RESUMEN
Thiomonas bacteria are ubiquitous at acid mine drainage sites and play key roles in the remediation of water at these locations by oxidizing arsenite to arsenate, favouring the sorption of arsenic by iron oxides and their coprecipitation. Understanding the adaptive capacities of these bacteria is crucial to revealing how they persist and remain active in such extreme conditions. Interestingly, it was previously observed that after exposure to arsenite, when grown in a biofilm, some strains of Thiomonas bacteria develop variants that are more resistant to arsenic. Here, we identified the mechanisms involved in the emergence of such variants in biofilms. We found that the percentage of variants generated increased in the presence of high concentrations of arsenite (5.33 mM), especially in the detached cells after growth under biofilm-forming conditions. Analysis of gene expression in the parent strain CB2 revealed that genes involved in DNA repair were upregulated in the conditions where variants were observed. Finally, we assessed the phenotypes and genomes of the subsequent variants generated to evaluate the number of mutations compared to the parent strain. We determined that multiple point mutations accumulated after exposure to arsenite when cells were grown under biofilm conditions. Some of these mutations were found in what is referred to as ICE19, a genomic island (GI) carrying arsenic-resistance genes, also harbouring characteristics of an integrative and conjugative element (ICE). The mutations likely favoured the excision and duplication of this GI. This research aids in understanding how Thiomonas bacteria adapt to highly toxic environments, and, more generally, provides a window to bacterial genome evolution in extreme environments.
Asunto(s)
Arsenitos/metabolismo , Biopelículas/crecimiento & desarrollo , Burkholderiales , Genoma Bacteriano/genética , Adaptación Fisiológica/genética , Arseniatos/metabolismo , Arsénico/metabolismo , Burkholderiales/genética , Burkholderiales/crecimiento & desarrollo , Burkholderiales/metabolismo , Reparación del ADN/genética , Elementos Transponibles de ADN/genética , Evolución Molecular , Perfilación de la Expresión Génica , Variación Genética/genética , Islas Genómicas/genética , Minería , Secuenciación Completa del GenomaRESUMEN
Bacteria of the genus Thiomonas are found ubiquitously in arsenic contaminated waters such as acid mine drainage (AMD), where they contribute to the precipitation and the natural bioremediation of arsenic. In these environments, these bacteria have developed a large range of resistance strategies among which the capacity to form particular biofilm structures. The biofilm formation is one of the most ubiquitous adaptive response observed in prokaryotes to various stresses, such as those induced in the presence of toxic compounds. This study focused on the process of biofilm formation in three Thiomonas strains (CB1, CB2 and CB3) isolated from the same AMD. The results obtained here show that these bacteria are all capable of forming biofilms, but the architecture and the kinetics of formation of these biofilms differ depending on whether arsenite is present in the environment and from one strain to another. Indeed, two strains favoured biofilm formation, whereas one favoured motility in the presence of arsenite. To identify the underlying mechanisms, the patterns of expression of some genes possibly involved in the process of biofilm formation were investigated in Thiomonas sp. CB2 in the presence and absence of arsenite, using a transcriptomic approach (RNA-seq). The findings obtained here shed interesting light on how the formation of biofilms, and the motility processes contribute to the adaptation of Thiomonas strains to extreme environments.
Asunto(s)
Arsenitos/metabolismo , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Burkholderiales/efectos de los fármacos , Burkholderiales/fisiología , Contaminantes Ambientales/metabolismo , Locomoción/efectos de los fármacos , Burkholderiales/genética , Farmacorresistencia Bacteriana , Perfilación de la Expresión GénicaRESUMEN
To explore the conservation of Src homology 3 (SH3) domain-mediated networks in evolution, we compared the specificity landscape of these domains among four yeast species, Saccharomyces cerevisiae, Ashbya gossypii, Candida albicans, and Schizosaccharomyces pombe, encompassing 400 million years of evolution. We first aligned and catalogued the families of SH3-containing proteins in these four species to determine the relationships between homologous domains. Then, we tagged and purified all soluble SH3 domains (82 in total) to perform a quantitative peptide assay (SPOT) for each SH3 domain. All SPOT readouts were hierarchically clustered and we observed that the organization of the SH3 specificity landscape in three distinct profile classes remains conserved across these four yeast species. We also produced a specificity profile for each SH3 domain from manually aligned top SPOT hits and compared the within-family binding motif consensus. This analysis revealed a striking example of binding motif divergence in a C. albicans Rvs167 paralog, which cannot be explained by overall SH3 sequence or interface residue divergence, and we validated this specificity change with a yeast two-hybrid (Y2H) assay. In addition, we show that position-weighted matrices (PWM) compiled from SPOT assays can be used for binding motif screening in potential binding partners and present cases where motifs are either conserved or lost among homologous SH3 interacting proteins. Finally, by comparing pairwise SH3 sequence identity to binding profile correlation we show that for ~75% of all analyzed families the SH3 specificity profile was remarkably conserved over a large evolutionary distance. Thus, a high sequence identity within an SH3 domain family predicts conserved binding specificity, whereas divergence in sequence identity often coincided with a change in binding specificity within this family. As such, our results are important for future studies aimed at unraveling complex specificity networks of peptide recognition domains in higher eukaryotes, including mammals.
Asunto(s)
Evolución Molecular , Proteínas Fúngicas/química , Levaduras/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Alineación de Secuencia , Técnicas del Sistema de Dos Híbridos , Dominios Homologos srcRESUMEN
The spatial and temporal regulation of actin polymerization is crucial for various cellular processes. Members of the Wiskott-Aldrich syndrome protein (WASP) family activate the Arp2/3-complex leading to actin polymerization. The yeast Saccharomyces cerevisiae contains only one WASP homolog, Las17, that requires additional factors for its regulation. Lsb1 and Lsb2/Pin3 are two yeast homologous proteins bearing an SH3 domain that were identified as Las17-binding proteins. Lsb2/Pin3 that promotes prion induction was suggested to link this prion formation to the actin cytoskeleton. However, the cellular role of Lsb1 and the molecular function of both Lsb1 and Lsb2 remain unknown. In this study, we show that Lsb1 and/or Lsb2 full-length proteins inhibit Las17-mediated actin polymerization in vitro, Lsb2 being a less potent inhibitor of Las17 activity compared to Lsb1. Addition of Lsb1 or Lsb2 to the corresponding full-length Lsb1/2 further inhibits Las17 activity. Lsb1 and Lsb2 form homo- and hetero-oligomeric complexes suggesting that these two proteins could regulate Las17 activity via dimerization or cooperative binding. In vivo, overexpressed Lsb1 and Lsb2 proteins cluster Las17-CFP in few cytoplasmic punctate structures that are also positive for other Arp2/3-dependent actin polymerization effectors like Sla1 or Abp1. But, only Lsb1 overexpression blocks the internalization step of receptor-mediated endocytosis. This shows a specific function of Lsb1 in endocytosis.
Asunto(s)
Actinas/química , Proteínas Portadoras/metabolismo , Endocitosis , Multimerización de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas Portadoras/química , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
SIRT1 regulates energy homeostasis by controlling the acetylation status and activity of a number of enzymes and transcriptional regulators. The fact that NAD(+) levels control SIRT1 activity confers a hypothetical basis for the design of new strategies to activate SIRT1 by increasing NAD(+) availability. Here we show that the deletion of the poly(ADP-ribose) polymerase-1 (PARP-1) gene, encoding a major NAD(+)-consuming enzyme, increases NAD(+) content and SIRT1 activity in brown adipose tissue and muscle. PARP-1(-/-) mice phenocopied many aspects of SIRT1 activation, such as a higher mitochondrial content, increased energy expenditure, and protection against metabolic disease. Also, the pharmacologic inhibition of PARP in vitro and in vivo increased NAD(+) content and SIRT1 activity and enhanced oxidative metabolism. These data show how PARP-1 inhibition has strong metabolic implications through the modulation of SIRT1 activity, a property that could be useful in the management not only of metabolic diseases, but also of cancer.
Asunto(s)
Mitocondrias/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Sirtuina 1/metabolismo , Tejido Adiposo Pardo/enzimología , Tejido Adiposo Pardo/metabolismo , Animales , Metabolismo Energético , Ratones , Ratones Noqueados , Músculo Esquelético/enzimología , Músculo Esquelético/metabolismo , NAD/metabolismo , Estrés Oxidativo , Fenotipo , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Interferencia de ARN , ARN Interferente Pequeño , Sirtuina 1/genéticaRESUMEN
SIRT1 is a NAD(+)-dependent enzyme that affects metabolism by deacetylating key transcriptional regulators of energy expenditure. Here, we tested whether deletion of PARP-2, an alternative NAD(+)-consuming enzyme, impacts on NAD(+) bioavailability and SIRT1 activity. Our results indicate that PARP-2 deficiency increases SIRT1 activity in cultured myotubes. However, this increase was not due to changes in NAD(+) levels, but to an increase in SIRT1 expression, as PARP-2 acts as a direct negative regulator of the SIRT1 promoter. PARP-2 deletion in mice increases SIRT1 levels, promotes energy expenditure, and increases mitochondrial content. Furthermore, PARP-2(-/-) mice were protected against diet-induced obesity. Despite being insulin sensitized, PARP-2(-/-) mice were glucose intolerant due to a defective pancreatic function. Hence, while inhibition of PARP activity promotes oxidative metabolism through SIRT1 activation, the use of PARP inhibitors for metabolic purposes will require further understanding of the specific functions of different PARP family members.
Asunto(s)
Poli(ADP-Ribosa) Polimerasas/metabolismo , Sirtuina 1/metabolismo , Animales , Línea Celular , Grasas de la Dieta/farmacología , Metabolismo Energético , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Intolerancia a la Glucosa , Humanos , Resistencia a la Insulina , Ratones , Ratones Noqueados , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño , Sirtuina 1/genéticaRESUMEN
The macroPARPs Parp-9 and Parp-14 are macro domain containing poly(ADP-ribose) polymerases involved in transcriptional regulation in response to immunoregulatory cytokines. Their genes reside in the same locus (16B3), and the Parp-9 gene lies head-to-head and shares its promoter with the gene encoding its partner, Bbap. Here, we provide a detailed analysis of Parp-9, Parp-14, and Bbap expression during mouse development and adulthood. Parp-9 is developmentally regulated, and prominently expressed in the thymus and specific regions of the brain and gut. In adults, highest expression is maintained in the thymus and intestine. Parp-14 is more weakly expressed, mainly in the thymus during development and in adulthood. In addition, we show that Bbap is essentially coexpressed with Parp-9 during development and in adult mouse. However, the different levels of their transcripts detected in the developing brain and gut suggest that Bbap and Parp-9 display both common and independent tissue-specific regulations.
Asunto(s)
Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Poli(ADP-Ribosa) Polimerasas/genética , Animales , Encéfalo/embriología , Encéfalo/enzimología , Encéfalo/metabolismo , Femenino , Hibridación in Situ , Mucosa Intestinal/metabolismo , Intestinos/embriología , Intestinos/enzimología , Isoenzimas/genética , Ratones , Embarazo , Timo/embriología , Timo/enzimología , Timo/metabolismoRESUMEN
The peroxisome proliferator-activated receptor-gamma (PPARgamma, NR1C3) in complex with the retinoid X receptor (RXR) plays a central role in white adipose tissue (WAT) differentiation and function, regulating the expression of key WAT proteins. In this report we show that poly(ADP-ribose) polymerase-2 (PARP-2), also known as an enzyme participating in the surveillance of the genome integrity, is a member of the PPARgamma/RXR transcription machinery. PARP-2(-/-) mice accumulate less WAT, characterized by smaller adipocytes. In the WAT of PARP-2(-/-) mice the expression of a number of PPARgamma target genes is reduced despite the fact that PPARgamma1 and -gamma2 are expressed at normal levels. Consistent with this, PARP-2(-/-) mouse embryonic fibroblasts fail to differentiate to adipocytes. In transient transfection assays, PARP-2 small interference RNA decreases basal activity and ligand-dependent activation of PPARgamma, whereas PARP-2 overexpression enhances the basal activity of PPARgamma, although it does not change the maximal ligand-dependent activation. In addition, we show a DNA-dependent interaction of PARP-2 and PPARgamma/RXR heterodimer by chromatin immunoprecipitation. In combination, our results suggest that PARP-2 is a novel cofactor of PPARgamma activity.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/fisiología , Regulación de la Expresión Génica , PPAR gamma/metabolismo , Poli(ADP-Ribosa) Polimerasas/fisiología , Receptores X Retinoide/metabolismo , Células 3T3-L1 , Tejido Adiposo/metabolismo , Animales , Diferenciación Celular , Dimerización , Fibroblastos/metabolismo , Heterocigoto , Ratones , Ratones Transgénicos , Modelos BiológicosRESUMEN
Besides the established central role of poly(ADP-ribose) polymerase-1 (Parp-1) and Parp-2 in the maintenance of genomic integrity, accumulating evidence indicates that poly(ADP-ribosyl)ation may modulate epigenetic modifications under physiological conditions. Here, we provide in vivo evidence for the pleiotropic involvement of Parp-2 in both meiotic and postmeiotic processes. We show that Parp-2-deficient mice exhibit severely impaired spermatogenesis, with a defect in prophase of meiosis I characterized by massive apoptosis at pachytene and metaphase I stages. Although Parp-2(-/-) spermatocytes exhibit normal telomere dynamics and normal chromosome synapsis, they display defective meiotic sex chromosome inactivation associated with derailed regulation of histone acetylation and methylation and up-regulated X- and Y-linked gene expression. Furthermore, a drastically reduced number of crossover-associated Mlh1 foci are associated with chromosome missegregation at metaphase I. Moreover, Parp-2(-/-) spermatids are severely compromised in differentiation and exhibit a marked delay in nuclear elongation. Altogether, our findings indicate that, in addition to its well known role in DNA repair, Parp-2 exerts essential functions during meiosis I and haploid gamete differentiation.
Asunto(s)
Meiosis/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Espermatogénesis/fisiología , Animales , Apoptosis , Segregación Cromosómica/genética , Cromosomas de los Mamíferos/genética , Infertilidad Masculina , Masculino , Metafase/fisiología , Ratones , Poli(ADP-Ribosa) Polimerasas/deficiencia , Cromosomas Sexuales/genética , Espermatocitos/citología , Telómero/metabolismo , Testículo/citologíaRESUMEN
Poly(ADP-ribosyl)ation is an immediate DNA damage-dependent posttranslational modification of histones and nuclear proteins that contributes to the survival of injured proliferating cells. Poly(ADP-ribose) polymerases (PARPs) now constitute a superfamily of 18 proteins, encoded by different genes and displaying a common conserved catalytic domain. PARP-1 (113kDa), the founding member, and PARP-2 (62kDa) are both involved in DNA-break sensing and signaling when single strand break repair (SSBR) or base excision repair (BER) pathways are engaged. The generation by homologous recombination of deficient mouse models have confirmed the caretaker function of PARP-1 and PARP-2 in mammalian cells under genotoxic stress. This review summarizes our present knowledge on their physiological role in the cellular response to DNA damage and on the genetic interactions between PARP-1, PARP-2, Atm that play an essential role during early embryogenesis.
Asunto(s)
Daño del ADN , Regulación del Desarrollo de la Expresión Génica , Poli(ADP-Ribosa) Polimerasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Dominio Catalítico , Proteínas de Ciclo Celular , Proliferación Celular , Reparación del ADN , Proteínas de Unión al ADN , Heterocigoto , Histonas/metabolismo , Humanos , Ratones , Modelos Biológicos , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Supresoras de TumorRESUMEN
The DNA damage-dependent poly(ADP-ribose) polymerases, PARP-1 and PARP-2, homo- and heterodimerize and are both involved in the base excision repair (BER) pathway. Here, we report that mice carrying a targeted disruption of the PARP-2 gene are sensitive to ionizing radiation. Following alkylating agent treatment, parp-2(-/-)-derived mouse embryonic fibroblasts exhibit increased post-replicative genomic instability, G(2)/M accumulation and chromosome mis-segregation accompanying kinetochore defects. Moreover, parp-1(-/-)parp-2(-/-) double mutant mice are not viable and die at the onset of gastrulation, demonstrating that the expression of both PARP-1 and PARP-2 and/or DNA-dependent poly(ADP-ribosyl) ation is essential during early embryogenesis. Interestingly, specific female embryonic lethality is observed in parp-1(+/-)parp-2(-/-) mutants at E9.5. Meta phase analyses of E8.5 embryonic fibroblasts highlight a specific instability of the X chromosome in those females, but not in males. Together, these results support the notion that PARP-1 and PARP-2 possess both overlapping and non-redundant functions in the maintenance of genomic stability.