The DNA transporter ComEC has metal-dependent nuclease activity that is important for natural transformation.
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ABSTRACT:
SUBMITTER: Silale A
PROVIDER: S-EPMC8579336 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Natural transformation enables the incorporation of exogenous DNA into host genomes and plays a fundamental role in the evolution of microbial populations. At the center of the natural transformation machinery, the ComEC protein mediates DNA import and serves potential functions in DNA recognition and single strand degradation. Despite its importance, the evolution of ComEC is not fully understood. Here, we aim to fill this knowledge gap by surveying putative ComEC proteins across 5,574 bacteria that span diverse phyla. We first derived the presence of a universal, core Competence domain through the analysis of ComEC proteins from known naturally competent species. Then, we followed this observation to identify Competence domain containing proteins (CDCPs) from all bacteria and used CDCPs as putative ComEC proteins for evolutionary analysis. A near universal presence of CDCPs was revealed, with 89% of the proteomes and 96% of the genomes encoding a single CDCP or a CDCP-like fragment. Two domains, DUF4131 and Lactamase_B, were found to commonly co-occur with the Competence domain. Ancestral state reconstruction of CDCPs over the bacterial species phylogeny suggested an origin of a Competence-only domain profile, while multiple gains and losses of the DUF4131 and Lactamase_B domains were observed among diverse bacterial lineages.
| S-EPMC6299819 | biostudies-literature
Project description:Bacterial competence, which can be natural or induced, allows the uptake of exogenous double stranded DNA (dsDNA) into a competent bacterium. This process is known as transformation. A multiprotein assembly binds and processes the dsDNA to import one strand and degrade another yet the underlying molecular mechanisms are relatively poorly understood. Here distant relationships of domains in Competence protein EC (ComEC) of Bacillus subtilis (Uniprot: P39695) were characterized. DNA-protein interactions were investigated in silico by analyzing models for structural conservation, surface electrostatics and structure-based DNA binding propensity; and by data-driven macromolecular docking of DNA to models. Our findings suggest that the DUF4131 domain contains a cryptic DNA-binding OB fold domain and that the β-lactamase-like domain is the hitherto cryptic competence nuclease. Proteins 2016; 84:1431-1442. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.
| S-EPMC5031224 | biostudies-literature
Project description:Transposon mutagenesis is a widely applied and powerful genetic tool for the discovery of genes associated with selected phenotypes. Chlamydia trachomatis is a clinically significant, obligate intracellular bacterium for which many conventional genetic tools and capabilities have been developed only recently. This report describes the successful development and application of a Himar transposon mutagenesis system for generating single-insertion mutant clones of C. trachomatis This system was used to generate a pool of 105 transposon mutant clones that included insertions in genes encoding flavin adenine dinucleotide (FAD)-dependent monooxygenase (C. trachomatis148 [ct148]), deubiquitinase (ct868), and competence-associated (ct339) proteins. A subset of Tn mutant clones was evaluated for growth differences under cell culture conditions, revealing that most phenocopied the parental strain; however, some strains displayed subtle and yet significant differences in infectious progeny production and inclusion sizes. Bacterial burden studies in mice also supported the idea that a FAD-dependent monooxygenase (ct148) and a deubiquitinase (ct868) were important for these infections. The ct339 gene encodes a hypothetical protein with limited sequence similarity to the DNA-uptake protein ComEC. A transposon insertion in ct339 rendered the mutant incapable of DNA acquisition during recombination experiments. This observation, along with in situ structural analysis, supports the idea that this protein is playing a role in the fundamental process of lateral gene transfer similar to that of ComEC. In all, the development of the Himar transposon system for Chlamydia provides an effective genetic tool for further discovery of genes that are important for basic biology and pathogenesis aspects.IMPORTANCEChlamydia trachomatis infections have an immense impact on public health; however, understanding the basic biology and pathogenesis of this organism has been stalled by the limited repertoire of genetic tools. This report describes the successful adaptation of an important tool that has been lacking in Chlamydia studies: transposon mutagenesis. This advance enabled the generation of 105 insertional mutants, demonstrating that numerous gene products are not essential for in vitro growth. Mammalian infections using these mutants revealed that several gene products are important for infections in vivo Moreover, this tool enabled the investigation and discovery of a gene critical for lateral gene transfer; a process fundamental to the evolution of bacteria and likely for Chlamydia as well. The development of transposon mutagenesis for Chlamydia has broad impact for the field and for the discovery of genes associated with selected phenotypes, providing an additional avenue for the discovery of molecular mechanisms used for pathogenesis and for a more thorough understanding of this important pathogen.
| S-EPMC6686042 | biostudies-literature
Project description:Genetic variants at the solute carrier family 39 member 8 (SLC39A8) gene locus are associated with the regulation of whole-blood manganese (Mn) and multiple physiological traits. SLC39A8 encodes ZIP8, a divalent metal ion transporter best known for zinc transport. Here, we hypothesized that ZIP8 regulates Mn homeostasis and Mn-dependent enzymes to influence metabolism. We generated Slc39a8-inducible global-knockout (ZIP8-iKO) and liver-specific-knockout (ZIP8-LSKO) mice and observed markedly decreased Mn levels in multiple organs and whole blood of both mouse models. By contrast, liver-specific overexpression of human ZIP8 (adeno-associated virus-ZIP8 [AAV-ZIP8]) resulted in increased tissue and whole blood Mn levels. ZIP8 expression was localized to the hepatocyte canalicular membrane, and bile Mn levels were increased in ZIP8-LSKO and decreased in AAV-ZIP8 mice. ZIP8-LSKO mice also displayed decreased liver and kidney activity of the Mn-dependent enzyme arginase. Both ZIP8-iKO and ZIP8-LSKO mice had defective protein N-glycosylation, and humans homozygous for the minor allele at the lead SLC39A8 variant showed hypogalactosylation, consistent with decreased activity of another Mn-dependent enzyme, β-1,4-galactosyltransferase. In summary, hepatic ZIP8 reclaims Mn from bile and regulates whole-body Mn homeostasis, thereby modulating the activity of Mn-dependent enzymes. This work provides a mechanistic basis for the association of SLC39A8 with whole-blood Mn, potentially linking SLC39A8 variants with other physiological traits.
| S-EPMC5451243 | biostudies-literature
Project description:Exonuclease 1 (Exo1) is an evolutionarily conserved eukaryotic nuclease that plays a multifaceted role in maintaining genome stability. The biochemical attributes of Exo1 have been extensively characterized via conventional assays. However, the key step governing its activation remains elusive. Extending the previous finding that Exo1 can digest a randomly selected single-stranded DNA (ssDNA) but not a poly(dT) oligonucleotide and using purified recombinant Exo1 and nuclease and electrophoretic mobility shift assays, here we determined that DNA hairpins with a stem size of 4 bp or longer are able to activate Exo1-mediated digestion of ssDNA. We further provide evidence suggesting that Exo1 uses an evolutionarily conserved residue, Lys185 This residue interacted with the phosphate group bridging the third and fourth nucleotide on the digestion strand of the substrate DNA for duplex recognition, critical for Exo1 activation on not only ssDNA but also dsDNA. Additionally, the defect of an exo1-K185A mutant in duplex digestion was partially rescued by longer overhanging DNA. However, we noted that the enhanced Exo1 nuclease activity by longer overhanging DNA is largely eliminated by replication protein A (RPA), likely because of the previously reported RPA activity that strips Exo1 off the ssDNA. We conclude that duplex DNA contact by Exo1 is a general mechanism that controls its activation and that this mechanism is particularly important for digestion of duplex DNA whose nascent ssDNA is bound by RPA.
| S-EPMC6663874 | biostudies-literature
Project description:Natural transformation has been described in bacterial species spread through nearly all major taxonomic groups. However, the current understanding of the structural components and the regulation of competence development is derived from only a few model organisms. Although natural transformation was discovered in members of the Actinobacteria (high GC Gram-positive bacteria) more than four decades ago, the structural components or the regulation of the competence system have not been studied in any representative of the entire phylum. In this report we identify a new role for a distinct type of pilus biogenesis genes (tad genes, for tight adherence), which so far have been connected only with biofilm formation, adherence and virulence traits. The tad-like genes found in the genome of Micrococcus luteus were shown to be required for genetic transformation in this actinobacterial species. We generated and analyzed individual knockout mutants for every open reading frame of the two predicted tad gene clusters as well as for a potential prepilin processing peptidase and identified the major component of the putative pili. By expressing a tagged variant of the major prepilin subunit and immunofluorescence microscopy we visualized filamentous structures extending from the cell surface. Our data indicate that the two tad gene islands complementarily contribute to the formation of a functional competence pilus in this organism. It seems likely that the involvement of tad genes in natural transformation is not unique only for M. luteus but may also prove to be the case in other representatives of the Actinobacteria, which contains important medically and biotechnologically relevant species.
| S-EPMC4322843 | biostudies-literature
Project description:Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) in association with CRISPR-associated (Cas) proteins constitutes a formidable defense system against mobile genetic elements in prokaryotes. In type I-C, the ribonucleoprotein surveillance complex comprises only three Cas proteins, namely, Cas5d, Csd1 and Csd2. Unlike type I-E that uses Cse3/CasE for metal-independent CRISPR RNA maturation, type I-C that lacks this deputes Cas5d to process the pre-crRNA. Here, we report the promiscuous DNase activity of Cas5d in presence of divalent metals. Remarkably, the active site that renders RNA hydrolysis may be tuned by metal to act on DNA substrates too. Further, the realization that Csd1 is a fusion of its functional homolog Cse1/CasA and Cse2/CasB forecasts that the stoichiometry of the constituents of the surveillance complex in type I-C may differ from type I-E. Although Csd2 seems to be inert, Csd1 too exhibits RNase and metal-dependent DNase activity. Thus, in addition to their proposed functions, the DNase activity of Cas5d and Csd1 may also enable them to be co-opted in adaptation and interference stages of CRISPR immunity wherein interaction with DNA substrates is involved.
| S-EPMC3973291 | biostudies-literature
Project description:Genetic transformation, in which cells internalize exogenous DNA and integrate it into their chromosome, is widespread in the bacterial kingdom. It involves a specialized membrane-associated machinery for binding double-stranded (ds) DNA and uptake of single-stranded (ss) fragments. In the human pathogen Streptococcus pneumoniae, this machinery is specifically assembled at competence. The EndA nuclease, a constitutively expressed virulence factor, is recruited during competence to play the key role of converting dsDNA into ssDNA for uptake. Here we use fluorescence microscopy to show that EndA is uniformly distributed in the membrane of noncompetent cells and relocalizes at midcell during competence. This recruitment requires the dsDNA receptor ComEA. We also show that under 'static' binding conditions, i.e., in cells impaired for uptake, EndA and ComEA colocalize at midcell, together with fluorescent end-labelled dsDNA (Cy3-dsDNA). We conclude that midcell clustering of EndA reflects its recruitment to the DNA uptake machinery rather than its sequestration away from this machinery to protect transforming DNA from extensive degradation. In contrast, a fraction of ComEA molecules were located at cell poles post-competence, suggesting the pole as the site of degradation of the dsDNA receptor. In uptake-proficient cells, we used Cy3-dsDNA molecules enabling expression of a GFP fusion upon chromosomal integration to identify transformed cells as GFP producers 60-70 min after initial contact between DNA and competent cells. Recording of images since initial cell-DNA contact allowed us to look back to the uptake period for these transformed cells. Cy3-DNA foci were thus detected at the cell surface 10-11 min post-initial contact, all exclusively found at midcell, strongly suggesting that active uptake of transforming DNA takes place at this position in pneumococci. We discuss how midcell uptake could influence homology search, and the likelihood that midcell uptake is characteristic of cocci and/or the growth phase-dependency of competence.
| S-EPMC3764208 | biostudies-literature
Project description:BackgroundRecent studies have demonstrated that several mineral products sold for medicinal purposes demonstrate antimicrobial activity, but little is known about the physicochemical properties involved in antibacterial activity.Methodology/principal findingsUsing in vitro mineral suspension testing, we have identified two natural mineral mixtures, arbitrarily designated BY07 and CB07, with antibacterial activity against a broad-spectrum of bacterial pathogens. Mineral-derived aqueous leachates also exhibited antibacterial activity, revealing that chemical, not physical, mineral characteristics were responsible for the observed activity. The chemical properties essential for bactericidal activity against Escherichia coli were probed by testing antibacterial activity in the presence of metal chelators, the hydroxyl radical scavenger, thiourea, and varying pH levels. Chelation of the BY07 minerals with EDTA or desferrioxamine eliminated or reduced BY07 toxicity, respectively, suggesting a role of an acid-soluble metal species, particularly Fe(3+) or other sequestered metal cations, in mineral toxicity. This conclusion was supported by NMR relaxation data, which indicated that BY07 and CB07 leachates contained higher concentrations of chemically accessible metal ions than leachates from non-bactericidal mineral samples.Conclusions/significanceWe conclude that the acidic environment of the hydrated minerals significantly contributes to antibacterial activity by increasing the availability and toxicity of metal ions. These findings provide impetus for further investigation of the physiological effects of mineral products and their applications in complementary antibacterial therapies.
| S-EPMC2830476 | biostudies-literature
Project description:Bacteria take up environmental DNA using dynamic appendages called type IV pili (T4P) to elicit horizontal gene transfer in a process called natural transformation. Natural transformation is widespread amongst bacteria yet determining how different factors universally contribute to or limit this process across species has remained challenging. Here we show that Acinetobacter baylyi, the most naturally transformable species, is highly transformable due to its ability to robustly bind nonspecific DNA via a dedicated orphan minor pilin, FimT. We show that, compared to its homologues, A. baylyi FimT contains multiple positively charged residues that additively promote DNA binding efficiency. Expression of A. baylyi FimT in a closely related Acinetobacter pathogen is sufficient to substantially improve its capacity for natural transformation, demonstrating that T4P-DNA binding is a rate-limiting step in this process. These results demonstrate the importance of T4P-DNA binding efficiency in driving natural transformation, establishing a key factor limiting horizontal gene transfer.
| S-EPMC11185590 | biostudies-literature