ABSTRACT
OBJECTIVE: Currently the majority of non-culturable microbes in sea water are yet to be discovered, Nanopore offers a solution to overcome the challenging tasks to identify the genomes and complex composition of oceanic microbiomes. In this study we evaluate the utility of Oxford Nanopore Technologies (ONT) sequencing to characterize microbial diversity in seawater from multiple locations. We compared the microbial species diversity of retrieved environmental samples from two different locations and time points. RESULTS: With only three ONT flow cells we were able to identify thousands of organisms, including bacteriophages, from which a large part at species level. It was possible to assemble genomes from environmental samples with Flye. In several cases this resulted in > 1 Mbp contigs and in the particular case of a Thioglobus singularis species it even produced a near complete genome. k-mer analysis reveals that a large part of the data represents species of which close relatives have not yet been deposited to the database. These results show that our approach is suitable for scalable genomic investigations such as monitoring oceanic biodiversity and provides a new platform for education in biodiversity.
Subject(s)
High-Throughput Nucleotide Sequencing , Nanopores , Pilot Projects , Seawater , Sequence Analysis, DNAABSTRACT
The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region-encoded transport system (reviewed in [1-5]). In addition, A. tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB/VirD4 transport system [6], and both assist there in the transformation of normal cells into tumor cells. The function of the 22 kDa VirF protein is not clear. Deletion of the virF gene in A. tumefaciens leads to diminished virulence [7, 8] and can be complemented by the expression of the virF gene in the host plant. This finding indicates that VirF functions within the plant cell [8]. Here, we report that the VirF protein is the first prokaryotic protein with an F box by which it can interact with plant homologs of the yeast Skp1 protein. The presence of the F box turned out to be essential for the biological function of VirF. F box proteins and Skp1p are both subunits of a class of E3 ubiquitin ligases referred to as SCF complexes. Thus, VirF may be involved in the targeted proteolysis of specific host proteins in early stages of the transformation process.
Subject(s)
Arabidopsis Proteins , Bacterial Proteins/metabolism , Plant Proteins/metabolism , Protein Serine-Threonine Kinases , Virulence Factors , Agrobacterium tumefaciens/genetics , Agrobacterium tumefaciens/pathogenicity , Amino Acid Sequence , Arabidopsis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/physiology , Base Sequence , Binding Sites , Cell Cycle Proteins , DNA, Plant , Molecular Sequence Data , Plant Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , S-Phase Kinase-Associated Proteins , SKP Cullin F-Box Protein Ligases , Saccharomyces cerevisiae , Sequence Homology, Amino Acid , VirulenceABSTRACT
The Agrobacterium VirB/D4 transport system mediates the transfer of a nucleoprotein T complex into plant cells, leading to crown gall disease. In addition, several Virulence proteins must somehow be transported to fulfill a function in planta. Here, we used fusions between Cre recombinase and VirE2 or VirF to directly demonstrate protein translocation into plant cells. Transport of the proteins was monitored by a Cre-mediated in planta recombination event resulting in a selectable phenotype and depended on the VirB/D4 transport system but did not require transferred DNA.
Subject(s)
Agrobacterium tumefaciens/metabolism , Arabidopsis/metabolism , Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Integrases/metabolism , Ion Channels , Protein Transport , Viral Proteins , Virulence Factors , Agrobacterium tumefaciens/genetics , Agrobacterium tumefaciens/pathogenicity , Arabidopsis/genetics , Arabidopsis/microbiology , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Drug Resistance , Integrases/genetics , Kanamycin/pharmacology , Plant Roots/metabolism , Plants, Genetically Modified , Plasmids , Polymerase Chain Reaction , Recombinant Fusion Proteins/metabolism , VirulenceABSTRACT
The VirA protein of Agrobacterium tumefaciens is thought to be a receptor for plant phenolic compounds such as acetosyringone. Although it is not known whether the interaction between VirA and the phenolics is direct or requires other phenolic-binding proteins, it is shown in this study that the first 280 amino acids of the VirA protein are not essential for the acetosyringone mediated vir gene induction response. Considering the fact that the cytoplasmic region between the amino acids 283 and 304 is highly conserved between the different VirA proteins, and that deletion of this region abolishes VirA activity, we suggest that the acetosyringone receptor domain is located in this cytoplasmic domain of the VirA protein.
Subject(s)
Acetophenones/pharmacology , Agrobacterium tumefaciens/genetics , Agrobacterium tumefaciens/pathogenicity , Bacterial Proteins/genetics , Escherichia coli Proteins , Gene Expression Regulation, Bacterial/drug effects , Receptors, Cell Surface , Virulence Factors , Amino Acid Sequence , Bacterial Proteins/biosynthesis , Base Sequence , Chemoreceptor Cells , DNA Mutational Analysis , Genes, Bacterial , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Molecular Sequence Data , Recombinant Fusion Proteins/biosynthesis , Sequence Deletion , Sequence Homology, Amino Acid , Signal Transduction/genetics , Structure-Activity Relationship , Transcriptional Activation , Virulence/geneticsABSTRACT
The virA gene of Agrobacterium tumefaciens encodes an inner membrane that mediates the transcriptional activation of virulence genes in response to plant signal molecules. We report here a functional analysis of the N-terminal, C-terminal and periplasmic domains of VirA in transmembrane signalling. First, we show that VirA has a transmembrane topology by analysis of the alkaline phosphatase activities, determined by several virA-phoA gene fusions. Second, we report here the construction of several virA-tar chimeric genes, in which the 3'-coding region of virA is conserved to study transmembrane signalling, as well as the construction of a set of virA deletion mutations. Results of analyses of vir induction behaviour and tumour inducing abilities of agrobacteria carrying these mutant genes do not support existing models for the chemoreceptor function of the VirA periplasmic domain. We demonstrate that the periplasmic domain of VirA can be either replaced by a corresponding region of the E.coli chemosensory protein Tar or even totally deleted from VirA without a loss of function. Here, we present a model of VirA which involves a receptor function for the second membrane-spanning domain and an intracellular signalling function for the cytoplasmic domain of VirA. In addition, we show that VirA plays a role in determining the sensitivity for pH and temperature in acetosyringone-mediated vir induction, and we propose a role for the VirA periplasmic domain in detection of the external pH conditions.
Subject(s)
Bacterial Proteins/genetics , Genes, Bacterial , Genes , Rhizobium/genetics , Virulence Factors , Amino Acid Sequence , Bacterial Proteins/biosynthesis , Bacterial Proteins/metabolism , Base Sequence , Chromosome Deletion , Molecular Sequence Data , Mutation , Oligonucleotide Probes , Plasmids , Protein ConformationABSTRACT
A gene library of the symbiotic 240-kb plasmid of Rhizobium leguminosarum strain 1001 was constructed in pUC18. The clones showing homology with a 6.6-kb fragment containing nodEFDABC from the Sym plasmid pRLlJI were detected by colony hybridization. Additional probes from the symbiotic region of pRLlJI were used to localize the corresponding genes on the map of pRle1001a. The relative positions of nod and nif gene clusters are different than those of pRLlJI. A comparison of the amino acid sequence for NodD from pRle1001a with NodD proteins from other Rhizobium species showed a high degree of sequence conservation at the amino terminus of the protein.
Subject(s)
Bacterial Proteins/genetics , Genes, Bacterial , Rhizobium leguminosarum/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Base Sequence , Cloning, Molecular , Computer Simulation , DNA, Bacterial , Molecular Sequence Data , Nitrogen Fixation/genetics , Plasmids , Restriction Mapping , Rhizobium leguminosarum/physiology , Sequence AlignmentABSTRACT
Stable cointegrates between incRh-1 octopine (Ach5) and nopaline (C58) Ti-plasmids, present in ten independently isolated Agrobacterium tumefaciens strains, showed identical restriction endonuclease patterns. Each cointegration event had taken place in the common sequence between the T-regions of both Ti-plasmids. This illustrates a high preference for this region when used in the formation of cointegrates. Four crown gall tissues, obtained after transformation of Nicotiana tabacum cells by one of the mutants, were analysed by using Southern blot analysis for their T-DNA structure. The borders of T-DNA frequently appeared to differ from T-DNA borders previously detected in tumour tissues that had been induced by Agrobacterium strain C58 or Ach5. Therefore, it was concluded that possibly a less stringent mechanism exists for the integration into plant DNA of T-DNA, derived from a composite (octopine/nopaline) T-region than for integration of T-DNA from a normal (octopine or nopaline) T-region.