ABSTRACT
Bacterial genetic diversity is often described solely using base-pair changes despite a wide variety of other mutation types likely being major contributors. Tandem duplication/amplifications are thought to be widespread among bacteria but due to their often-intractable size and instability, comprehensive studies of these mutations are rare. We define a methodology to investigate amplifications in bacterial genomes based on read depth of genome sequence data as a proxy for copy number. We demonstrate the approach with Bordetella pertussis, whose insertion sequence element-rich genome provides extensive scope for amplifications to occur. Analysis of data for 2430 B. pertussis isolates identified 272 putative amplifications, of which 94â% were located at 11 hotspot loci. We demonstrate limited phylogenetic connection for the occurrence of amplifications, suggesting unstable and sporadic characteristics. Genome instability was further described in vitro using long-read sequencing via the Nanopore platform, which revealed that clonally derived laboratory cultures produced heterogenous populations rapidly. We extended this research to analyse a population of 1000 isolates of another important pathogen, Mycobacterium tuberculosis. We found 590 amplifications in M. tuberculosis, and like B. pertussis, these occurred primarily at hotspots. Genes amplified in B. pertussis include those involved in motility and respiration, whilst in M. tuberuclosis, functions included intracellular growth and regulation of virulence. Using publicly available short-read data we predicted previously unrecognized, large amplifications in B. pertussis and M. tuberculosis. This reveals the unrecognized and dynamic genetic diversity of B. pertussis and M. tuberculosis, highlighting the need for a more holistic understanding of bacterial genetics.
Subject(s)
Bordetella pertussis/genetics , Genetic Variation , Mycobacterium tuberculosis/genetics , Bordetella pertussis/classification , Genes, Bacterial/genetics , Genome, Bacterial , Genomic Instability , Mutation , Mycobacterium tuberculosis/classification , Phylogeny , Virulence/genetics , Whooping Cough/microbiologyABSTRACT
Despite sharing many of the traits that have allowed the genus Bacillus to gain recognition for its agricultural relevance, the genus Lysinibacillus is not as well-known and studied. The present study employs in vitro, in vivo, in planta, and in silico approaches to characterize Lysinibacillus fusiformis strain S4C11, isolated from the roots of an apple tree in northern Italy. The in vitro and in vivo assays demonstrated that strain S4C11 possesses an antifungal activity against different fungal pathogens, and is capable of interfering with the germination of Botrytis cinerea conidia, as well as of inhibiting its growth through the production of volatile organic molecules. In planta assays showed that the strain possesses the ability to promote plant growth, that is not host-specific, both in controlled conditions and in a commercial nursery. Biocontrol assays carried out against phytopathogenic viruses gave contrasting results, suggesting that the strain does not activate the host's defense pathways. The in silico analyses were carried out by sequencing the genome of the strain through an innovative approach that combines Illumina and High-Definition Mapping methods, allowing the reconstruction of a main chromosome and two plasmids from strain S4C11. The analysis of the genes encoded by the genome contributed to the characterization of the strain, detecting genes related to the biocontrol effect detected in the experimental trials.
Subject(s)
Bacillaceae/physiology , Antibiosis , Bacillaceae/genetics , Bacillaceae/isolation & purification , Botrytis/growth & development , Botrytis/physiology , Computer Simulation , Genome, Bacterial , Italy , Malus/microbiology , Plant Diseases/microbiology , Plant Roots/microbiologyABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution and comprehensiveness. To help translate these methods to routine research and clinical practice, we developed a sequence-resolved benchmark set for identification of both false-negative and false-positive germline large insertions and deletions. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle Consortium integrated 19 sequence-resolved variant calling methods from diverse technologies. The final benchmark set contains 12,745 isolated, sequence-resolved insertion (7,281) and deletion (5,464) calls ≥50 base pairs (bp). The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.51 Gbp and 5,262 insertions and 4,095 deletions supported by ≥1 diploid assembly. We demonstrate that the benchmark set reliably identifies false negatives and false positives in high-quality SV callsets from short-, linked- and long-read sequencing and optical mapping.
Subject(s)
Germ-Line Mutation/genetics , INDEL Mutation/genetics , Diploidy , Genomic Structural Variation , Humans , Molecular Sequence Annotation , Sequence Analysis, DNAABSTRACT
The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity in DNA binding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and by UV cross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaB-DnaC complex at a DnaA box. Because a decreased ATP concentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature.
Subject(s)
Bacterial Proteins/metabolism , DNA Helicases/metabolism , DNA, Bacterial/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , DnaB Helicases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Alleles , Amino Acid Substitution , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Conserved Sequence , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Replication , DNA, Bacterial/chemistry , DNA, Single-Stranded/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DnaB Helicases/chemistry , DnaB Helicases/genetics , Enzyme Stability , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Hot Temperature/adverse effects , Kinetics , Mutation , Protein Interaction Domains and Motifs , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Replication OriginABSTRACT
Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites-the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.
Subject(s)
Caffeine/pharmacology , Glucose Transporter Type 1/metabolism , Glucose/metabolism , 3-O-Methylglucose/metabolism , Adenosine Triphosphate/metabolism , Binding Sites , Biological Transport/drug effects , Biological Transport/physiology , Cytochalasin B/metabolism , Erythrocyte Membrane/metabolism , Erythrocytes/drug effects , Erythrocytes/metabolism , Humans , Signal Transduction/drug effectsABSTRACT
GLUT1, the primary glucose transport protein in human erythrocytes [red blood cells (RBCs)], also transports oxidized vitamin C [dehydroascorbic acid (DHA)]. A recent study suggests that RBC GLUT1 transports DHA as its primary substrate and that only a subpopulation of GLUT1 transports sugars. This conclusion is based on measurements of cellular glucose and DHA equilibrium spaces, rather than steady-state transport rates. We have characterized RBC transport of DHA and 3-O-methylglucose (3-OMG), a transported, nonmetabolizable sugar. Steady-state 3-OMG and DHA uptake in the absence of intracellular substrate are characterized by similar Vmax (0.16 ± 0.01 and 0.13 ± 0.02 mmol·l(-1)·min(-1), respectively) and apparent Km (1.4 ± 0.2 and 1.6 ± 0.7 mM, respectively). 3-OMG and DHA compete for uptake, with Ki(app) of 0.7 ± 0.4 and 1.1 ± 0.1 mM, respectively. Uptake measurements using RBC inside-out-membrane vesicles demonstrate that 3-OMG and DHA compete at the cytoplasmic surface of the membrane, with Ki(app) of 0.7 ± 0.1 and 0.6 ± 0.1 mM, respectively. Intracellular 3-OMG stimulates unidirectional uptake of 3-OMG and DHA. These findings indicate that DHA and 3-OMG bind at mutually exclusive sites at exo- and endofacial surfaces of GLUT1 and are transported via the same GLUT1 complex.
Subject(s)
3-O-Methylglucose/metabolism , Dehydroascorbic Acid/metabolism , Erythrocyte Membrane/metabolism , Glucose Transporter Type 1/metabolism , Glucose/metabolism , Binding Sites , Binding, Competitive , Biological Transport , Carbon Radioisotopes , Humans , Kinetics , Protein Binding , TritiumABSTRACT
Homologous recombination is essential for productive DNA replication particularly under stress conditions. We previously demonstrated a stress-induced recruitment of Rad51 to mitochondria and a critical need for its activity in the maintenance of mitochondrial DNA (mtDNA) copy number. Using the human osteosarcoma cell line U20S, we show in the present study that recruitment of Rad51 to mitochondria under stress conditions requires ongoing mtDNA replication. Additionally, Rad51 levels in mitochondria increase in cells recovering from mtDNA depletion. Our findings highlight an important new role for Rad51 in supporting mtDNA replication, and further promote the idea that recombination is indispensable for sustaining DNA synthesis under conditions of replication stress.
Subject(s)
DNA Replication , DNA, Mitochondrial/biosynthesis , Mitochondria/metabolism , Rad51 Recombinase/metabolism , Stress, Physiological , Cell Line , HumansABSTRACT
Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free repair of DNA double strand breaks. Rad51 is the central catalyst of HR in all eukaryotes, and to this point studies of human Rad51 have focused exclusively on events occurring within the nucleus. However, substantial amounts of HR proteins exist in the cytoplasm, yet the function of these protein pools has not been addressed. Here, we provide the first demonstration that Rad51 and the related HR proteins Rad51C and Xrcc3 exist in human mitochondria. We show stress-induced increases in both the mitochondrial levels of each protein and, importantly, the physical interaction between Rad51 and mitochondrial DNA (mtDNA). Depletion of Rad51, Rad51C, or Xrcc3 results in a dramatic decrease in mtDNA copy number as well as the complete suppression of a characteristic oxidative stress-induced copy number increase. Our results identify human mtDNA as a novel Rad51 substrate and reveal an important role for HR proteins in the maintenance of the human mitochondrial genome.
Subject(s)
DNA, Mitochondrial/genetics , Genome , Rad51 Recombinase/genetics , Rad51 Recombinase/physiology , Cell Cycle , Cell Line, Tumor , Cytoplasm/metabolism , DNA Breaks, Double-Stranded , DNA Damage , DNA-Binding Proteins/metabolism , Humans , Mitochondria/metabolism , Oxidative Stress , Rad51 Recombinase/metabolism , Subcellular Fractions/metabolismABSTRACT
Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damage-induced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damage-induced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.
Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , DNA Damage , DNA, Neoplasm/genetics , DNA-Binding Proteins/metabolism , Rad51 Recombinase/metabolism , Active Transport, Cell Nucleus , Cell Nucleus/genetics , Cell Nucleus/radiation effects , Cytoplasm/genetics , Cytoplasm/radiation effects , DNA, Neoplasm/metabolism , DNA-Binding Proteins/genetics , Genes, BRCA2 , HCT116 Cells , HeLa Cells , Humans , Immunoblotting , Infrared Rays , Nuclear Localization Signals , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rad51 Recombinase/genetics , Reverse Transcriptase Polymerase Chain Reaction , Subcellular FractionsABSTRACT
The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counterselected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the dnaC gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of Escherichia coli and related enterobacteria. The method consists of using a strain carrying a large deletion of the dnaC gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-beta-d-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a dnaC mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes.