RESUMO
Extracellular vesicles (EVs) are critical elements of cell-cell communication. Here, we characterized the outer membrane vesicles (OMVs) released by specific clones of Escherichia coli isolated from the Long-Term Evolution Experiment after 50,000 generations (50K) of adaptation to glucose minimal medium. Compared with their ancestor, the evolved clones produce small OMVs but also larger ones which display variable amounts of both OmpA and LPS. Tracking ancestral, fluorescently labelled OMVs revealed that they fuse with both ancestral- and 50K-evolved cells, albeit in different proportions. We quantified that less than 2% of the cells from one 50K-evolved clone acquired the fluorescence delivered by OMVs from the ancestral strain but that one cell concomitantly fuses with several OMVs. Globally, our results showed that OMV production in E. coli is a phenotype that varies along bacterial evolution and question the contribution of OMVs-mediated interactions in bacterial adaptation.
Assuntos
Escherichia coli , Vesículas Extracelulares , Escherichia coli/genética , Proteínas da Membrana Bacteriana Externa/genéticaRESUMO
The microbiota constitutes an important part of the holobiont in which extracellular vesicles (EVs) are key players in health, especially regarding inter- and intra-kingdom communications. Analysis of EVs from the red blood cell concentrates of healthy donors revealed variable amounts of OmpA and LPS in 12 of the 14 analyzed samples, providing indirect experimental evidence of the presence of microbiota EVs in human circulating blood in the absence of barrier disruption. To investigate the role of these microbiota EVs, we tracked the fusion of fluorescent Escherichia coli EVs with blood mononuclear cells and showed that, in the circulating blood, these EVs interacted almost exclusively with monocytes. This study demonstrates that bacterial EVs constitute critical elements of the host-microbiota cellular communication. The analysis of bacterial EVs should thus be systematically included in any characterization of human EVs.
Assuntos
Vesículas Extracelulares , Microbiota , Humanos , Nível de Saúde , Eritrócitos , Monócitos , Escherichia coliRESUMO
Insertion sequences (IS) are ubiquitous bacterial mobile genetic elements, and the mutations they cause can be deleterious, neutral, or beneficial. The long-term dynamics of IS elements and their effects on bacteria are poorly understood, including whether they are primarily genomic parasites or important drivers of adaptation by natural selection. Here, we investigate the dynamics of IS elements and their contribution to genomic evolution and fitness during a long-term experiment with Escherichia coli. IS elements account for ~35% of the mutations that reached high frequency through 50,000 generations in those populations that retained the ancestral point-mutation rate. In mutator populations, IS-mediated mutations are only half as frequent in absolute numbers. In one population, an exceptionally high ~8-fold increase in IS150 copy number is associated with the beneficial effects of early insertion mutations; however, this expansion later slowed down owing to reduced IS150 activity. This population also achieves the lowest fitness, suggesting that some avenues for further adaptation are precluded by the IS150-mediated mutations. More generally, across all populations, we find that higher IS activity becomes detrimental to adaptation over evolutionary time. Therefore, IS-mediated mutations can both promote and constrain evolvability.
Assuntos
Bactérias/genética , Elementos de DNA Transponíveis/genética , Evolução Molecular , Mutagênese Insercional , Adaptação Fisiológica/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Aptidão Genética , Genoma Bacteriano , Taxa de Mutação , Fenótipo , Seleção GenéticaRESUMO
Over millennia, life has been exposed to ionizing radiation from cosmic rays and natural radioisotopes. Biological experiments in underground laboratories have recently demonstrated that the contemporary terrestrial radiation background impacts the physiology of living organisms, yet the evolutionary consequences of this biological stress have not been investigated. Explaining the mechanisms that give rise to the results of underground biological experiments remains difficult, and it has been speculated that hereditary mechanisms may be involved. Here, we have used evolution experiments in standard and very low-radiation backgrounds to demonstrate that environmental ionizing radiation does not significantly impact the evolutionary trajectories of E. coli bacterial populations in a 500 generations evolution experiment.
Assuntos
Radiação de Fundo/efeitos adversos , Escherichia coli/genética , Escherichia coli/efeitos da radiação , Evolução Molecular , Radiação Cósmica/efeitos adversos , Relação Dose-Resposta à Radiação , Escherichia coli/crescimento & desenvolvimento , Aptidão Genética/efeitos da radiação , MutaçãoRESUMO
DNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes' local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.
Assuntos
Cromossomos Bacterianos/genética , DNA Bacteriano/metabolismo , DNA Super-Helicoidal , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Transcrição Gênica , Proteínas de Bactérias/metabolismo , Simulação por Computador , Escherichia coli/genética , Perfilação da Expressão Gênica , Genes Bacterianos , Modelos Biológicos , Regiões Promotoras Genéticas , Processos Estocásticos , TranscriptomaRESUMO
Negative frequency-dependent selection (NFDS) is an important mechanism for species coexistence and for the maintenance of genetic polymorphism. Long-term coexistence nevertheless requires NFDS interactions to be resilient to further evolution of the interacting species or genotypes. For closely related genotypes, NFDS interactions have been shown to be preserved through successive rounds of evolution in coexisting lineages. On the contrary, the evolution of NFDS interactions between distantly related species has received less attention. Here, we tracked the co-evolution of Escherichia coli and Citrobacter freundii that initially differ in their ecological characteristics. We showed that these two bacterial species engaged in an NFDS interaction particularly resilient to further evolution: despite a very strong asymmetric rate of adaptation, their coexistence was maintained owing to an NFDS pattern where fitness increases steeply as the frequency decreases towards zero. Using a model, we showed how and why such NFDS pattern can emerge. These findings provide a robust explanation for the long-term maintenance of species at very low frequencies.
Assuntos
Bactérias , Ecologia , Polimorfismo Genético , Bactérias/genética , Citrobacter freundii/genética , Escherichia coli/genética , Seleção GenéticaRESUMO
Transcription regulatory networks (TRNs) are of central importance for both short-term phenotypic adaptation in response to environmental fluctuations and long-term evolutionary adaptation, with global regulatory genes often being targets of natural selection in laboratory experiments. Here, we combined evolution experiments, whole-genome resequencing, and molecular genetics to investigate the driving forces, genetic constraints, and molecular mechanisms that dictate how bacteria can cope with a drastic perturbation of their TRNs. The crp gene, encoding a major global regulator in Escherichia coli, was deleted in four different genetic backgrounds, all derived from the Long-Term Evolution Experiment (LTEE) but with different TRN architectures. We confirmed that crp deletion had a more deleterious effect on growth rate in the LTEE-adapted genotypes; and we showed that the ptsG gene, which encodes the major glucose-PTS transporter, gained CRP (cyclic AMP receptor protein) dependence over time in the LTEE. We then further evolved the four crp-deleted genotypes in glucose minimal medium, and we found that they all quickly recovered from their growth defects by increasing glucose uptake. We showed that this recovery was specific to the selective environment and consistently relied on mutations in the cis-regulatory region of ptsG, regardless of the initial genotype. These mutations affected the interplay of transcription factors acting at the promoters, changed the intrinsic properties of the existing promoters, or produced new transcription initiation sites. Therefore, the plasticity of even a single promoter region can compensate by three different mechanisms for the loss of a key regulatory hub in the E. coli TRN.
Assuntos
Evolução Biológica , Proteína Receptora de AMP Cíclico/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Regiões Promotoras Genéticas , Escherichia coli , Deleção de Genes , Mutação , FenótipoRESUMO
Understanding the extreme variation among bacterial genomes remains an unsolved challenge in evolutionary biology, despite long-standing debate about the relative importance of natural selection, mutation, and random drift. A potentially important confounding factor is the variation in mutation rates between lineages and over evolutionary history, which has been documented in several species. Mutation accumulation experiments have shown that hypermutability can erode genomes over short timescales. These results, however, were obtained under conditions of extremely weak selection, casting doubt on their general relevance. Here, we circumvent this limitation by analyzing genomes from mutator populations that arose during a long-term experiment with Escherichia coli, in which populations have been adaptively evolving for >50,000 generations. We develop an analytical framework to quantify the relative contributions of mutation and selection in shaping genomic characteristics, and we validate it using genomes evolved under regimes of high mutation rates with weak selection (mutation accumulation experiments) and low mutation rates with strong selection (natural isolates). Our results show that, despite sustained adaptive evolution in the long-term experiment, the signature of selection is much weaker than that of mutational biases in mutator genomes. This finding suggests that relatively brief periods of hypermutability can play an outsized role in shaping extant bacterial genomes. Overall, these results highlight the importance of genomic draft, in which strong linkage limits the ability of selection to purge deleterious mutations. These insights are also relevant to other biological systems evolving under strong linkage and high mutation rates, including viruses and cancer cells.
Assuntos
Escherichia coli/genética , Evolução Molecular , Genoma Bacteriano , Seleção Genética , Escherichia coli/fisiologia , Mutação , Taxa de Mutação , FilogeniaRESUMO
Adaptive diversification is an essential evolutionary process, one that produces phenotypic innovations including the colonization of available ecological niches. Bacteria can diverge in sympatry when ecological opportunities allow, but the underlying genetic mechanisms are often unknown. Perhaps, the longest-lasting adaptive diversification seen in the laboratory occurred during the long-term evolution experiment, in which 12 populations of Escherichia coli have been evolving independently for more than 65,000 generations from a common ancestor. In one population, two lineages, S and L, emerged at ~6500 generations and have dynamically coexisted ever since by negative frequency-dependent interactions mediated, in part, by acetate secretion by L. Mutations in spoT, arcA, and gntR promoted the emergence of the S lineage, although they reproduced only partially its phenotypic traits. Here, we characterize the evolved mechanism of acetate consumption by the S lineage that enabled invasion and coexistence with the L lineage. We identified an additional mutation in acs that, together with the arcA mutation, drove an early restructuring of the transcriptional control of central metabolism in S, leading to improved acetate consumption. Pervasive epistatic interactions within the S genome contributed to the exploitation of this new ecological opportunity. The emergence and maintenance of this long-term polymorphism is a complex multi-step process.
Assuntos
Adaptação Biológica , Evolução Biológica , Escherichia coli/genética , Mutação , Ácido Acético/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Fenótipo , Polimorfismo Genético , Pirofosfatases/genética , Proteínas Repressoras/genéticaRESUMO
Ecological opportunities promote population divergence into coexisting lineages. However, the genetic mechanisms that enable new lineages to exploit these opportunities are poorly understood except in cases of single mutations. We examined how two Escherichia coli lineages diverged from their common ancestor at the outset of a long-term coexistence. By sequencing genomes and reconstructing the genetic history of one lineage, we showed that three mutations together were sufficient to produce the frequency-dependent fitness effects that allowed this lineage to invade and stably coexist with the other. These mutations all affected regulatory genes and collectively caused substantial metabolic changes. Moreover, the particular derived alleles were critical for the initial divergence and invasion, indicating that the establishment of this polymorphism depended on specific epistatic interactions.
Assuntos
Epistasia Genética , Escherichia coli/genética , Escherichia coli/fisiologia , Mutação , Polimorfismo Genético , Alelos , Escherichia coli/metabolismo , Evolução Molecular , Genes Bacterianos , Genes Reguladores , Aptidão Genética , Genótipo , Glucose/metabolismo , Interações MicrobianasRESUMO
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen that resists the acidic gastric environment, colonizes the gut epithelium, and causes hemorrhagic colitis and hemolytic-uremic syndrome, especially in children. The genomic island OI-47 of E. coli O157:H7 contains a gene, z1528, encoding an EAL-domain protein potentially involved in c-di-GMP hydrolysis that is absent in non-pathogenic E. coli. This gene, designated vmpA, is co-transcribed with ycdT, which is present in non pathogenic E. coli and encodes a diguanylate cyclase involved in c-di-GMP synthesis. To test for vmpA function, we constructed a vmpA knockout mutant. We also overexpressed vmpA, purified the VmpA protein and assayed for its activity in vitro. We found that VmpA possesses c-di-GMP phosphodiesterase activity and that the vmpA mutation results in increased biofilm formation, and reduced swimming motility, which is consistent with the function determined in vitro. Unexpectedly, suppressor mutations arise frequently in the vmpA background suggesting that VmpA plays an important regulatory role in E. coli O157:H7. These findings represent an example of remarkable flexibility in the organization of c-di-GMP signaling pathways in closely related species.
Assuntos
GMP Cíclico/análogos & derivados , Escherichia coli O157/fisiologia , Diester Fosfórico Hidrolases/deficiência , Sequência de Aminoácidos , Biofilmes/crescimento & desenvolvimento , Movimento Celular/fisiologia , GMP Cíclico/metabolismo , Escherichia coli O157/enzimologia , Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Ilhas Genômicas , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Hibridização de Ácido Nucleico , Diester Fosfórico Hidrolases/metabolismo , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
Closely related organisms usually occupy similar ecological niches, leading to intense competition and even extinction. Such competition also can promote rapid phenotypic evolution and ecological divergence. This process may end with the stable occupation of distinct niches or, alternatively, may entail repeated bouts of evolution. Here we examine two Escherichia coli lineages, called L and S, that coexisted for more than 30,000 generations after diverging from a common ancestor. Both lineages underwent sustained phenotypic evolution based on global transcription and resource utilization profiles, with L seeming to encroach over time on the catabolic profile of S. Reciprocal invasion experiments with L and S clones from the same or different generations revealed evolutionary changes in their interaction, including an asymmetry that confirmed the encroachment by L on the niche of the S lineage. In general, L and S clones from the same generation showed negative frequency-dependent effects, consistent with stable coexistence. However, L clones could invade S clones from both earlier and later generations, whereas S clones could invade only L clones from earlier generations. In this system, the long-term coexistence of competing lineages evidently depended on successive rounds of evolution, rather than on initial divergence followed by a static equilibrium.
Assuntos
Ecologia , Escherichia coli/genética , Evolução Molecular , Análise por Conglomerados , Escherichia coli/crescimento & desenvolvimento , Perfilação da Expressão GênicaRESUMO
Microbiology research has recently undergone major developments that have led to great progress towards obtaining an integrated view of microbial cell function. Microbial genetics, high-throughput technologies and systems biology have all provided an improved understanding of the structure and function of bacterial genomes and cellular networks. However, integrated evolutionary perspectives are needed to relate the dynamics of adaptive changes to the phenotypic and genotypic landscapes of living organisms. Here, we review evolution experiments, carried out both in vivo with microorganisms and in silico with artificial organisms, that have provided insights into bacterial adaptation and emphasize the potential of bacterial regulatory networks to evolve.
Assuntos
Adaptação Biológica , Bactérias/genética , Fenômenos Fisiológicos Bacterianos , Evolução Biológica , Biologia Computacional/métodos , Técnicas Microbiológicas/métodosRESUMO
The phenotypic plasticity of global regulatory networks provides bacteria with rapid acclimation to a wide range of environmental conditions, while genetic changes in those networks provide additional flexibility as bacteria evolve across long time scales. We previously identified mutations in the global regulator-encoding gene fis that enhanced organismal fitness during a long-term evolution experiment with Escherichia coli. To gain insight into the effects of these mutations, we produced two-dimensional protein gels with strains carrying different fis alleles, including a beneficial evolved allele and one with an in-frame deletion. We found that Fis controls the expression of the major porin-encoding gene ompF in the E. coli B-derived ancestral strain used in the evolution experiment, a relationship that has not been described before. We further showed that this regulatory connection evolved over two different time scales, perhaps explaining why it was not observed before. On the longer time scale, we showed that this regulation of ompF by Fis is absent from the more widely studied K-12 strain and thus is specific to the B strain. On a shorter time scale, this regulatory linkage was lost during 20,000 generations of experimental evolution of the B strain. Finally, we mapped the Fis binding sites in the ompF regulatory region, and we present a hypothetical model of ompF expression that includes its other known regulators.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Fator Proteico para Inversão de Estimulação/metabolismo , Regulação da Expressão Gênica , Porinas/biossíntese , Sequência de Bases , Eletroforese em Gel Bidimensional , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/deficiência , Fator Proteico para Inversão de Estimulação/genética , Deleção de Genes , Dados de Sequência Molecular , Mutação , Proteoma/análiseRESUMO
Infection with Shiga-toxin producing Escherichia coli (STEC) may result in the development of the haemolytic-uremic syndrome (HUS), the main cause of acute renal failure in children. While O157:H7 STEC are associated with large outbreaks of HUS, it is difficult to predict whether a non-O157:H7 isolate can be pathogenic for humans. The mucosal innate immune response plays a central role in the pathogenesis of HUS; therefore, we compared the induction of IL-8 and CCL20 in human colon epithelial cells infected with strains belonging to different serotypes, isolated from cattle or from HUS patients. No correlation was observed between strain virulence and chemokine gene expression. Rather, the genetic background of the strains seems to determine the chemokine gene expression profile. Investigating the contribution of different bacterial factors in this process, we show that the type III secretion system of O157:H7 bacteria, but not the intimate adhesion, is required to stimulate the cells. In addition, H7, H10, and H21 flagellins are potent inducers of chemokine gene expression when synthesized in large amount.
Assuntos
Quimiocinas/biossíntese , Infecções por Escherichia coli/imunologia , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/imunologia , Adesinas Bacterianas/imunologia , Animais , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Linhagem Celular , Quimiocinas/genética , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Flagelina/imunologia , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras/imunologia , Fosfoproteínas/genética , Toxina Shiga I/genética , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Fatores de Virulência/genéticaRESUMO
In aquatic environments, biofilms constitute an ecological niche where Legionella pneumophila persists as sessile cells. However, very little information on the sessile mode of life of L. pneumophila is currently available. We report here the development of a model biofilm of L. pneumophila strain Lens and the first transcriptome analysis of L. pneumophila biofilm cells. Global gene expression analysis of sessile cells as compared to two distinct populations of planktonic cells revealed that a substantial proportion of L. pneumophila genes is differentially expressed, as 2.3 % of the 2932 predicted genes exhibited at least a twofold change in gene expression. Comparison with previous results defining the gene expression profile of replicative- and transmissive-phase Legionella suggests that sessile cells resemble bacteria in the replicative phase. Further analysis of the most strongly regulated genes in sessile cells identified two induced gene clusters. One contains genes that encode alkyl hydroperoxide reductases known to act against oxidative stress. The second encodes proteins similar to PvcA and PvcB that are involved in siderophore biosynthesis in Pseudomonas aeruginosa. Since iron has been reported to modify biofilm formation in other species, we further focused on iron control of gene expression and biofilm formation. Among the genes showing the greatest differences in expression between planktonic cells and biofilm, only pvcA and pvcB were regulated by iron concentration. A DeltapvcA L. pneumophila mutant showed no changes in biofilm formation compared to the wild-type, suggesting that the pvcA product is not mandatory for biofilm formation. However, biofilm formation by L. pneumophila wild-type and a DeltapvcA strain was clearly inhibited in iron-rich conditions.
Assuntos
Biofilmes/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Legionella pneumophila/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Vias Biossintéticas/genética , Deleção de Genes , Genes Bacterianos/genética , Legionella pneumophila/genética , Estresse Oxidativo/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sideróforos/biossíntese , Sideróforos/genéticaRESUMO
Shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli (EHEC) binds to endothelial cells expressing globotriaosylceramide-3 (Gb-3) and induces cell death by inhibiting translation. Nonetheless, the effects of Stx on human enterocytes, which lacks receptor Gb-3, remain less known. In this study, we questioned whether EHEC-derived Stx may modulate cellular signalization in the Gb-3-negative human epithelial cell line T84. Stx produced by EHEC was fixed and internalized by the cells. A weak activation of NF-kappaB was observed in T84 cells after EHEC infection. Cells infected with an isogenic mutant lacking stx1 and stx2, the genes encoding Stx, displayed an increased NF-kappaB DNA-binding activity. Consequently, the NF-kappaB-dependent CCL20 and IL-8 gene transcription and chemokine production were enhanced in T84 cells infected with the Stx mutant in comparison to the wild-type strain. Investigating the mechanism by which Stx modulates NF-kappaB activation, we showed that the PI3K/Akt signaling pathway was not induced by EHEC but was enhanced by the strain lacking Stx. Pharmacological inhibition of the PI3K/Akt signalization in EHEC DeltaStx-infected T84 cells yielded to a complete decrease of NF-kappaB activation and CCL20 and IL-8 mRNA expression. This demonstrates that the induction of the PI3K/Akt/NF-kappaB pathway is potentially induced by EHEC, but is inhibited by Stx in Gb-3-negative epithelial cells. Thus, Stx is an unrecognized modulator of the innate immune response of human enterocytes.
Assuntos
Escherichia coli/patogenicidade , Mucosa Intestinal/imunologia , NF-kappa B/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Toxina Shiga/toxicidade , Células Cultivadas , Quimiocina CCL20 , Quimiocinas CC/antagonistas & inibidores , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Interleucina-18/antagonistas & inibidores , Interleucina-18/genética , Interleucina-18/metabolismo , Mucosa Intestinal/química , Mucosa Intestinal/microbiologia , Proteínas Inflamatórias de Macrófagos/antagonistas & inibidores , Proteínas Inflamatórias de Macrófagos/genética , Proteínas Inflamatórias de Macrófagos/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Toxina Shiga/genética , Toxina Shiga/metabolismo , Triexosilceramidas/análiseRESUMO
Shiga-toxin (Stx) is the cardinal virulence factor of enterohemorrhagic Escherichia coli (EHEC). The genes encoding Stx are carried by a lambdoid phage integrated in the bacterial genome and are fully expressed after a bacterial SOS response induced by DNA-damaging agents. Because nitric oxide (NO) is an essential mediator of the innate immune response of infected colonic mucosa, we aimed to determine its role in Stx production by EHEC. Here we demonstrate that chemical or cellular sources of NO inhibit spontaneous and mitomycin C-induced stx mRNA expression and Stx synthesis, without altering EHEC viability. The synthesis of stx phage is also reduced by NO. This inhibitory effect apparently occurs through the NO-mediated sensitization of EHEC because mutation of the NO sensor nitrite-sensitive repressor results in loss of NO inhibiting activity on stx expression. Thus our findings identify NO as an inhibitor of stx expressing-phage propagation and Stx release and thus as a potential protective factor limiting the development of hemolytic syndromes.
Assuntos
Escherichia coli O157/metabolismo , Escherichia coli O157/patogenicidade , Óxido Nítrico/farmacologia , Toxina Shiga/antagonistas & inibidores , Bacteriófago lambda/efeitos dos fármacos , Bacteriófago lambda/genética , Linhagem Celular Tumoral , Técnicas de Cocultura , Neoplasias do Colo/patologia , Células Epiteliais/metabolismo , Escherichia coli O157/efeitos dos fármacos , Escherichia coli O157/genética , Escherichia coli O157/virologia , Regulação Bacteriana da Expressão Gênica , Genes Virais , Humanos , Mitomicina/farmacologia , Mutagênese Insercional , Doadores de Óxido Nítrico/farmacologia , RNA Mensageiro/metabolismo , Toxina Shiga/biossínteseRESUMO
Lantibiotics are antimicrobial peptides from the bacteriocin family, secreted by Gram-positive bacteria. These peptides differ from other bacteriocins by the presence of (methyl)lanthionine residues, which result from enzymatic modification of precursor peptides encoded by structural genes. Several groups of lantibiotics have been distinguished, the largest of which is the lacticin 481 group. This group consists of at least 16 members, including lacticin 481, streptococcin A-FF22, mutacin II, nukacin ISK-1, and salivaricins. We present the first review devoted to this lantibiotic group, knowledge of which has increased significantly within the last few years. After updating the group composition and defining the common properties of these lantibiotics, we highlight the most recent developments. The latter concern: transcriptional regulation of the lantibiotic genes; understanding the biosynthetic machinery, in particular the ability to perform in vitro prepeptide maturation; characterization of a novel type of immunity protein; and broad application possibilities. This group differs in many aspects from the best known lantibiotic group (nisin group), but shares properties with less-studied groups such as the mersacidin, cytolysin and lactocin S groups.
Assuntos
Antibacterianos , Bacteriocinas , Ácidos/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Bacteriocinas/química , Bacteriocinas/genética , Bacteriocinas/metabolismo , Bacteriocinas/farmacologia , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Dados de Sequência Molecular , Família Multigênica , Probióticos , Engenharia de Proteínas , Percepção de Quorum , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
We previously characterized three Lactococcus lactis promoters, P170, P1 and P3, which are induced by low pH. Here, we identified a novel 14 bp regulatory DNA region centred at around -41.5 and composed of three tetranucleotide sequences, boxes A, C and D. Boxes A and C contribute to P1 activity, whereas box D and the position of boxes ACD (renamed ACiD-box) are essential to P1 activity and acid response. We also identified a trans -acting protein, RcfB, which is involved in P170 and P1 basal activity and is essential for their pH induction. The regulator belongs to the Crp-Fnr family of transcription regulators. Overexpression of rcfB resulted in increased beta-galactosidase activities and lantibiotic lacticin 481 production from P170- and P1-controlled genes, respectively, in acid condition. RcfB is thus probably activated when cells encounter an acid environment. rcfB is co-transcribed with genes encoding an universal stress-like protein and a multidrug transporter. RcfB plays a role in acid adaptation, as the survival rate of an rcfB mutant after a lethal acid challenge was 130-fold lower than that of the wild-type strain, when the bacteria were first grown in acidic medium. The groESL promoter includes a sequence resembling an ACiD-box and the chaperone GroEL production is partly RcfB dependent in acid condition. Our results suggest that the ACiD-box could be the DNA target site of RcfB.