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1.
PLoS Genet ; 19(2): e1010347, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36763677

RESUMEN

Recombination is often suppressed at sex-determining loci in plants and animals, and at self-incompatibility or mating-type loci in plants and fungi. In fungal ascomycetes, recombination suppression around the mating-type locus is associated with pseudo-homothallism, i.e. the production of self-fertile dikaryotic sexual spores carrying the two opposite mating types. This has been well studied in two species complexes from different families of Sordariales: Podospora anserina and Neurospora tetrasperma. However, it is unclear whether this intriguing association holds in other species. We show here that Schizothecium tetrasporum, a fungus from a third family in the order Sordariales, also produces mostly self-fertile dikaryotic spores carrying the two opposite mating types. This was due to a high frequency of second meiotic division segregation at the mating-type locus, indicating the occurrence of a single and systematic crossing-over event between the mating-type locus and the centromere, as in P. anserina. The mating-type locus has the typical Sordariales organization, plus a MAT1-1-1 pseudogene in the MAT1-2 haplotype. High-quality genome assemblies of opposite mating types and segregation analyses revealed a suppression of recombination in a region of 1.47 Mb around the mating-type locus. We detected three evolutionary strata, indicating a stepwise extension of recombination suppression. The three strata displayed no rearrangement or transposable element accumulation but gene losses and gene disruptions were present, and precisely at the strata margins. Our findings indicate a convergent evolution of self-fertile dikaryotic sexual spores across multiple ascomycete fungi. The particular pattern of meiotic segregation at the mating-type locus was associated with recombination suppression around this locus, that had extended stepwise. This association between pseudo-homothallism and recombination suppression across lineages and the presence of gene disruption at the strata limits are consistent with a recently proposed mechanism of sheltering deleterious alleles to explain stepwise recombination suppression.


Asunto(s)
Ascomicetos , Sordariales , Genes del Tipo Sexual de los Hongos/genética , Reproducción/genética , Ascomicetos/genética , Sordariales/genética , Recombinación Genética/genética , Esporas
2.
Mol Phylogenet Evol ; 189: 107938, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37820761

RESUMEN

The order Sordariales is taxonomically diverse, and harbours many species with different lifestyles and large economic importance. Despite its importance, a robust genome-scale phylogeny, and associated comparative genomic analysis of the order is lacking. In this study, we examined whole-genome data from 99 Sordariales, including 52 newly sequenced genomes, and seven outgroup taxa. We inferred a comprehensive phylogeny that resolved several contentious relationships amongst families in the order, and cleared-up intrafamily relationships within the Podosporaceae. Extensive comparative genomics showed that genomes from the three largest families in the dataset (Chaetomiaceae, Podosporaceae and Sordariaceae) differ greatly in GC content, genome size, gene number, repeat percentage, evolutionary rate, and genome content affected by repeat-induced point mutations (RIP). All genomic traits showed phylogenetic signal, and ancestral state reconstruction revealed that the variation of the properties stems primarily from within-family evolution. Together, the results provide a thorough framework for understanding genome evolution in this important group of fungi.


Asunto(s)
Genómica , Sordariales , Humanos , Filogenia , Genómica/métodos , Genoma , Sordariales/genética , Secuencia de Bases , Evolución Molecular
3.
Mol Biol Evol ; 38(6): 2475-2492, 2021 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-33555341

RESUMEN

Sex chromosomes often carry large nonrecombining regions that can extend progressively over time, generating evolutionary strata of sequence divergence. However, some sex chromosomes display an incomplete suppression of recombination. Large genomic regions without recombination and evolutionary strata have also been documented around fungal mating-type loci, but have been studied in only a few fungal systems. In the model fungus Podospora anserina (Ascomycota, Sordariomycetes), the reference S strain lacks recombination across a 0.8-Mb region around the mating-type locus. The lack of recombination in this region ensures that nuclei of opposite mating types are packaged into a single ascospore (pseudohomothallic lifecycle). We found evidence for a lack of recombination around the mating-type locus in the genomes of ten P. anserina strains and six closely related pseudohomothallic Podospora species. Importantly, the size of the nonrecombining region differed between strains and species, as indicated by the heterozygosity levels around the mating-type locus and experimental selfing. The nonrecombining region is probably labile and polymorphic, differing in size and precise location within and between species, resulting in occasional, but infrequent, recombination at a given base pair. This view is also supported by the low divergence between mating types, and the lack of strong linkage disequilibrium, chromosomal rearrangements, transspecific polymorphism and genomic degeneration. We found a pattern suggestive of evolutionary strata in P. pseudocomata. The observed heterozygosity levels indicate low but nonnull outcrossing rates in nature in these pseudohomothallic fungi. This study adds to our understanding of mating-type chromosome evolution and its relationship to mating systems.


Asunto(s)
Evolución Biológica , Cromosomas Fúngicos , Genes del Tipo Sexual de los Hongos , Podospora/genética , Recombinación Genética , Conversión Génica , Heterocigoto , Autofecundación
4.
Fungal Genet Biol ; 161: 103711, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35597448

RESUMEN

The Crippled Growth (CG) cell degeneration of the model ascomycete Podospora anserina (strain S) is controlled by a prion-like element and has been linked to the self-activation of the PaMpk1 MAP kinase cascade. Here, we report on the identification of the "86-11" locus containing twelve genes, ten of which are involved either in setting up the self-activation loop of CG or in inhibiting this loop, as demonstrated by targeted gene deletion. Interestingly, deletion of the whole locus results only in the elimination of CG and in no detectable additional physiological defect. Sequence comparison shows that these ten genes belong to four different families, each one endowed with a specific activity: two encode factors activating the loop, a third one encodes a factor crucial for inhibition of the loop and the fourth one participates in inhibiting the loop in a pathway parallel to the one controlled by the previously described PDC1 gene. Intriguingly, a very distant homologue of this "86-11" locus is present at the syntenic position in Podospora comata (strain T) that do not present Crippled Growth. Introgression of the P. comata strain T locus in P. anserina strain S and the P. anserina strain S in P. comata strain T showed that both drive CG in the P. anserina strain S genetic background, but not in the genetic background of strain P. comata T, indicating that genetic determinants outside the twelve-gene locus are responsible for lack of CG in P. comata strain T. Our data question the role of this twelve-gene locus in the physiology of P. anserina.


Asunto(s)
Familia de Multigenes , Podospora , Eliminación de Gen , Sistema de Señalización de MAP Quinasas , Podospora/genética , Podospora/crecimiento & desarrollo
5.
Environ Microbiol ; 23(3): 1594-1607, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33393164

RESUMEN

Secreted proteins are key players in fungal physiology and cell protection against external stressing agents and antifungals. Oak stress-induced protein 1 (OSIP1) is a fungal-specific protein with unknown function. By using Podospora anserina and Phanerochaete chrysosporium as models, we combined both in vivo functional approaches and biophysical characterization of OSIP1 recombinant protein. The P. anserina OSIP1Δ mutant showed an increased sensitivity to the antifungal caspofungin compared to the wild type. This correlated with the production of a weakened extracellular exopolysaccharide/protein matrix (ECM). Since the recombinant OSIP1 from P. chrysosporium self-assembled as fibers and was capable of gelation, it is likely that OSIP1 is linked to ECM formation that acts as a physical barrier preventing drug toxicity. Moreover, compared to the wild type, the OSIP1Δ mutant was more sensitive to oak extractives including chaotropic phenols and benzenes. It exhibited a strongly modified secretome pattern and an increased production of proteins associated to the cell-wall integrity signalling pathway, when grown on oak sawdust. This demonstrates that OSIP1 has also an important role in fungal resistance to extractive-induced stress.


Asunto(s)
Phanerochaete , Podospora , Antifúngicos/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Phanerochaete/metabolismo , Transducción de Señal
6.
Am J Med Genet A ; 185(5): 1494-1497, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33522073

RESUMEN

First trimester ultrasound screening is an essential fetal examination performed generally at 11-13 weeks of gestation (WG). However, it does not allow for an accurate description of all fetal organs, partly due to their development in progress. Meanwhile, increased nuchal translucency (INT) is a widely used marker known to be associated with chromosomal deleterious rearrangements. We report on a 14 WG fetus with an association of INT and univentricular congenital heart malformation (CHM) leading to chorionic villous sampling (CVS). Cytogenetic investigations performed using array-Comparative Genomic Hybridization (CGH) and fluorescence in situ hybridization (FISH) demonstrated a 1.17 Mb deletion in 16q24.1 encompassing FOXF1 arisen de novo on maternal inherited chromosome. Fetopathological study confirmed CHM with hypoplastic left heart syndrome (HLHS) associating aortic atresia, mitral stenosis, and left ventricular hypoplasia and revealed in addition specific lung lesions corresponding to alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). This is so far the first case of first trimester prenatal diagnosis of ACDMPV due to the deletion of FOXF1 gene. An interpretation of the complex genomic data generated by ultrasound markers is facilitated considerably by the genotype-phenotype correlations on fetopathological examination.


Asunto(s)
Deleción Cromosómica , Factores de Transcripción Forkhead/genética , Predisposición Genética a la Enfermedad , Síndrome de Circulación Fetal Persistente/diagnóstico , Alveolos Pulmonares/anomalías , Cromosomas Humanos Par 16/genética , Hibridación Genómica Comparativa , Diagnóstico Precoz , Femenino , Estudios de Asociación Genética , Humanos , Hibridación Fluorescente in Situ , Recién Nacido , Síndrome de Circulación Fetal Persistente/genética , Síndrome de Circulación Fetal Persistente/patología , Embarazo , Diagnóstico Prenatal , Alveolos Pulmonares/patología , Venas Pulmonares/anomalías , Venas Pulmonares/diagnóstico por imagen , Venas Pulmonares/crecimiento & desarrollo , Venas Pulmonares/patología , Eliminación de Secuencia
7.
Mol Genet Genomics ; 294(1): 177-190, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30288581

RESUMEN

Mechanisms involved in fine adaptation of fungi to their environment include differential gene regulation associated with single nucleotide polymorphisms and indels (including transposons), horizontal gene transfer, gene copy amplification, as well as pseudogenization and gene loss. The two Podospora genome sequences examined here emphasize the role of pseudogenization and gene loss, which have rarely been documented in fungi. Podospora comata is a species closely related to Podospora anserina, a fungus used as model in several laboratories. Comparison of the genome of P. comata with that of P. anserina, whose genome is available for over 10 years, should yield interesting data related to the modalities of genome evolution between these two closely related fungal species that thrive in the same types of biotopes, i.e., herbivore dung. Here, we present the genome sequence of the mat + isolate of the P. comata reference strain T. Comparison with the genome of the mat + isolate of P. anserina strain S confirms that P. anserina and P. comata are likely two different species that rarely interbreed in nature. Despite having a 94-99% of nucleotide identity in the syntenic regions of their genomes, the two species differ by nearly 10% of their gene contents. Comparison of the species-specific gene sets uncovered genes that could be responsible for the known physiological differences between the two species. Finally, we identified 428 and 811 pseudogenes (3.8 and 7.2% of the genes) in P. anserina and P. comata, respectively. Presence of high numbers of pseudogenes supports the notion that difference in gene contents is due to gene loss rather than horizontal gene transfers. We propose that the high frequency of pseudogenization leading to gene loss in P. anserina and P. comata accompanies specialization of these two fungi. Gene loss may be more prevalent during the evolution of other fungi than usually thought.


Asunto(s)
Proteínas Fúngicas/genética , Podospora/genética , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Mapeo Cromosómico , Evolución Molecular , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Especiación Genética , Podospora/clasificación , Seudogenes , Análisis de Secuencia de ARN
8.
Dev Biol ; 421(2): 126-138, 2017 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-27979655

RESUMEN

Filamentous ascomycetes produce complex multicellular structures during sexual reproduction. Little is known about the genetic pathways enabling the construction of such structures. Here, with a combination of classical and reverse genetic methods, as well as genetic mosaic and graft analyses, we identify and provide evidence for key roles for two genes during the formation of perithecia, the sexual fruiting bodies, of the filamentous fungus Podospora anserina. Data indicate that the proteins coded by these two genes function cell-non-autonomously and that their activity depends upon conserved cysteines, making them good candidate for being involved in the transmission of a reactive oxygen species (ROS) signal generated by the PaNox1 NADPH oxidase inside the maturing fruiting body towards the PaMpk1 MAP kinase, which is located inside the underlying mycelium, in which nutrients are stored. These data provide important new insights to our understanding of how fungi build multicellular structures.


Asunto(s)
Cuerpos Fructíferos de los Hongos/crecimiento & desarrollo , Cuerpos Fructíferos de los Hongos/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Podospora/crecimiento & desarrollo , Podospora/genética , Transducción de Señal/genética , Secuencia de Aminoácidos , Western Blotting , Celulosa/farmacología , Secuencia Conservada , Cisteína/metabolismo , Evolución Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/metabolismo , Mosaicismo , Micelio/metabolismo , Fenotipo , Fosforilación/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Vacuolas/metabolismo
9.
Biochem Biophys Res Commun ; 503(2): 703-709, 2018 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-29932913

RESUMEN

We reported recently that the Parkinsonism-associated protein DJ-1 and its bacterial homologs Hsp31, YhbO and YajL function as deglycases that repair proteins and nucleotides from endogeneous glycation by glyoxal and methylglyoxal, two reactive by-products of glucose metabolism responsible for up to 60% of glycation damage. Here, we show that DJ-1, deglycase 1 and deglycase 2 repair glyoxal- and methylglyoxal-glycated substrates, whereas deglycase 3 principally repairs glyoxal-glycated substrates. Moreover, deglycase 1 and 2 are overexpressed in stationary phase, whereas deglycase 3 is steadily expressed throughout bacterial growth. Finally, deglycase mutants overexpress glyoxalases, aldoketoreductases, glutathione-S-transferase and efflux pumps to alleviate carbonyl stress. In the discussion, we present an overview of the multiple functions of DJ-1 proteins. Our thourough work on deglycases provides compelling evidence that their previously reported glyoxalase III activity merely reflects their deglycase activity. Moreover, for their deglycase activity the Maillard deglycases likely recruit: i) their chaperone activity to interact with glycated proteins, ii) glyoxalase 1 activity to catalyze the rearrangement of Maillard products (aminocarbinols and hemithioacetals) into amides and thioesters, respectively, iii) their protease activity to cleave amide bonds of glycated arginine, lysine and guanine, and iv) glyoxalase 2 activity to cleave thioester bonds of glycated cysteine. Finally, because glycation affects many cellular processes, the discovery of the Maillard deglycases, awaited since 1912, likely constitutes a major advance for medical research, including ageing, cancer, atherosclerosis, neurodegenerative, post-diabetic, renal and autoimmune diseases.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Proteínas Ribosómicas/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación , Glioxal/metabolismo , Humanos , Piruvaldehído/metabolismo
10.
Appl Environ Microbiol ; 83(2)2017 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-27836848

RESUMEN

Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi. IMPORTANCE: Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO.


Asunto(s)
Deshidrogenasas de Carbohidratos/genética , Celulosa/metabolismo , Proteínas Fúngicas/genética , Podospora/enzimología , Podospora/genética , Deshidrogenasas de Carbohidratos/metabolismo , Activación Enzimática/genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Fenotipo , Filogenia , Podospora/metabolismo
11.
Biochem Biophys Res Commun ; 470(2): 282-286, 2016 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-26774339

RESUMEN

YhbO and YajL belong to the PfpI/Hsp31/DJ-1 superfamily. Both proteins are involved in protection against environmental stresses. Here, we show that, like DJ-1 and Hsp31, they repair glyoxal- and methylglyoxal-glycated proteins. YhbO and YajL repair glycated serum albumin, collagen, glyceraldehyde-3-phosphate dehydrogenase, and fructose biphosphate aldolase. Bacterial extracts from deglycase mutants display increased glycation levels, whereas deglycase overexpression decreases protein glycation. Moreover, yhbO and yajL mutants display decreased viability in methylglyoxal- or glucose-containing media. Finally, the apparent glyoxalase activities of YhbO and YajL reflect their deglycase activities.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Glioxal/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Piruvaldehído/metabolismo , Proteínas Ribosómicas/metabolismo , Citoprotección/fisiología , Productos Finales de Glicación Avanzada/metabolismo
12.
Fungal Genet Biol ; 94: 1-10, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27353975

RESUMEN

In filamentous fungi, entrance into stationary phase is complex as it is accompanied by several differentiation and developmental processes, including the synthesis of pigments, aerial hyphae, anastomoses and sporophores. The regulatory networks that control these processes are still incompletely known. The analysis of the "Impaired in the development of Crippled Growth (IDC)" mutants of the model filamentous ascomycete Podospora anserina has already yielded important information regarding the pathway regulating entrance into stationary phase. Here, the genes affected in two additional IDC mutants are identified as orthologues of the Saccharomyces cerevisiae WHI2 and PSR1 genes, known to regulate stationary phase in this yeast, arguing for a conserved role of these proteins throughout the evolution of ascomycetes.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Redes Reguladoras de Genes , Micelio/genética , Podospora/genética , Proteínas Fúngicas/genética , Prueba de Complementación Genética , Mutación , Micelio/crecimiento & desarrollo , Fosforilación , Podospora/crecimiento & desarrollo
13.
Birth Defects Res A Clin Mol Teratol ; 106(4): 298-303, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26931099

RESUMEN

BACKGROUND: Monochorionic twins are generally considered as a monozygotic twin pregnancy. However, several cases of monochorial dizygotic twin pregnancies have been reported. CASE REPORT: We report on a rare case of monochorionic dizygotic twin pregnancy conceived after induced ovulation in a 32-year-old woman. The diagnosis was made on morphological ultrasound examination at 18+4 weeks of gestation, showing two fetuses with discordant sex. The amniocentesis was declined by the patient. RESULTS: The monochorionic status was confirmed after a histopathalogical study of the placenta. At delivery, both a phenotypically normal boy and a phenotypically normal girl without sexual abnormality were observed. This analysis also revealed the presence of vascular anastomoses between both fetal circulations. Postnatal cytogenetic analyses indicated the presence of a chimerism in peripheral blood lymphocytes. This chimerism was not observed in cells obtained from a buccal swab. Molecular determination of zygosity confirmed the existence of the confined peripheral blood chimerism with the presence of four parental alleles. CONCLUSION: We report on a case of monochorionic dizygotic twin pregnancy. This observation underlies the need to carefully assess twin pregnancies, especially when obtained after assisted reproductive technology.


Asunto(s)
Quimerismo , Gemelos Dicigóticos , Adulto , Femenino , Humanos , Masculino , Inducción de la Ovulación , Embarazo
14.
Microbiology (Reading) ; 161(11): 2220-31, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26377309

RESUMEN

YajL is the closest prokaryotic homologue of Parkinson's disease-associated DJ-1, a protein of undefined function involved in the oxidative stress response. We reported recently that YajL and DJ-1 protect cells against oxidative stress-induced protein aggregation by acting as covalent chaperones for the thiol proteome, including the NuoG subunit of NADH dehydrogenase 1, and that NADH dehydrogenase 1 activity is negligible in the yajL mutant. We report here that this mutant compensates for low NADH dehydrogenase activity by utilizing NADH-independent alternative dehydrogenases, including pyruvate oxidase PoxB and d-amino acid dehydrogenase DadA, and mixed acid aerobic fermentations characterized by acetate, lactate, succinate and ethanol excretion. The yajL mutant has a low adenylate energy charge favouring glycolytic flux, and a high NADH/NAD ratio favouring fermentations over pyruvate dehydrogenase and the Krebs cycle. DNA array analysis showed upregulation of genes involved in glycolytic and pentose phosphate pathways and alternative respiratory pathways. Moreover, the yajL mutant preferentially catabolized pyruvate-forming amino acids over Krebs cycle-related amino acids, and thus the yajL mutant utilizes pyruvate-centred respiro-fermentative metabolism to compensate for the NADH dehydrogenase 1 defect and constitutes an interesting model for studying eukaryotic respiratory complex I deficiencies, especially those associated with Alzheimer's and Parkinson's diseases.


Asunto(s)
Escherichia coli/enzimología , Escherichia coli/metabolismo , NADH Deshidrogenasa/deficiencia , Proteínas Ribosómicas/deficiencia , Aerobiosis , Proteínas de Escherichia coli , Fermentación , Perfilación de la Expresión Génica , Humanos , Análisis de Flujos Metabólicos , Análisis por Micromatrices , Modelos Biológicos , Datos de Secuencia Molecular , Trastornos Parkinsonianos/patología , Análisis de Secuencia de ADN
15.
Microb Cell Fact ; 14: 104, 2015 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-26178240

RESUMEN

BACKGROUND: Lactococcus lactis, a lactic acid bacterium traditionally used to ferment milk and manufacture cheeses, is also, in the biotechnology field, an interesting host to produce proteins of medical interest, as it is "Generally Recognized As Safe". Furthermore, as L. lactis naturally secretes only one major endogenous protein (Usp45), the secretion of heterologous proteins in this species facilitates their purification from a protein-poor culture medium. Here, we developed and optimized protein production and secretion in L. lactis to obtain proteins of high quality, both correctly folded and pure to a high extent. As proteins to be produced, we chose the two transmembrane members of the HtrA protease family in Staphylococcus aureus, an important extra-cellular pathogen, as these putative surface-exposed antigens could constitute good targets for vaccine development. RESULTS: A recombinant ORF encoding a C-terminal, soluble, proteolytically inactive and tagged form of each staphylococcal HtrA protein was cloned into a lactococcal expression-secretion vector. After growth and induction of recombinant gene expression, L. lactis was able to produce and secrete each recombinant rHtrA protein as a stable form that accumulated in the culture medium in similar amounts as the naturally secreted endogenous protein, Usp45. L. lactis growth in fermenters, in particular in a rich optimized medium, led to higher yields for each rHtrA protein. Protein purification from the lactococcal culture medium was easily achieved in one step and allowed recovery of highly pure and stable proteins whose identity was confirmed by mass spectrometry. Although rHtrA proteins were monomeric, they displayed the same secondary structure content, thermal stability and chaperone activity as many other HtrA family members, indicating that they were correctly folded. rHtrA protein immunogenicity was established in mice. The raised polyclonal antibodies allowed studying the expression and subcellular localization of wild type proteins in S. aureus: although both proteins were expressed, only HtrA1 was found to be, as predicted, exposed at the staphylococcal cell surface suggesting that it could be a better candidate for vaccine development. CONCLUSIONS: In this study, an efficient process was developed to produce and secrete putative staphylococcal surface antigens in L. lactis and to purify them to homogeneity in one step from the culture supernatant. This allowed recovering fully folded, stable and pure proteins which constitute promising vaccine candidates to be tested for protection against staphylococcal infection. L. lactis thus proved to be an efficient and competitive cell factory to produce proteins of high quality for medical applications.


Asunto(s)
Antígenos Bacterianos/química , Proteínas Bacterianas/química , Vacunas Bacterianas/química , Lactococcus lactis/genética , Péptido Hidrolasas/química , Staphylococcus aureus/enzimología , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/aislamiento & purificación , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Humanos , Lactococcus lactis/metabolismo , Ratones , Péptido Hidrolasas/genética , Péptido Hidrolasas/inmunología , Péptido Hidrolasas/aislamiento & purificación , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus/química , Staphylococcus aureus/inmunología
16.
Genome Biol Evol ; 16(3)2024 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-38386982

RESUMEN

The filamentous fungus Podospora anserina is a model organism used extensively in the study of molecular biology, senescence, prion biology, meiotic drive, mating-type chromosome evolution, and plant biomass degradation. It has recently been established that P. anserina is a member of a complex of 7 closely related species. In addition to P. anserina, high-quality genomic resources are available for 2 of these taxa. Here, we provide chromosome-level annotated assemblies of the 4 remaining species of the complex, as well as a comprehensive data set of annotated assemblies from a total of 28 Podospora genomes. We find that all 7 species have genomes of around 35 Mb arranged in 7 chromosomes that are mostly collinear and less than 2% divergent from each other at genic regions. We further attempt to resolve their phylogenetic relationships, finding significant levels of phylogenetic conflict as expected from a rapid and recent diversification.


Asunto(s)
Podospora , Podospora/genética , Filogenia , Reproducción , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo
17.
J Fungi (Basel) ; 10(1)2024 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-38276025

RESUMEN

The ascomycete Podospora anserina is a heterothallic filamentous fungus found mainly on herbivore dung. It is commonly used in laboratories as a model system, and its complete life cycle lasting eight days is well mastered in vitro. The main objective of our team is to understand better the global process of fruiting body development, named perithecia, induced normally in this species by fertilization. Three allelic mutants, named pfd3, pfd9, and pfd23 (for "promoting fruiting body development") obtained by UV mutagenesis, were selected in view of their abilities to promote barren perithecium development without fertilization. By complete genome sequencing of pfd3 and pfd9, and mutant complementation, we identified point mutations in the mcm1 gene as responsible for spontaneous perithecium development. MCM1 proteins are MADS box transcription factors that control diverse developmental processes in plants, metazoans, and fungi. We also identified using the same methods a mutation in the VelC gene as responsible for spontaneous perithecium development in the vacua mutant. The VelC protein belongs to the velvet family of regulators involved in the control of development and secondary metabolite production. A key role of MCM1 and VelC in coordinating the development of P. anserina perithecia with gamete formation and fertilization is highlighted.

18.
J Bacteriol ; 195(6): 1167-78, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23292772

RESUMEN

YajL is the most closely related Escherichia coli homolog of Parkinsonism-associated protein DJ-1, a protein with a yet-undefined function in the oxidative-stress response. YajL protects cells against oxidative-stress-induced protein aggregation and functions as a covalent chaperone for the thiol proteome, including FeS proteins. To clarify the cellular responses to YajL deficiency, transcriptional profiling of the yajL mutant was performed. Compared to the parental strain, the yajL mutant overexpressed genes coding for chaperones, proteases, chemical chaperone transporters, superoxide dismutases, catalases, peroxidases, components of thioredoxin and glutaredoxin systems, iron transporters, ferritins and FeS cluster biogenesis enzymes, DNA repair proteins, RNA chaperones, and small regulatory RNAs. It also overexpressed the RNA polymerase stress sigma factors sigma S (multiple stresses) and sigma 32 (protein stress) and activated the OxyR and SoxRS oxidative-stress transcriptional regulators, which together trigger the global stress response. The yajL mutant also overexpressed genes involved in septation and adopted a shorter and rounder shape characteristic of stressed bacteria. Biochemical experiments showed that this upregulation of many stress genes resulted in increased expression of stress proteins and improved biochemical function. Thus, protein defects resulting from the yajL mutation trigger the onset of a robust and global stress response in a prokaryotic model of DJ-1-associated Parkinsonism.


Asunto(s)
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/biosíntesis , Estrés Oxidativo/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Reparación del ADN , ADN Bacteriano/metabolismo , Escherichia coli/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hierro/metabolismo , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/genética , Mutación , Proteínas Oncogénicas/genética , Oxidación-Reducción , Trastornos Parkinsonianos/genética , Proteína Desglicasa DJ-1
19.
J Biol Chem ; 287(8): 5861-70, 2012 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-22157000

RESUMEN

YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently reported the aggregation of proteins in a yajL mutant in an oxidative stress-dependent manner and that YajL exhibits chaperone activity. Here, we show that YajL displays covalent chaperone and weak protein oxidoreductase activities that are dependent on its exposed cysteine 106. It catalyzes reduced RNase oxidation and scrambled RNase isomerization and insulin reduction and forms mixed disulfides with many cellular proteins upon oxidative stress. The formation of mixed disulfides was detected by immunoblotting bacterial extracts with anti-YajL antibodies under nonreducing conditions. Disulfides were purified from bacterial extracts on a YajL affinity column, separated by nonreducing-reducing SDS-PAGE, and identified by mass spectrometry. Covalent YajL substrates included ribosomal proteins, aminoacyl-tRNA synthetases, chaperones, catalases, peroxidases, and other proteins containing cysteines essential for catalysis or FeS cluster binding, such as glyceraldehyde-3-phosphate dehydrogenase, aldehyde dehydrogenase, aconitase, and FeS cluster-containing subunits of respiratory chains. In addition, we show that DJ-1 also forms mixed disulfides with cytoplasmic proteins upon oxidative stress. These results shed light on the oxidative stress-dependent chaperone function of YajL and identify YajL substrates involved in translation, stress protection, protein solubilization, and metabolism. They reveal a crucial role for cysteine 106 and suggest that DJ-1 also functions as a covalent chaperone. These findings are consistent with several defects observed in yajL or DJ-1 mutants, including translational defects, protein aggregation, oxidative stress sensitivity, and metabolic deficiencies.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Chaperonas Moleculares/metabolismo , Proteínas Oncogénicas/química , Proteoma/metabolismo , Proteínas Ribosómicas/metabolismo , Homología de Secuencia de Aminoácido , Compuestos de Sulfhidrilo/metabolismo , Disulfuros/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutación , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Estrés Oxidativo , Oxidorreductasas/metabolismo , Proteína Desglicasa DJ-1 , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética
20.
J Biol Chem ; 285(14): 10328-36, 2010 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-20124404

RESUMEN

YajL is the closest prokaryotic homolog of the parkinsonism-associated protein DJ-1 (40% sequence identity and similar three-dimensional structure), a protein of unknown function involved in the cellular response to oxidative stress. We report here that a yajL mutant of Escherichia coli displays an increased sensitivity to oxidative stress. It also exhibits a protein aggregation phenotype in aerobiosis, but not in anaerobiosis or in aerobic cells overexpressing superoxide dismutase, suggesting that protein aggregation depends on the presence of reactive oxygen species produced by respiratory chains. The protein aggregation phenotype of the yajL mutant, which can be rescued by the wild-type yajL gene, but not by the corresponding cysteine 106 mutant allele, is similar to that of multiple mutants deficient in superoxide dismutases and catalases, although intracellular hydrogen peroxide levels were not increased in the yajL mutant, suggesting that protein aggregation in this strain does not result from a hydrogen peroxide detoxification defect. Aggregation-prone proteins included 17 ribosomal proteins, the ATP synthase beta subunit, flagellin, and the outer membrane proteins OmpA and PAL; all of them are part of multiprotein complexes, suggesting that YajL might be involved in optimal expression of these complexes, especially during oxidative stress. YajL stimulated the renaturation of urea-unfolded citrate synthase and the solubilization of the urea-unfolded ribosomal proteins S1 and L3 and was more efficient as a chaperone in its oxidized form than in its reduced form. The mRNA levels of several aggregated proteins of the yajL mutant were severely affected, suggesting that YajL also acts at the level of gene expression. These two functions of YajL might explain the protein aggregation phenotype of the yajL mutant.


Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Mutación/genética , Proteínas Oncogénicas/química , Estrés Oxidativo , Aerobiosis , Anaerobiosis , Catalasa/metabolismo , Citrato (si)-Sintasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Peróxido de Hidrógeno/metabolismo , Conformación Proteica , Proteína Desglicasa DJ-1 , Pliegue de Proteína , Especies Reactivas de Oxígeno/metabolismo , Proteína Ribosomal L3 , Proteínas Ribosómicas/metabolismo , Superóxido Dismutasa/metabolismo
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