scholarly journals Genetic Control of Oxidative Mutagenesis in Sulfolobus acidocaldarius

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Rupal Jain ◽  
Samuel Dhiman ◽  
Dennis W. Grogan
Keyword(s):  
Dna Polymerase ◽  
Genome Stability ◽  
Dna Oxidation ◽  
Sulfolobus Acidocaldarius ◽  
Hyperthermophilic Archaea ◽  
Genome Replication ◽  
Content Type ◽  
Cellular Survival ◽  
Genes Encoding

ABSTRACT To identify DNA oxidation defenses of hyperthermophilic archaea, we deleted genes encoding the putative 7,8-dihydro-8-oxoguanine (oxoG)-targeted N-glycosylase of Sulfolobus acidocaldarius (ogg; Saci_01367), the Y family DNA polymerase (dbh; Saci_0554), or both and measured the effects on cellular survival, replication accuracy, and oxoG bypass in vivo. Spontaneous G·C-to-T·A transversions were elevated in all Δogg and Δdbh constructs, and the Δogg Δdbh double mutant lost viability at a higher rate than isogenic wild-type (WT) and ogg strains. The distribution of G·C-to-T·A transversions within mutation detector genes suggested that reactivity of G toward oxidation and the effect on translation contribute heavily to the pattern of mutations that are recovered. An impact of the Ogg protein on the overall efficiency of bypassing oxoG in transforming DNA was evident only in the absence of Dbh, and Ogg status did not affect the accuracy of bypass. Dbh function, in contrast, dramatically influenced both the efficiency and accuracy of oxoG bypass. Thus, Ogg and Dbh were found to work independently to avoid mutagenesis by oxoG, and inactivating this simple but effective defense system by deleting both genes imposed a severe mutational burden on S. acidocaldarius cells. IMPORTANCE Hyperthermophilic archaea are expected to have effective (and perhaps atypical) mechanisms to limit the genetic consequences of DNA damage, but few gene products have been demonstrated to have genome-preserving functions in vivo. This study confirmed by genetic criteria that the S. acidocaldarius Ogg protein avoids the characteristic mutagenesis of G oxidation. This enzyme and the bypass polymerase Dbh have similar impacts on genome stability but work independently and may comprise most of the DNA oxidation defense of S. acidocaldarius. The critical dependence of accurate oxoG bypass on the accessory DNA polymerase Dbh further argues that some form of polymerase exchange is important for accurate genome replication in Sulfolobus, and perhaps in related hyperthermophilic archaea.

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

scholarly journals Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo

2013 ◽  
Vol 87 (17) ◽  
pp. 9780-9787 ◽  
Author(s):  
M. Redrejo-Rodriguez ◽  
J. M. Rodriguez ◽  
C. Suarez ◽  
J. Salas ◽  
M. L. Salas
Keyword(s):  
Dna Polymerase ◽  
Viral Genome ◽  
Genome Stability ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus

scholarly journals How a Genetically Stable Extremophile Evolves: Modes of Genome Diversification in the Archaeon Sulfolobus acidocaldarius

2017 ◽  
Vol 199 (17) ◽  
Author(s):  
Dominic Mao ◽  
Dennis W. Grogan
Keyword(s):  
Dna Repair ◽  
Genetic Variation ◽  
Tandem Repeats ◽  
Local Population ◽  
Genome Structure ◽  
Sulfolobus Acidocaldarius ◽  
Hyperthermophilic Archaea ◽  
Wild Type ◽  
Induced Mutations ◽  
Content Type

ABSTRACT In order to analyze in molecular terms how Sulfolobus genomes diverge, damage-induced mutations and natural polymorphisms (PMs) were identified in laboratory constructs and wild-type isolates, respectively, of Sulfolobus acidocaldarius. Among wild-type isolates drawn from one local population, pairwise nucleotide divergence averaged 4 × 10−6, which is about 0.15% of the corresponding divergence reported for Sulfolobus islandicus. The most variable features of wild-type S. acidocaldarius genomes were homopolymer (mononucleotide) tracts and longer tandem repeats, consistent with the spontaneous mutations that occur under laboratory conditions. Natural isolates, however, also revealed large insertions/deletions and inversions, which did not occur in any of the laboratory-manipulated strains. Several of the large insertions/deletions could be attributed to the integration or excision of mobile genetic elements (MGEs), and each MGE represented a distinct system of site-specific recombination. The mode of recombination associated with one MGE, a provirus related to Sulfolobus turreted icosahedral virus, was also seen in certain chromosomal inversions. Artificially induced mutations, non-MGE insertions/deletions, and small PMs exhibited different distributions over the genome, suggesting that large-scale patterning of Sulfolobus genomes begins early in the divergence process. Unlike induced mutations, natural base pair substitutions occurred in clusters, and one cluster exhibited properties expected of nonreciprocal recombination (gene conversion) between dispersed imperfect repeats. Taken together, the results identify simple replication errors, slipped-strand events promoted by tandem repeats, homologous recombination, and rearrangements promoted by MGEs as the primary sources of genetic variation for this extremely acidophilic archaeon in its geothermal environment. IMPORTANCE The optimal growth temperatures of hyperthermophilic archaea accelerate DNA decomposition, which is expected to make DNA repair especially important for their genetic stability, yet these archaea lack certain broadly conserved types of DNA repair proteins. In this study, the genome of the extreme thermoacidophile Sulfolobus acidocaldarius was found to be remarkably stable, accumulating few mutations in many (though not all) laboratory manipulations and in natural populations. Furthermore, all the genetic processes that were inferred to diversify these genomes also operate in mesophilic bacteria and eukaryotes. This suggests that a common set of mechanisms produces most of the genetic variation in all microorganisms, despite the fundamental differences in physiology, DNA repair systems, and genome structure represented in the three domains of life.

scholarly journals The Tat Substrate SufI Is Critical for the Ability of Yersinia pseudotuberculosis To Cause Systemic Infection

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Ummehan Avican ◽  
Tugrul Doruk ◽  
Yngve Östberg ◽  
Anna Fahlgren ◽  
Åke Forsberg
Keyword(s):  
Stress Responses ◽  
Pathogenic Bacteria ◽  
Yersinia Pseudotuberculosis ◽  
Systemic Infection ◽  
Mesenteric Lymph Nodes ◽  
Content Type ◽  
Twin Arginine Translocation ◽  
Genes Encoding ◽  
Folded Proteins

ABSTRACT The twin arginine translocation (Tat) system targets folded proteins across the inner membrane and is crucial for virulence in many important human-pathogenic bacteria. Tat has been shown to be required for the virulence of Yersinia pseudotuberculosis, and we recently showed that the system is critical for different virulence-related stress responses as well as for iron uptake. In this study, we wanted to address the role of the Tat substrates in in vivo virulence. Therefore, 22 genes encoding potential Tat substrates were mutated, and each mutant was evaluated in a competitive oral infection of mice. Interestingly, a ΔsufI mutant was essentially as attenuated for virulence as the Tat-deficient strain. We also verified that SufI was Tat dependent for membrane/periplasmic localization in Y. pseudotuberculosis. In vivo bioluminescent imaging of orally infected mice revealed that both the ΔsufI and ΔtatC mutants were able to colonize the cecum and Peyer's patches (PPs) and could spread to the mesenteric lymph nodes (MLNs). Importantly, at this point, neither the ΔtatC mutant nor the ΔsufI mutant was able to spread systemically, and they were gradually cleared. Immunostaining of MLNs revealed that both the ΔtatC and ΔsufI mutants were unable to spread from the initial infection foci and appeared to be contained by neutrophils, while wild-type bacteria readily spread to establish multiple foci from day 3 postinfection. Our results show that SufI alone is required for the establishment of systemic infection and is the major cause of the attenuation of the ΔtatC mutant.

scholarly journals Adaptive Strategies and Pathogenesis of Clostridium difficile fromIn VivoTranscriptomics

2013 ◽  
Vol 81 (10) ◽  
pp. 3757-3769 ◽  
Author(s):  
Claire Janoir ◽  
Cécile Denève ◽  
Sylvie Bouttier ◽  
Frédéric Barbut ◽  
Sandra Hoys ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Stress Responses ◽  
Differentially Expressed ◽  
Content Type ◽  
Host Interactions ◽  
Colonization Factor ◽  
Genes Encoding ◽  
Colonization Process

ABSTRACTClostridium difficileis currently the major cause of nosocomial intestinal diseases associated with antibiotic therapy in adults. In order to improve our knowledge ofC. difficile-host interactions, we analyzed the genome-wide temporal expression ofC. difficile630 genes during the first 38 h of mouse colonization to identify genes whose expression is modulatedin vivo, suggesting that they may play a role in facilitating the colonization process. In the ceca of theC. difficile-monoassociated mice, 549 genes of theC. difficilegenome were differentially expressed compared to their expression duringin vitrogrowth, and they were distributed in several functional categories. Overall, our results emphasize the roles of genes involved in host adaptation. Colonization results in a metabolic shift, with genes responsible for the fermentation as well as several other metabolic pathways being regulated inversely to those involved in carbon metabolism. In addition, several genes involved in stress responses, such as ferrous iron uptake or the response to oxidative stress, were regulatedin vivo. Interestingly, many genes encoding conserved hypothetical proteins (CHP) were highly and specifically upregulatedin vivo. Moreover, genes for all stages of sporulation were quickly inducedin vivo, highlighting the observation that sporulation is central to the persistence ofC. difficilein the gut and to its ability to spread in the environment. Finally, we inactivated two genes that were differentially expressedin vivoand evaluated the relative colonization fitness of the wild-type and mutant strains in coinfection experiments. We identified a CHP as a putative colonization factor, supporting the suggestion that thein vivotranscriptomic approach can unravel newC. difficilevirulence genes.

scholarly journals Gene Models, Expression Repertoire, and Immune Response of Plasmodium vivax Reticulocyte Binding Proteins

2015 ◽  
Vol 84 (3) ◽  
pp. 677-685 ◽  
Author(s):  
Jenni Hietanen ◽  
Anongruk Chim-ong ◽  
Thanprakorn Chiramanewong ◽  
Jakub Gruszczyk ◽  
Wanlapa Roobsoong ◽  
...  
Keyword(s):  
Plasmodium Vivax ◽  
Significant Negative Correlation ◽  
Protein Coding ◽  
Content Type ◽  
Immune Pressure ◽  
The Family ◽  
Genes Encoding ◽  
Gene Models

Members of thePlasmodium vivaxreticulocyte binding protein (PvRBP) family are believed to mediate specific invasion of reticulocytes byP. vivax. In this study, we performed molecular characterization of genes encoding members of this protein family. Through cDNA sequencing, we constructed full-length gene models and verified genes that are protein coding and those that are pseudogenes. We also used quantitative PCR to measure theirin vivotranscript abundances in clinicalP. vivaxisolates. Like genes encoding related invasion ligands ofP. falciparum,Pvrbpexpression levels vary broadly across different parasite isolates. Through antibody measurements, we found that host immune pressure may be the driving force behind the distinctly high diversity of one of the family members, PvRBP2c. Mild yet significant negative correlation was found between parasitemia and the PvRBP2b antibody level, suggesting that antibodies to the protein may interfere with invasion.

scholarly journals Evolutionary Adaptation of an AraC-Like Regulatory Protein in Citrobacter rodentium and Escherichia Species

2015 ◽  
Vol 83 (4) ◽  
pp. 1384-1395 ◽  
Author(s):  
Aimee Tan ◽  
Nicola K. Petty ◽  
Dianna Hocking ◽  
Vicki Bennett-Wood ◽  
Matthew Wakefield ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Regulatory Protein ◽  
Gene Repertoire ◽  
Citrobacter Rodentium ◽  
Environmental Strain ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding

The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogenCitrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and thegrlRAoperon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genusEscherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated thatregAhas been horizontally transferred toEscherichiaspp. andC. rodentium. Comparative studies of two RegA homologues, namely, those fromC. rodentiumandE. coliSMS-3-5, a multiresistant environmental strain ofE. coli, showed that the two regulators acted similarlyin vitrobut differed in terms of their abilities to activate the virulence ofC. rodentiumin vivo, which evidently was due to their differential activation ofgrlRA. Our data indicate that RegA fromC. rodentiumhas strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence byC. rodentiumand on the evolution of virulence-regulatory genes of bacterial pathogens in general.

scholarly journals Identification and Characterization of a Novel Aspergillus fumigatus Rhomboid Family Putative Protease, RbdA, Involved in Hypoxia Sensing and Virulence

2016 ◽  
Vol 84 (6) ◽  
pp. 1866-1878 ◽  
Author(s):  
Yakir Vaknin ◽  
Falk Hillmann ◽  
Rossana Iannitti ◽  
Netali Ben Baruch ◽  
Hana Sandovsky-Losica ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Regulatory Element ◽  
Invasive Pulmonary Aspergillosis ◽  
Hyphal Growth ◽  
Fungal Virulence ◽  
Th17 Response ◽  
Content Type ◽  
Genes Encoding ◽  
Element Binding Protein

Aspergillus fumigatusis the most common pathogenic mold infecting humans and a significant cause of morbidity and mortality in immunocompromised patients. In invasive pulmonary aspergillosis,A. fumigatusspores are inhaled into the lungs, undergoing germination and invasive hyphal growth. The fungus occludes and disrupts the blood vessels, leading to hypoxia and eventual tissue necrosis. The ability of this mold to adapt to hypoxia is regulated in part by the sterol regulatory element binding protein (SREBP) SrbA and the DscA to DscD Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. Loss of the genes encoding these proteins results in avirulence. To identify novel regulators of hypoxia sensing, we screened theNeurospora crassagene deletion library under hypoxia and identified a novel rhomboid family protease essential for hypoxic growth. Deletion of theA. fumigatusrhomboid homologrbdAresulted in an inability to grow under hypoxia, hypersensitivity to CoCl2, nikkomycin Z, fluconazole, and ferrozine, abnormal swollen tip morphology, and transcriptional dysregulation—accurately phenocopying deletion ofsrbA. In vivo,rbdAdeletion resulted in increased sensitivity to phagocytic killing, a reduced inflammatory Th1 and Th17 response, and strongly attenuated virulence. Phenotypic rescue of the ΔrbdAmutant was achieved by expression and nuclear localization of the N terminus of SrbA, including its HLH domain, further indicating that RbdA and SrbA act in the same signaling pathway. In summary, we have identified RbdA, a novel putative rhomboid family protease inA. fumigatusthat mediates hypoxia adaptation and fungal virulence and that is likely linked to SrbA cleavage and activation.

scholarly journals Complex Regulation Pathways of AmpC-Mediated β-Lactam Resistance in Enterobacter cloacae Complex

2015 ◽  
Vol 59 (12) ◽  
pp. 7753-7761 ◽  
Author(s):  
François Guérin ◽  
Christophe Isnard ◽  
Vincent Cattoir ◽  
Jean Christophe Giard
Keyword(s):  
Drug Targets ◽  
Genetic Regulation ◽  
Enterobacter Cloacae ◽  
Opportunistic Pathogen ◽  
Regulation Mechanism ◽  
Content Type ◽  
Genes Encoding ◽  
High Concentrations ◽  
High Level

ABSTRACTEnterobacter cloacaecomplex (ECC), an opportunistic pathogen causing numerous infections in hospitalized patients worldwide, is able to resist β-lactams mainly by producing the AmpC β-lactamase enzyme. AmpC expression is highly inducible in the presence of some β-lactams, but the underlying genetic regulation, which is intricately linked to peptidoglycan recycling, is still poorly understood. In this study, we constructed different mutant strains that were affected in genes encoding enzymes suspected to be involved in this pathway. As expected, the inactivation ofampC,ampR(which encodes the regulator protein ofampC), andampG(encoding a permease) abolished β-lactam resistance. Reverse transcription-quantitative PCR (qRT-PCR) experiments combined with phenotypic studies showed that cefotaxime (at high concentrations) and cefoxitin induced the expression ofampCin different ways: one involving NagZ (aN-acetyl-β-d-glucosaminidase) and another independent of NagZ. Unlike the model established forPseudomonas aeruginosa, inactivation of DacB (also known as PBP4) was not responsible for a constitutiveampCoverexpression in ECC, whereas it caused AmpC-mediated high-level β-lactam resistance, suggesting a post-transcriptional regulation mechanism. Global transcriptomic analysis by transcriptome sequencing (RNA-seq) of adacBdeletion mutant confirmed these results. Lastly, analysis of 37 ECC clinical isolates showed that amino acid changes in the AmpD sequence were likely the most crucial event involved in the development of high-level β-lactam resistancein vivoas opposed toP. aeruginosawheredacBmutations have been commonly found. These findings bring new elements for a better understanding of β-lactam resistance in ECC, which is essential for the identification of novel potential drug targets.

scholarly journals Evaluation of CpxRA as a Therapeutic Target for UropathogenicEscherichia coliInfections

2018 ◽  
Vol 86 (3) ◽  
pp. e00798-17 ◽  
Author(s):  
Lana Dbeibo ◽  
Julia J. van Rensburg ◽  
Sara N. Smith ◽  
Kate R. Fortney ◽  
Dharanesh Gangaiah ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Virulence Determinants ◽  
Membrane Repair ◽  
Single Strain ◽  
Content Type ◽  
Phosphatase Inhibitors ◽  
Genes Encoding ◽  
Tract Infections

ABSTRACTCpxRA is an envelope stress response system found in all members of the familyEnterobacteriaceae; CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR, a transcription factor. CpxR also accepts phosphate groups from acetyl phosphate, a glucose metabolite. Activation of CpxR increases the transcription of genes encoding membrane repair and downregulates virulence determinants. We hypothesized that activation of CpxR could serve as an antimicrobial/antivirulence strategy and discovered compounds that activate CpxR inEscherichia coliby inhibiting CpxA phosphatase activity. As a prelude to testing such compoundsin vivo, here we constructedcpxA(in the presence of glucose, CpxR is activated because of a lack of CpxA phosphatase) andcpxR(system absent) deletion mutants of uropathogenicE. coli(UPEC) CFT073. By RNA sequencing, few transcriptional differences were noted between thecpxRmutant and its parent, but in thecpxAmutant, several UPEC virulence determinants were downregulated, including thefimandpapoperons, and it exhibited reduced mannose-sensitive hemagglutination of guinea pig red blood cellsin vitro. In competition experiments with mice, both mutants were less fit than the parent in the urine, bladder, and kidney; these fitness defects were complemented intrans. Unexpectedly, in single-strain challenges, only thecpxAmutant was attenuated for virulence in the kidney but not in the bladder or urine. For thecpxAmutant, this may be due to the preferential use of amino acids over glucose as a carbon source in the bladder and urine by UPEC. These studies suggest that CpxA phosphatase inhibitors may have some utility for treating complex urinary tract infections.

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