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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 -
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 -
Genes Oct 2020Mycorrhizal desert truffles such as , , and , form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild...
Mycorrhizal desert truffles such as , , and , form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an -mediated genetic transformation system in . This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 μM of acetosyringone, attaining a bacterial optical density of 0.3 OD; transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for , and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species.
Topics: Agrobacterium tumefaciens; Ascomycota; Cistaceae; Genetic Engineering; Mycelium; Mycorrhizae; Transformation, Genetic
PubMed: 33143066
DOI: 10.3390/genes11111293 -
MBio May 2014The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by...
UNLABELLED
The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales. To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales- and Rhodobacterales-specific l,d-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d-amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli.
IMPORTANCE
Many rod-shaped bacteria, including pathogens such as Brucella and Mycobacteriu, grow by adding new material to their cell poles, and yet the proteins and mechanisms contributing to this process are not yet well defined. The polarly growing plant pathogen Agrobacterium tumefaciens was used as a model bacterium to explore these polar growth mechanisms. The results obtained indicate that polar growth in this organism is facilitated by repurposed cell division components and an otherwise obscure class of alternative peptidoglycan transpeptidases (l,d-transpeptidases). This growth results in dynamically changing cell widths as the poles expand to maturity and contrasts with the tightly regulated cell widths characteristic of canonical rod-shaped growth. Furthermore, the abundance and/or activity of l,d-transpeptidases appears to associate with polar growth strategies, suggesting that these enzymes may serve as attractive targets for specifically inhibiting growth of Rhizobiales, Actinomycetales, and other polarly growing bacterial pathogens.
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Cell Division; Cytoskeletal Proteins; Gene Expression; Peptidoglycan; Peptidyl Transferases; Phylogeny; Protein Transport
PubMed: 24865559
DOI: 10.1128/mBio.01219-14 -
The FEBS Journal Dec 2015Classical zinc finger (ZF) domains were thought to be confined to the eukaryotic kingdom until the transcriptional regulator Ros protein was identified in... (Comparative Study)
Comparative Study Review
Classical zinc finger (ZF) domains were thought to be confined to the eukaryotic kingdom until the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. The Ros Cys2 His2 ZF binds DNA in a peculiar mode and folds in a domain significantly larger than its eukaryotic counterpart consisting of 58 amino acids (the 9-66 region) arranged in a βββαα topology, and stabilized by a conserved, extensive, 15-residue hydrophobic core. The prokaryotic ZF domain, then, shows some intriguing new features that make it interestingly different from its eukaryotic counterpart. This review will focus on the prokaryotic ZFs, summarizing and discussing differences and analogies with the eukaryotic domains and providing important insights into their structure/function relationships.
Topics: Agrobacterium tumefaciens; Eukaryotic Cells; Humans; Prokaryotic Cells; Zinc Fingers
PubMed: 26365095
DOI: 10.1111/febs.13503 -
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 -
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 -
MBio Jun 2022Many pathogens or symbionts of animals and plants contain multiple replicons, a configuration called a multipartite genome. Multipartite genomes enable those species to...
Many pathogens or symbionts of animals and plants contain multiple replicons, a configuration called a multipartite genome. Multipartite genomes enable those species to replicate their genomes faster and better adapt to new niches. Despite their prevalence, the mechanisms by which multipartite genomes are stably maintained are poorly understood. Agrobacterium tumefaciens is a plant pathogen that contains four replicons: a circular chromosome (Ch1), a linear chromosome (Ch2), and two large plasmids. Recent work indicates that their replication origins are clustered at the cell poles in a manner that depends on their ParB family centromeric proteins: ParB1 for Ch1 and individual RepB paralogs for Ch2 and the plasmids. However, understanding of these interactions and how they contribute to genome maintenance is limited. By combining genome-wide chromosome conformation capture (Hi-C) assays, chromatin-immunoprecipitation sequencing (ChIP-seq), and live cell fluorescence microscopy, we provide evidence here that centromeric clustering is mediated by interactions between these centromeric proteins. We further show that the disruption of centromere clustering results in the loss of replicons. Our data establish the role of centromeric clustering in multipartite genome stability. About 10% of sequenced bacteria have multiple replicons, also known as multipartite genomes. How these multipartite genomes are maintained is still poorly understood. Here, we use Agrobacterium tumefaciens as a model and show that the replication origins of the four replicons are clustered through direct interactions between the centromeric proteins; disruption of origin clustering leads to the loss of replicons. Thus, our study provided evidence that centromeric clustering is important for maintaining multipartite genomes.
Topics: Agrobacterium tumefaciens; Animals; Bacterial Proteins; Centromere; Plasmids; Replicon
PubMed: 35536004
DOI: 10.1128/mbio.00508-22 -
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 -
Applied and Environmental Microbiology Aug 2019is a rhizosphere bacterium that can infect wound sites on plants. The bacterium transfers a segment of DNA (T-DNA) from the Ti plasmid to the plant host cell via a type...
is a rhizosphere bacterium that can infect wound sites on plants. The bacterium transfers a segment of DNA (T-DNA) from the Ti plasmid to the plant host cell via a type IV secretion system where the DNA becomes integrated into the host cell chromosomes. The expression of T-DNA in the plant results in tumor formation. Although the binding of the bacteria to plant surfaces has been studied previously, there is little work on possible interactions of the bacteria with the plant cell wall. Seven of the 48 genes encoding putative glycoside hydrolases (, , , , , , and ) in the genome of C58 were found to play a role in virulence on tomato and Two of these genes ( and ; and ) encode enzymes capable of digesting polygalacturonic acid and, thus, may play a role in the digestion of pectin. One gene (; ) encodes an arabinosylfuranosidase, which could remove arabinose from the ends of polysaccharide chains. Two genes ( and ; and ) encode proteins with β-glycosidase activity and could digest a variety of plant cell wall oligosaccharides and polysaccharides. One gene (; ) encodes a putative xylanase, which may play a role in the digestion of xylan. Another gene (; ) encodes a protein with α-galactosidase activity and may be involved in the breakdown of arabinogalactans. Limited digestion of the plant cell wall by may be involved in tumor formation on tomato and is used in the construction of genetically engineered plants, as it is able to transfer DNA to plant hosts. Knowledge of the mechanisms of DNA transfer and the genes required will aid in the understanding of this process. Manipulation of glycoside hydrolases may increase transformation and widen the host range of the bacterium. also causes disease (crown gall tumors) on a variety of plants, including stone fruit trees, grapes, and grafted ornamentals such as roses. It is possible that compounds that inhibit glycoside hydrolases could be used to control crown gall disease caused by .
Topics: Agrobacterium tumefaciens; Bacterial Proteins; Crassulaceae; Genes, Bacterial; Glycoside Hydrolases; Solanum lycopersicum; Plant Diseases; Plant Tumors; Virulence
PubMed: 31126942
DOI: 10.1128/AEM.00603-19