RESUMO
The nosocomial bacterium Acinetobacter baumannii is protected from antibiotic treatment by acquiring antibiotic resistances and by forming biofilms. Cell attachment, one of the first steps in biofilm formation, is normally induced by environmental metabolites. We hypothesized that vanillic acid (VA), the oxidized form of vanillin and a widely available metabolite, may play a role in A. baumannii cell attachment. We first discovered that A. baumannii actively breaks down VA through the evolutionarily conserved vanABKP genes. These genes are under the control of the repressor VanR, which we show binds directly to VanR binding sites within the vanABKP genes bidirectional promoter. VA in turn counteracts VanR inhibition. We identified a VanR binding site and searched for it throughout the genome, especially in pili encoding promoter genes. We found a VanR binding site in the pilus encoding csu operon promoter and showed that VanR binds specifically to it. As expected, a strain lacking VanR overproduces Csu pili and makes robust biofilms. Our study uncovers the role that VA plays in facilitating the attachment of A. baumannii cells to surfaces, a crucial step in biofilm formation. These findings provide valuable insights into a previously obscure catabolic pathway with significant clinical implications.
Assuntos
Acinetobacter baumannii , Aderência Bacteriana , Proteínas de Bactérias , Biofilmes , Fímbrias Bacterianas , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Ácido Vanílico , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/genética , Acinetobacter baumannii/efeitos dos fármacos , Ácido Vanílico/metabolismo , Ácido Vanílico/farmacologia , Biofilmes/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/genética , Óperon , Sítios de Ligação , Benzaldeídos/metabolismo , Benzaldeídos/farmacologiaRESUMO
Infections caused by Acinetobacter baumannii, a Gram-negative opportunistic pathogen, are difficult to eradicate due to the bacterium's propensity to quickly gain antibiotic resistances and form biofilms, a protective bacterial multicellular community. The A. baumannii DNA damage response (DDR) mediates the antibiotic resistance acquisition and regulates RecA in an atypical fashion; both RecALow and RecAHigh cell types are formed in response to DNA damage. The findings of this study demonstrate that the levels of RecA can influence formation and dispersal of biofilms. RecA loss results in surface attachment and prominent biofilms, while elevated RecA leads to diminished attachment and dispersal. These findings suggest that the challenge to treat A. baumannii infections may be explained by the induction of the DDR, common during infection, as well as the delicate balance between maintaining biofilms in low RecA cells and promoting mutagenesis and dispersal in high RecA cells. This study underscores the importance of understanding the fundamental biology of bacteria to develop more effective treatments for infections.
Assuntos
Acinetobacter baumannii , Acinetobacter baumannii/metabolismo , Dano ao DNA , Biofilmes , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Farmacorresistência Bacteriana MúltiplaRESUMO
Bacterial recombinational repair of double-strand breaks often begins with creation of initiating 3' single-stranded DNA (ssDNA) tails on each side of a double-strand break (DSB). Importantly, if the RecBCD pathway is followed, RecBCD creates a gap between the sequences at 3' ends of the initiating strands. The gap flanks the DSB and extends at least to the nearest Chi site on each strand. Once the initiating strands form ssDNA-RecA filaments, each ssDNA-RecA filament searches for homologous double-stranded DNA (dsDNA) to use as a template for the DNA synthesis needed to fill the gap created by RecBCD. Our experimental results show that the DNA synthesis requires formation of a heteroduplex dsDNA that pairs >20 contiguous bases in the initiating strand with sequence matched bases in a strand from the original dsDNA. To trigger synthesis, the heteroduplex must be near the 3' end of the initiating strand. Those experimentally determined requirements for synthesis combined with the Chi site dependence of the function of RecBCD and the distribution of Chi sites in bacterial genomes could allow the RecBCD pathway to avoid some genomic rearrangements arising from directly induced DSBs; however, the same three factors could promote other rearrangements.
Assuntos
DNA/genética , Exodesoxirribonuclease V/genética , Genoma Bacteriano/genética , Sequência de Bases , DNA/biossíntese , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Escherichia coli/genética , Ácidos Nucleicos Heteroduplexes/genética , Recombinases Rec A/genética , Recombinação GenéticaRESUMO
DNA recombination resulting from RecA-mediated strand exchange aided by RecBCD proteins often enables accurate repair of DNA double-strand breaks. However, the process of recombinational repair between short DNA regions of accidental similarity can lead to fatal genomic rearrangements. Previous studies have probed how effectively RecA discriminates against interactions involving a short similar sequence that is embedded in otherwise dissimilar sequences but have not yielded fully conclusive results. Here, we present results of in vitro experiments with fluorescent probes strategically located on the interacting DNA fragments used for recombination. Our findings suggest that DNA synthesis increases the stability of the recombination products. Fluorescence measurements can also probe the homology dependence of the extension of invading DNA strands in D-loops formed by RecA-mediated strand exchange. We examined the slow extension of the invading strand in a D-loop by DNA polymerase (Pol) IV and the more rapid extension by DNA polymerase LF-Bsu We found that when DNA Pol IV extends the invading strand in a D-loop formed by RecA-mediated strand exchange, the extension afforded by 82 bp of homology is significantly longer than the extension on 50 bp of homology. In contrast, the extension of the invading strand in D-loops by DNA LF-Bsu Pol is similar for intermediates with ≥50 bp of homology. These results suggest that fatal genomic rearrangements due to the recombination of small regions of accidental homology may be reduced if RecA-mediated strand exchange is immediately followed by DNA synthesis by a slow polymerase.
Assuntos
DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Recombinação Homóloga , Recombinases Rec A/química , Sondas de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Recombinases Rec A/genética , Recombinases Rec A/metabolismoRESUMO
The evolutionarily conserved Escherichia coli translesion DNA polymerase IV (DinB) is one of three enzymes that can bypass potentially deadly DNA lesions on the template strand during DNA replication. Remarkably, however, DinB is the only known translesion DNA polymerase active in RecA-mediated strand exchange during error-prone double-strand break repair. In this process, a single-stranded DNA (ssDNA)-RecA nucleoprotein filament invades homologous dsDNA, pairing the ssDNA with the complementary strand in the dsDNA. When exchange reaches the 3' end of the ssDNA, a DNA polymerase can add nucleotides onto the end, using one strand of dsDNA as a template and displacing the other. It is unknown what makes DinB uniquely capable of participating in this reaction. To explore this topic, we performed molecular modeling of DinB's interactions with the RecA filament during strand exchange, identifying key contacts made with residues in the DinB fingers domain. These residues are highly conserved in DinB, but not in other translesion DNA polymerases. Using a novel FRET-based assay, we found that DinB variants with mutations in these conserved residues are less effective at stabilizing RecA-mediated strand exchange than native DinB. Furthermore, these variants are specifically deficient in strand displacement in the absence of RecA filament. We propose that the amino acid patch of highly conserved residues in DinB-like proteins provides a mechanistic explanation for DinB's function in strand exchange and improves our understanding of recombination by providing evidence that RecA plays a role in facilitating DinB's activity during strand exchange.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Bacteriano/química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Recombinases Rec A/química , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Recombinases Rec A/metabolismoRESUMO
Acinetobacter baumannii is a Gram-negative opportunistic pathogen that is known to survive harsh environmental conditions and is a leading cause of hospital-acquired infections. Specifically, multicellular communities (known as biofilms) of A. baumannii can withstand desiccation and survive on hospital surfaces and equipment. Biofilms are bacteria embedded in a self-produced extracellular matrix composed of proteins, sugars, and/or DNA. Bacteria in a biofilm are protected from environmental stresses, including antibiotics, which provides the bacteria with selective advantage for survival. Although some gene products are known to play roles in this developmental process in A. baumannii, mechanisms and signaling remain mostly unknown. Here, we find that Lon protease in A. baumannii affects biofilm development and has other important physiological roles, including motility and the cell envelope. Lon proteases are found in all domains of life, participating in regulatory processes and maintaining cellular homeostasis. These data reveal the importance of Lon protease in influencing key A. baumannii processes to survive stress and to maintain viability.IMPORTANCEAcinetobacter baumannii is an opportunistic pathogen and is a leading cause of hospital-acquired infections. A. baumannii is difficult to eradicate and to manage, because this bacterium is known to robustly survive desiccation and to quickly gain antibiotic resistance. We sought to investigate biofilm formation in A. baumannii, since much remains unknown about biofilm formation in this bacterium. Biofilms, which are multicellular communities of bacteria, are surface attached and difficult to eliminate from hospital equipment and implanted devices. Our research identifies multifaceted physiological roles for the conserved bacterial protease Lon in A. baumannii These roles include biofilm formation, motility, and viability. This work broadly affects and expands understanding of the biology of A. baumannii, which will permit us to find effective ways to eliminate the bacterium.
Assuntos
Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/fisiologia , Biofilmes/crescimento & desenvolvimento , Locomoção , Protease La/metabolismo , Viabilidade Microbiana , Estresse FisiológicoRESUMO
In the nosocomial opportunistic pathogen Acinetobacter baumannii, RecA-dependent mutagenesis, which causes antibiotic resistance acquisition, is linked to the DNA damage response (DDR). Notably, unlike the Escherichia coli paradigm, recA and DDR gene expression in A. baumannii is bimodal. Namely, there is phenotypic variation upon DNA damage, which may provide a bet-hedging strategy for survival. Thus, understanding recA gene regulation is key to elucidate the yet unknown DDR regulation in A. baumannii Here, we identify a structured 5' untranslated region (UTR) in the recA transcript which serves as a cis-regulatory element. We show that a predicted stem-loop structure in this 5' UTR affects mRNA half-life and underlies bimodal gene expression and thus phenotypic variation in response to ciprofloxacin treatment. We furthermore show that the stem-loop structure of the recA 5' UTR influences intracellular RecA protein levels and, in vivo, impairing the formation of the stem-loop structure of the recA 5' UTR lowers cell survival of UV treatment and decreases rifampin resistance acquisition from DNA damage-induced mutagenesis. We hypothesize that the 5' UTR allows for stable recA transcripts during stress, including antibiotic treatment, enabling cells to maintain suitable RecA levels for survival. This innovative strategy to regulate the DDR in A. baumannii may contribute to its success as a pathogen.IMPORTANCEAcinetobacter baumannii is an opportunistic pathogen quickly gaining antibiotic resistances. Mutagenesis and antibiotic resistance acquisition are linked to the DNA damage response (DDR). However, how the DDR is regulated in A. baumannii remains unknown, since unlike most bacteria, A. baumannii does not follow the regulation of the Escherichia coli paradigm. In this study, we have started to uncover the mechanisms regulating the novel A. baumannii DDR. We have found that a cis-acting 5' UTR regulates recA transcript stability, RecA protein levels, and DNA damage-induced phenotypic variation. Though 5' UTRs are known to provide stability to transcripts in bacteria, this is the first example in which it regulates a bimodal DDR response through recA transcript stabilization, potentially enabling cells to have enough RecA for survival and genetic variability.
Assuntos
Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , Recombinases Rec A/química , Recombinases Rec A/genética , Regiões 5' não Traduzidas , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/efeitos da radiação , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fenótipo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Recombinases Rec A/metabolismo , Rifampina/farmacologia , Estresse Fisiológico , Raios UltravioletaRESUMO
The multidrug-resistant, opportunistic pathogen, Acinetobacter baumannii, has spread swiftly through hospitals worldwide. Previously, we demonstrated that A. baumannii regulates the expression of various genes in response to DNA damage. Some of these regulated genes, especially those encoding the multiple error-prone DNA polymerases, can be implicated in induced mutagenesis, leading to antibiotic resistance. Here, we further explore the DNA damage-inducible system at the single cell level using chromosomal transcriptional reporters for selected DNA damage response genes. We found the genes examined respond in a bimodal fashion to ciprofloxacin treatment, forming two phenotypic subpopulations: induced and uninduced. This bimodal response to ciprofloxacin treatment in A. baumannii is unique and quite different than the Escherichia coli paradigm. The subpopulations are not genetically different, with each subpopulation returning to a starting state and differentiating with repeated treatment. We then identified a palindromic motif upstream of certain DNA damage response genes, and have shown alterations to this sequence to diminish the bimodal induction in response to DNA damaging treatment. Lastly, we are able to show a biological advantage for a bimodal response, finding that one subpopulation survives ciprofloxacin treatment better than the other.
Assuntos
Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Ciprofloxacina/farmacologia , Dano ao DNA , Sequências Repetidas Invertidas , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Dano ao DNA/efeitos dos fármacos , Farmacorresistência Bacteriana , Genes Reporter/genética , Genoma Bacteriano , Metanossulfonato de Metila/farmacologia , Testes de Sensibilidade Microbiana , Fenótipo , Resposta SOS em GenéticaRESUMO
Escherichia coli strains overproducing DinB undergo survival loss; however, the mechanisms regulating this phenotype are poorly understood. Here we report a genetic selection revealing DinB residues essential to effect this loss-of-survival phenotype. The selection uses strains carrying both an antimutator allele of DNA polymerase III (Pol III) α-subunit (dnaE915) and either chromosomal or plasmid-borne dinB alleles. We hypothesized that dnaE915 cells would respond to DinB overproduction differently from dnaE(+) cells because the dnaE915 allele is known to have an altered genetic interaction with dinB(+) compared to its interaction with dnaE(+). Notably, we observe a loss-of-survival phenotype in dnaE915 strains with either a chromosomal catalytically inactive dinB(D103N) allele or a low-copy-number plasmid-borne dinB(+) upon DNA damage treatment. Furthermore, we find that the loss-of-survival phenotype occurs independently of DNA damage treatment in a dnaE915 strain expressing the catalytically inactive dinB(D103N) allele from a low-copy-number plasmid. The selective pressure imposed resulted in suppressor mutations that eliminated growth defects. The dinB intragenic mutations examined were either base pair substitutions or those that we inferred to be loss of function (i.e., deletions and insertions). Further analyses of selected novel dinB alleles, generated by single-base-pair substitutions in the dnaE915 strain, indicated that these no longer effect loss of survival upon overproduction in dnaE(+) strains. These mutations are mapped to specific areas of DinB; this permits us to gain insights into the mechanisms underlying the DinB-mediated overproduction loss-of-survival phenotype.
Assuntos
Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/fisiologia , Expressão Gênica , Viabilidade Microbiana , Seleção Genética , Alelos , Análise Mutacional de DNA , Proteínas de Escherichia coli/química , Modelos Moleculares , Mutagênese Insercional , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Plasmídeos , Mutação Puntual , Conformação Proteica , Deleção de Sequência , Supressão GenéticaRESUMO
Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen that survives desiccation and quickly acquires resistance to multiple antibiotics. Escherichia coli gains antibiotic resistances by expressing genes involved in a global response to DNA damage. Therefore, we asked whether A. baumannii does the same through a yet undetermined DNA damage response akin to the E. coli paradigm. We found that recA and all of the multiple error-prone DNA polymerase V (Pol V) genes, those organized as umuDC operons and unlinked, are induced upon DNA damage in a RecA-mediated fashion. Consequently, we found that the frequency of rifampin-resistant (Rif(r)) mutants is dramatically increased upon UV treatment, alkylation damage, and desiccation, also in a RecA-mediated manner. However, in the recA insertion knockout strain, in which we could measure the recA transcript, we found that recA was induced by DNA damage, while uvrA and one of the unlinked umuC genes were somewhat derepressed in the absence of DNA damage. Thus, the mechanism regulating the A. baumannii DNA damage response is likely different from that in E. coli. Notably, it appears that the number of DNA Pol V genes may directly contribute to desiccation-induced mutagenesis. Sequences of the rpoB gene from desiccation-induced Rif(r) mutants showed a signature that was consistent with E. coli DNA polymerase V-generated base-pair substitutions and that matched that of sequenced A. baumannii clinical Rif(r) isolates. These data strongly support an A. baumannii DNA damage-inducible response that directly contributes to antibiotic resistance acquisition, particularly in hospitals where A. baumannii desiccates and tenaciously survives on equipment and surfaces.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Farmacorresistência Bacteriana/genética , Recombinases Rec A/genética , Acinetobacter baumannii/genética , Adenosina Trifosfatases/genética , Alquilação , Dano ao DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Dessecação , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Técnicas de Inativação de Genes , Mutação , Rifampina/farmacologia , Resposta SOS em Genética/efeitos dos fármacos , Raios UltravioletaRESUMO
The activity of DinB is governed by the formation of a multiprotein complex (MPC) with RecA and UmuD. We identified two highly conserved surface residues in DinB, cysteine 66 (C66) and proline 67 (P67). Mapping on the DinB tertiary structure suggests these are noncatalytic, and multiple-sequence alignments indicate that they are unique among DinB-like proteins. To investigate the role of the C66-containing surface in MPC formation, we constructed the dinB(C66A) derivative. We found that DinB(C66A) copurifies with its interacting partners, RecA and UmuD, to a greater extent than DinB. Notably, copurification of RecA with DinB is somewhat enhanced in the absence of UmuD and is further increased for DinB(C66A). In vitro pulldown assays also indicate that DinB(C66A) binds RecA and UmuD better than DinB. We note that the increased affinity of DinB(C66A) for UmuD is RecA dependent. Thus, the C66-containing binding surface appears to be critical to modulate interaction with UmuD, and particularly with RecA. Expression of dinB(C66A) from the chromosome resulted in detectable differences in dinB-dependent lesion bypass fidelity and homologous recombination. Study of this DinB derivative has revealed a key surface on DinB, which appears to modulate the strength of MPC binding, and has suggested a binding order of RecA and UmuD to DinB. These findings will ultimately permit the manipulation of these enzymes to deter bacterial antibiotic resistance acquisition and to gain insights into cancer development in humans.
Assuntos
DNA Polimerase beta/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Recombinases Rec A/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , DNA Polimerase beta/metabolismo , DNA Polimerase Dirigida por DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Recombinases Rec A/genética , Alinhamento de SequênciaRESUMO
The wood cockroach Cryptocercus punctulatus nests as family units inside decayed wood, a substrate known for its high microbial load. We tested the hypothesis that defecation within their nests, a common occurrence in this species, reduces the probability of fungal development. Conidia of the entomopathogenic fungus, Metarhizium anisopliae, were incubated with crushed feces and subsequently plated on potato dextrose agar. Relative to controls, the viability of fungal conidia was significantly reduced following incubation with feces and was negatively correlated with incubation time. Although the cockroach's hindgut contained abundant ß-1,3-glucanase activity, its feces had no detectable enzymatic function. Hence, these enzymes are unlikely the source of the fungistasis. Instead, the antifungal compound(s) of the feces involved heat-sensitive factor(s) of potential microbial origin. When feces were boiled or when they were subjected to ultraviolet radiation and subsequently incubated with conidia, viability was "rescued" and germination rates were similar to those of controls. Filtration experiments indicate that the fungistatic activity of feces results from chemical interference. Because Cryptocercidae cockroaches have been considered appropriate models to make inferences about the factors fostering the evolution of termite sociality, we suggest that nesting in microbe-rich environments likely selected for the coupling of intranest defecation and feces fungistasis in the common ancestor of wood cockroaches and termites. This might in turn have served as a preadaptation that prevented mycosis as these phylogenetically related taxa diverged and evolved respectively into subsocial and eusocial organizations.
Assuntos
Baratas/microbiologia , Baratas/fisiologia , Fezes/microbiologia , Metarhizium/fisiologia , Comportamento de Nidação , Animais , Antifúngicos/farmacologia , Defecação , Fezes/enzimologia , Metarhizium/efeitos dos fármacos , Madeira/microbiologiaRESUMO
Acinetobacter baumannii is a Gram-negative nosocomial opportunistic pathogen frequently found in hospital settings, causing high incidence of in-hospital infections. It belongs to the ESKAPE group of pathogens (the "A" stands for A. baumannii), which are known to easily develop antibiotic resistances. It is crucial to create a molecular toolkit to investigate its basic biology, such as gene regulation. Despite A. baumannii having been a threat for almost two decades, an efficient and high-throughput plasmid system that can replicate in A. baumannii has not yet been developed. This study adapts an existing toolkit for Escherichia coli to meet A. baumannii's unique requirements and expands it by constructing a plasmid-based CRISPR interference (CRISPRi) system to generate gene knockdowns in A. baumannii.
Assuntos
Acinetobacter baumannii , Acinetobacter baumannii/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Escherichia coli/genética , Técnicas de Silenciamento de Genes , Expressão GênicaRESUMO
Translesion synthesis (TLS) by Y-family DNA polymerases is a chief mechanism of DNA damage tolerance. Such TLS can be accurate or error-prone, as it is for bypass of a cyclobutane pyrimidine dimer by DNA polymerase eta (XP-V or Rad30) or bypass of a (6-4) TT photoproduct by DNA polymerase V (UmuD'2C), respectively. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, the biological rationale for this striking conservation has remained enigmatic. Here we report that the Escherichia coli dinB gene is required for resistance to some DNA-damaging agents that form adducts at the N2-position of deoxyguanosine (dG). We show that DinB (DNA polymerase IV) catalyses accurate TLS over one such N2-dG adduct (N2-furfuryl-dG), and that DinB and its mammalian orthologue, DNA polymerase kappa, insert deoxycytidine (dC) opposite N2-furfuryl-dG with 10-15-fold greater catalytic proficiency than opposite undamaged dG. We also show that mutating a single amino acid, the 'steric gate' residue of DinB (Phe13 --> Val) and that of its archaeal homologue Dbh (Phe12 --> Ala), separates the abilities of these enzymes to perform TLS over N2-dG adducts from their abilities to replicate an undamaged template. We propose that DinB and its orthologues are specialized to catalyse relatively accurate TLS over some N2-dG adducts that are ubiquitous in nature, that lesion bypass occurs more efficiently than synthesis on undamaged DNA, and that this specificity may be achieved at least in part through a lesion-induced conformational change.
Assuntos
Aminoácidos/metabolismo , Adutos de DNA/metabolismo , Dano ao DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Aminoácidos/genética , Animais , Pareamento Incorreto de Bases/genética , Catálise , Sequência Conservada , DNA/biossíntese , Adutos de DNA/genética , Dano ao DNA/efeitos dos fármacos , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Desoxiguanosina/genética , Desoxiguanosina/metabolismo , Proteínas de Escherichia coli/genética , Cinética , Mutagênicos/farmacologia , Mutação/genética , Especificidade por Substrato , Moldes GenéticosRESUMO
Microplastics have become one of the most serious global threats to animal and human health. While their presence has been documented in all Earth water ecosystems, including remote mountain lakes, the observation that the abundance of microplastics is largely different across nearby lakes has rarely been examined. As part of a citizen science initiative, this study analyzed for the first time the abundance of microplastics in the surface of 35 glacial lakes of Sierra Nevada National Park in Southern Spain with the objective of determining the local factors that control their abundance. First, we described the shape, size, color and nature of microplastics. Second, we tested whether the number of microplastics differed between basins and analyzed environmental and morphometrical features of lakes affecting their abundance. We found that microplastics were common in most lakes, with a maximum abundance of 21.3 particles per liter that akin to some of the most microplastic polluted lakes worldwide. Fragments were the predominant shape (59.7%) followed by fibers (38.8%) and very scarce spheres (1.5%). Microplastics were observed for all size-fractions, but the abundance of particles <45 µm was higher, what advocates for the use of low pore-size filters to prevent underestimation of microplastics. While the mean abundance of microplastics did not differ among basins, their quantity was related to the presence of meadows surrounding the lakes. This result indicates that while atmospheric transport of microsplastics may equally reach all basins, differences in microplastics among nearby-lakes has an anthropic origin caused by mountaineers who find lakes with ample meadows much more attractive to visit relative to barren lakes. The staggering number in these remote lakes, headwaters of rivers that feed drinking reservoirs, is a major concern that warrants further investigation and the strict compliance with waste management laws to reduce the harmful impacts of microplastic contamination.
Assuntos
Microplásticos , Poluentes Químicos da Água , Animais , Ecossistema , Monitoramento Ambiental , Humanos , Lagos , Plásticos , Espanha , Poluentes Químicos da Água/análiseRESUMO
Sources contributing to specific concentration of microplastics and fibers are still not completely understood. This study aimed at assessing the concentrations of microplastics (2-5000 µm) and fibers (18-5667 µm) in three areas of distinct influences in the Douro river, Porto, Portugal: (i) a countryside area; (ii) a wastewater treatment effluent release zone; and (iii) an area in proximity to a boat dock and maintenance station. Nile Red staining coupled with microscopy allowed the identification of small microplastics (≥2 µm) with a median concentration of the three areas of 231 MP L-1. Most were fragments (69%). Sizes <40 µm were the most abundant (84%). Highest concentrations of microplastics were found near the boat dock/maintenance and lowest in the countryside area. Fibers were mostly natural (non-synthetic, 63%). Highest concentrations of fibers were found in the area influenced by the wastewater effluent, especially of synthetic fibers, and lowest in the countryside area. Concentration of all fibers and synthetic fibers was 46 F L-1 and 6 F L-1, respectively. High concentrations of microplastics and fiber contamination suggest that the wastewater treatment plant effluent and boat dock/maintenance are the likely sources originating hotspot areas.
RESUMO
Microplastics became an unprecedented challenge and mapping their contamination all over the world is needed in order to establish baseline levels and identify the polymers in order to enhance adequate legislation and policy. The main objective of this study is to demonstrate the existence of microplastic pollution on three beaches on the coast of Granada (Spain), namely La Herradura, Motril Beach and La Rábita, characterizing the particles and the relationships in their distribution. This may contribute supporting the studies carried out at a national level in accordance with the Directive on Marine Strategy (2008/56/EC). The results showed a greater median concentration of particles/kg of dry sediment in La Herradura (45.0⯱â¯24.7) than in Motril (31.5⯱â¯21.5) and La Rábita (22.0⯱â¯23.2). These data revealed a higher contamination by microplastics in an enclosed bay-type beach (La Herradura) in comparison with open delta-type beaches. The predominant morphologies were microspheres and fragments, with maximum median concentrations of 38.0⯱â¯23.7 and 6.0⯱â¯0.7 particles/kg, respectively. The distribution and size of the particles is affected by the geomorphological and sedimentary characteristics of these beaches, which are different from any other in Spain and in the Mediterranean in general. The beaches of Granada showed more microplastic contamination than Greek or Slovenian beaches, but less than other Spanish beaches. In this area of the Mediterranean, the presence of microplastics can be affected by the wind, sea currents or methodological aspects such as the pore size of the filters used. All of these factors were analysed when comparing the beaches of Granada with other Mediterranean beaches. This study shows that there is contamination by microplastics on the beaches of Granada, which have been little explored until now due to the difficult geological and granulometric characteristics, and gives support to other national studies.
RESUMO
NusA, a modulator of RNA polymerase, interacts with the DNA polymerase DinB. An increased level of expression of dinB or umuDC suppresses the temperature sensitivity of the nusA11 strain, requiring the catalytic activities of these proteins. We propose that NusA recruits translesion DNA synthesis (TLS) polymerases to RNA polymerases stalled at gaps, coupling TLS to transcription.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sítios de Ligação , Dano ao DNA , Replicação do DNA , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Conformação Molecular , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Ligação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Elongação da TranscriçãoRESUMO
We characterized the nanLET operon in Bacteroides fragilis, whose products are required for the utilization of the sialic acid N-acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is nanL, which codes for an aldolase that cleaves NANA into N-acetyl mannosamine (manNAc) and pyruvate. The next gene, nanE, codes for a manNAc/N-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is nanT, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of B. fragilis to grow on NANA. Although B. fragilis does not normally grow with manNAc as the sole carbon source, we isolated a B. fragilis mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the nanE epimerase gene or the rokA hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus, B. fragilis possesses a new pathway of NANA utilization, which we show is also found in other Bacteroides species.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteroides fragilis/enzimologia , Bacteroides fragilis/metabolismo , Carboidratos Epimerases/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Proteínas de Bactérias/genética , Bacteroides fragilis/genética , Carboidratos Epimerases/genética , Hexosaminas/metabolismo , Modelos Biológicos , MutaçãoRESUMO
Contrary to the generally held notions about microbial survival, the recently published paper by Johnson et al., 'Ancient bacteria show evidence of DNA repair', presents evidence suggesting that non-spore-forming bacteria in ancient samples are apparently alive, as judged by intact DNA, and fare better than spores. The data presented in this work raise intriguing questions about the nature of bacteria in many of the ancient samples reported to date: are they spores, persisters, sessile vegetative cells or do they make up a slow-growing population?