae causes cell enlargement without DNA damage in recipient cells. In a rich medium, harboring only functional Tae was able to maintain competitiveness among and its own sibling cells. Growth suppression and the competitive advantage of were abrogated when recipient cells produced the Tae-specific immunity protein Tai. Given that Tae is highly conserved among strains, the combination of Tae and Tde effectors could allow to better compete with various competitors by increasing its survival during changing environmental conditions. The T6SS encodes multiple effectors with diverse functions, but little is known about the biological significance of harboring such a repertoire of effectors. We reported that the T6SS antibacterial activity of the plant pathogen can be enhanced under carbon starvation or when recipient cell wall peptidoglycan is disturbed. This led to a newly discovered role for the T6SS peptidoglycan amidase Tae effector in providing a growth advantage dependent on the growth status of the target cell. This is in contrast to the Tde DNase effectors that are dominant during carbon starvation. Our study suggests that combining Tae and other effectors could allow to increase its competitiveness among changing environmental conditions.
Topics: Agrobacterium tumefaciens; Anti-Bacterial Agents; Bacterial Proteins; Cell Wall; Deoxyribonucleases; Escherichia coli; Peptidoglycan; Type VI Secretion Systems
PubMed: 33168638
DOI: 10.1128/JB.00490-20 -
Advances in Genetics 2022Several species of the genus represent unique bacterial pathogens able to genetically transform plants, by transferring and integrating a segment of their own DNA... (Review)
Review
Several species of the genus represent unique bacterial pathogens able to genetically transform plants, by transferring and integrating a segment of their own DNA (T-DNA, transferred DNA) in their host genome. Whereas in nature this process results in uncontrolled growth of the infected plant cells (tumors), this capability of has been widely used as a crucial tool to generate transgenic plants, for research and biotechnology. The virulence of relies on a series of virulence genes, mostly encoded on a large plasmid (Ti-plasmid, tumor inducing plasmid), involved in the different steps of the DNA transfer to the host cell genome: activation of bacterial virulence, synthesis and export of the T-DNA and its associated proteins, intracellular trafficking of the T-DNA and effector proteins in the host cell, and integration of the T-DNA in the host genomic DNA. Multiple interactions between these bacterial encoded proteins and host factors occur during the infection process, which determine the outcome of the infection. Here, we review our current knowledge of the mechanisms by which bacterial and plant factors control virulence and host plant susceptibility.
Topics: Virulence; Agrobacterium tumefaciens; Plants, Genetically Modified; Plasmids; Bacteria; Bacterial Proteins; Virulence Factors
PubMed: 37283660
DOI: 10.1016/bs.adgen.2022.08.001 -
Current Topics in Microbiology and... 2018The Agrobacterium tumefaciens VirB/VirD4 translocation machine is a member of a superfamily of translocators designated as type IV secretion systems (T4SSs) that... (Review)
Review
The Agrobacterium tumefaciens VirB/VirD4 translocation machine is a member of a superfamily of translocators designated as type IV secretion systems (T4SSs) that function in many species of gram-negative and gram-positive bacteria. T4SSs evolved from ancestral conjugation systems for specialized purposes relating to bacterial colonization or infection. A. tumefaciens employs the VirB/VirD4 T4SS to deliver oncogenic DNA (T-DNA) and effector proteins to plant cells, causing the tumorous disease called crown gall. This T4SS elaborates both a cell-envelope-spanning channel and an extracellular pilus for establishing target cell contacts. Recent mechanistic and structural studies of the VirB/VirD4 T4SS and related conjugation systems in Escherichia coli have defined T4SS architectures, bases for substrate recruitment, the translocation route for DNA substrates, and steps in the pilus biogenesis pathway. In this review, we provide a brief history of A. tumefaciens VirB/VirD4 T4SS from its discovery in the 1980s to its current status as a paradigm for the T4SS superfamily. We discuss key advancements in defining VirB/VirD4 T4SS function and structure, and we highlight the power of in vivo mutational analyses and chimeric systems for identifying mechanistic themes and specialized adaptations of this fascinating nanomachine.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Mutagenesis; Recombinant Fusion Proteins; Type IV Secretion Systems
PubMed: 29808338
DOI: 10.1007/82_2018_94