RESUMO
Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Glutamato-Amônia Ligase/deficiência , Glutamina/sangue , Hiperamonemia/genética , NAD/sangue , NAD/deficiência , Linfócitos B/citologia , Técnicas de Cultura de Células , Suplementos Nutricionais , Fibroblastos/citologia , Glutamato-Amônia Ligase/genética , Humanos , Mutação PuntualRESUMO
5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common inherited disorder of folate metabolism and causes severe hyperhomocysteinaemia. To better understand the relationship between mutation and function, we performed molecular genetic analysis of 76 MTHFR deficient patients, followed by extensive enzymatic characterization of fibroblasts from 72 of these. A deleterious mutation was detected on each of the 152 patient alleles, with one allele harboring two mutations. Sixty five different mutations (42 novel) were detected, including a common splicing mutation (c.1542G>A) found in 21 alleles. Using an enzyme assay in the physiological direction, we found residual activity (1.7%-42% of control) in 42 cell lines, of which 28 showed reduced affinity for nicotinamide adenine dinucleotide phosphate (NADPH), one reduced affinity for methylenetetrahydrofolate, five flavin adenine dinucleotide-responsiveness, and 24 abnormal kinetics of S-adenosylmethionine inhibition. Missense mutations causing virtually absent activity were found exclusively in the N-terminal catalytic domain, whereas missense mutations in the C-terminal regulatory domain caused decreased NADPH binding and disturbed inhibition by S-adenosylmethionine. Characterization of patients in this way provides a basis for improved diagnosis using expanded enzymatic criteria, increases understanding of the molecular basis of MTHFR dysfunction, and points to the possible role of cofactor or substrate in the treatment of patients with specific mutations.
Assuntos
Estudos de Associação Genética , Homocistinúria/diagnóstico , Homocistinúria/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Espasticidade Muscular/diagnóstico , Espasticidade Muscular/genética , Alelos , Processamento Alternativo , Ativação Enzimática , Éxons , Fibroblastos/metabolismo , Homocistinúria/metabolismo , Humanos , Íntrons , Cinética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Espasticidade Muscular/metabolismo , Mutação , Polimorfismo de Nucleotídeo Único , Estabilidade Proteica , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/genética , Transtornos Psicóticos/metabolismoRESUMO
Inherited disorders of vitamin B12 (cobalamin) have provided important clues to how this vitamin, which is essential for hematological and neurological function, is transported and metabolized. We describe a new disease that results in failure to release vitamin B12 from lysosomes, which mimics the cblF defect caused by LMBRD1 mutations. Using microcell-mediated chromosome transfer and exome sequencing, we identified causal mutations in ABCD4, a gene that codes for an ABC transporter, which was previously thought to have peroxisomal localization and function. Our results show that ABCD4 colocalizes with the lysosomal proteins LAMP1 and LMBD1, the latter of which is deficient in the cblF defect. Furthermore, we show that mutations altering the putative ATPase domain of ABCD4 affect its function, suggesting that the ATPase activity of ABCD4 may be involved in intracellular processing of vitamin B12.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Anormalidades Múltiplas/genética , Erros Inatos do Metabolismo/genética , Mutação , Vitamina B 12/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Anormalidades Múltiplas/enzimologia , Estudos de Casos e Controles , Células Cultivadas , Análise Mutacional de DNA , Fibroblastos/metabolismo , Expressão Gênica , Genes Recessivos , Estudos de Associação Genética , Humanos , Recém-Nascido , Proteínas de Membrana Lisossomal/metabolismo , Erros Inatos do Metabolismo/enzimologia , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Plant-beneficial fluorescent Pseudomonas spp. play important ecological roles. Here, their evolutionary history was investigated by a multilocus approach targeting genes involved in synthesis of secondary antimicrobial metabolites implicated in biocontrol of phytopathogens. Some of these genes were proposed to be ancestral, and this was investigated using a worldwide collection of 30 plant-colonizing fluorescent pseudomonads, based on phylogenetic analysis of 14 loci involved in production of 2,4-diacetylphloroglucinol (phlACBDE, phlF, intergenic locus phlA/phlF), hydrogen cyanide (hcnABC, anr) or global regulation of secondary metabolism (gacA, gacS, rsmZ). The 10 housekeeping loci rrs, dsbA, gyrB, rpoD, fdxA, recA, rpoB, rpsL, rpsG, and fusA served as controls. Each strain was readily distinguished from the others when considering allelic combinations for these 14 biocontrol-relevant loci. Topology comparisons based on Shimodaira-Hasegawa tests showed extensive incongruence when comparing single-locus phylogenetic trees with one another, but less when comparing (after sequence concatenation) trees inferred for genes involved in 2,4-diacetylphloroglucinol synthesis, hydrogen cyanide synthesis, or secondary metabolism global regulation with trees for housekeeping genes. The 14 loci displayed linkage disequilibrium, as housekeeping loci did, and all 12 protein-coding loci were subjected to purifying selection except for one positively-selected site in HcnA. Overall, the evolutionary history of Pseudomonas genes involved in synthesis of secondary antimicrobial metabolites important for biocontrol functions is in fact similar to that of housekeeping genes, and results suggest that they are ancestral in pseudomonads producing hydrogen cyanide and 2,4-diacetylphloroglucinol.
Assuntos
Anti-Infecciosos/metabolismo , Vias Biossintéticas/genética , Cianetos/metabolismo , Floroglucinol/metabolismo , Pseudomonas fluorescens/genética , Antibiose/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , Teorema de Bayes , Códon/genética , Evolução Molecular , Genes Bacterianos , Genes Essenciais , Funções Verossimilhança , Desequilíbrio de Ligação , Modelos Genéticos , Tipagem de Sequências Multilocus , Filogenia , Plantas/microbiologia , Pseudomonas fluorescens/metabolismoRESUMO
Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.
Assuntos
Proteínas de Bactérias/genética , Cofator PQQ/biossíntese , Fosfatos/metabolismo , Pseudomonas/classificação , Pseudomonas/genética , Análise por Conglomerados , Primers do DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Dados de Sequência Molecular , Filogenia , Raízes de Plantas/microbiologia , Pseudomonas/isolamento & purificação , Pseudomonas/metabolismo , Rizosfera , Análise de Sequência de DNA , Microbiologia do Solo , Triticum/microbiologiaRESUMO
Fluorescent Pseudomonas strains producing the antimicrobial secondary metabolite 2,4-diacetylphloroglucinol (Phl) play a prominent role in the biocontrol of plant diseases. A subset of Phl-producing fluorescent Pseudomonas strains, which can additionally synthesize the antimicrobial compound pyoluteorin (Plt), appears to cluster separately from other fluorescent Pseudomonas spp. based on 16S rRNA gene analysis and shares at most 98.4% 16S rRNA gene sequence identity with any other Pseudomonas species. In this study, a polyphasic approach based on molecular and phenotypic methods was used to clarify the taxonomy of representative Phl(+) Plt(+) strains isolated from tobacco, cotton or wheat on different continents. Phl(+) Plt(+) strains clustered separately from their nearest phylogenetic neighbors (i.e. species from the 'P. syringae', 'P. fluorescens' and 'P. chlororaphis' species complexes) based on rpoB, rpoD or gyrB phylogenies. DNA-DNA hybridization experiments clarified that Phl(+) Plt(+) strains formed a tight genomospecies that was distinct from P. syringae, P. fluorescens, or P. chlororaphis type strains. Within Phl(+) strains, the Phl(+) Plt(+) strains were differentiated from other biocontrol fluorescent Pseudomonas strains that produced Phl but not Plt, based on phenotypic and molecular data. Discriminative phenotypic characters were also identified by numerical taxonomic analysis and siderotyping. Altogether, this polyphasic approach supported the conclusion that Phl(+) Plt(+) fluorescent Pseudomonas strains belonged to a novel species for which the name Pseudomonas protegens is proposed, with CHA0(T) (=CFBP 6595(T), =DSM 19095(T)) as the type strain.
Assuntos
Antibacterianos/metabolismo , Controle Biológico de Vetores , Fenóis/metabolismo , Doenças das Plantas/prevenção & controle , Pseudomonas/classificação , Pirróis/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Impressões Digitais de DNA , DNA Girase/genética , DNA Bacteriano/genética , Genes Bacterianos/genética , Gossypium/microbiologia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fenótipo , Floroglucinol/análogos & derivados , Floroglucinol/metabolismo , Filogenia , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Pseudomonas/genética , Pseudomonas/metabolismo , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Sideróforos/classificação , Microbiologia do Solo , Nicotiana/microbiologia , Triticum/microbiologiaRESUMO
There is a significant potential to improve the plant-beneficial effects of root-colonizing pseudomonads by breeding wheat genotypes with a greater capacity to sustain interactions with these bacteria. However, the interaction between pseudomonads and crop plants at the cultivar level, as well as the conditions which favor the accumulation of beneficial microorganisms in the wheat rhizosphere, is largely unknown. Therefore, we characterized the three Swiss winter wheat (Triticum aestivum) cultivars Arina, Zinal, and Cimetta for their ability to accumulate naturally occurring plant-beneficial pseudomonads in the rhizosphere. Cultivar performance was measured also by the ability to select for specific genotypes of 2,4-diacetylphloroglucinol (DAPG) producers in two different soils. Cultivar-specific differences were found; however, these were strongly influenced by the soil type. Denaturing gradient gel electrophoresis (DGGE) analysis of fragments of the DAPG biosynthetic gene phlD amplified from natural Pseudomonas rhizosphere populations revealed that phlD diversity substantially varied between the two soils and that there was a cultivar-specific accumulation of certain phlD genotypes in one soil but not in the other. Furthermore, the three cultivars were tested for their ability to benefit from Pseudomonas inoculants. Interestingly, Arina, which was best protected against Pythium ultimum infection by inoculation with Pseudomonas fluorescens biocontrol strain CHA0, was the cultivar which profited the least from the bacterial inoculant in terms of plant growth promotion in the absence of the pathogen. Knowledge gained of the interactions between wheat cultivars, beneficial pseudomonads, and soil types allows us to optimize cultivar-soil combinations for the promotion of growth through beneficial pseudomonads. Additionally, this information can be implemented by breeders into a new and unique breeding strategy for low-input and organic conditions.
Assuntos
Agricultura/métodos , Raízes de Plantas/microbiologia , Pseudomonas/fisiologia , Microbiologia do Solo , Triticum/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Cruzamento/métodos , Análise por Conglomerados , Primers do DNA/genética , Eletroforese em Gel de Gradiente Desnaturante , Dados de Sequência Molecular , Floroglucinol/análogos & derivados , Floroglucinol/metabolismo , Filogenia , Raízes de Plantas/metabolismo , Análise de Sequência de DNA , Especificidade da Espécie , Suíça , Triticum/crescimento & desenvolvimentoRESUMO
Work on soils suppressive to Thielaviopsis basicola-mediated tobacco black root rot has focused on antagonistic pseudomonads to date. The role of non-Pseudomonas rhizosphere populations has been neglected, and whether they differ in black root rot-suppressive versus -conducive soils is unknown. To assess this possibility, tobacco was grown in a suppressive and a conducive soil of similar physicochemical properties, and rhizobacterial community composition was compared using a 16S rRNA taxonomic microarray. The microarray contains 1033 probes and targets 19 bacterial phyla. Among them, 398 probes were designed for Proteobacteria, Firmicutes, Actinomycetes, Cyanobacteria and Bacteroidetes genera/species known to include strains relevant for plant protection or plant growth promotion. Hierarchical clustering as well as principal component analysis of microarray data discriminated clearly between black root rot-suppressive and -conducive soils. In contrast, T. basicola inoculation had no impact on rhizobacterial community composition. In addition to fluorescent Pseudomonas, the taxa Azospirillum, Gluconacetobacter, Burkholderia, Comamonas and Sphingomonadaceae, which are known to comprise strains with plant-beneficial properties, were more prevalent in the suppressive soil. Mycobacterium, Bradyrhizobium, Rhodobacteraceae, Rhodospirillum and others were more prevalent in the conducive soil. For selected taxa, microarray results were largely corroborated by quantitative PCR and cloning/sequencing. In conclusion, this work identified novel bacterial taxa that could serve as indicators of disease suppressiveness in soil-quality assessments, and it extends the range of bacterial taxa hypothesized to participate in black root rot suppression.
Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Biodiversidade , Nicotiana/microbiologia , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Microbiologia do Solo , Bactérias/genética , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Análise em Microsséries , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
In the rhizosphere, biocontrol pseudomonads producing 2,4-diacetylphloroglucinol (Phl) can protect plants from soil-borne pathogens. DGGE of phlD has been proposed to monitor these bacteria, but two distinct protocols were needed for analysis of both the 'Pseudomonas fluorescens' species complex and the strains from rrs restriction group ARDRA-1. Here, a single DGGE protocol performed on 668-bp GC-clamp-containing phlD amplicons was effective with both types of pseudomonads, and 36 reference biocontrol strains from the 'P. fluorescens' complex or group ARDRA-1 gave a total of 11 distinct DGGE bands. phlD amplicons with at least two to seven nucleotidic differences could be discriminated, and the discrimination level was similar to that of phlD restriction analysis with four enzymes. Multiple phlD-DGGE bands were obtained when studying rhizosphere soil containing indigenous phlD+ pseudomonads, and phlD diversity was higher when DGGE was implemented after incubation of tobacco rhizosphere extracts in semi-selective medium (MPN approach) in comparison with approaches based on direct analysis of rhizosphere DNA extracts or assessment of phlD+colonies. phlD-DGGE profiles differed for a soil suppressive and a soil conducive to black root rot of tobacco, and each soil yielded new phlD sequences. In conclusion, this DGGE protocol was useful for monitoring indigenous rhizosphere consortia of phlD+ pseudomonads.
Assuntos
Proteínas de Bactérias/genética , Impressões Digitais de DNA/métodos , Eletroforese em Gel de Poliacrilamida , Desnaturação de Ácido Nucleico , Pseudomonas/classificação , Microbiologia do Solo , DNA Bacteriano/química , DNA Bacteriano/genética , Dados de Sequência Molecular , Floroglucinol/análogos & derivados , Floroglucinol/metabolismo , Filogenia , Raízes de Plantas/microbiologia , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento , Pseudomonas/metabolismo , Análise de Sequência de DNA , Homologia de Sequência , Nicotiana/microbiologiaRESUMO
The genetic and evolutionary relationship among 2,4-diacetylphloroglucinol (Phl)-producing pseudomonads that protect plants from soil-borne pathogens were investigated by multilocus sequence typing. A total of 65 pseudomonads consisting of 58 Phl-positive biocontrol strains of worldwide origin and seven Phl-negative representatives of characterized Pseudomonas species were compared using 10 housekeeping genes (i.e. rrs, dsbA, gyrB, rpoD, fdxA, recA, rpoB, fusA, rpsL and rpsG). Multilocus sequence typing differentiated 51 strains among 58 Phl-positive pseudomonads and proved to be as discriminative as enterobacterial repetitive intergenic consensus polymerase chain reaction profiling. As phylogenetic trees inferred from each locus were rather incongruent with one another, we derived the topology from all concatenated loci, which led to the identification of six main groups of Phl-producing Pseudomonas spp. Taxonomically, these groups could correspond to at least six different species. Linkage disequilibrium analysis pointed to a rather clonal structure, even when the analysis was restricted to Phl-producing pseudomonads from a same geographic location or a same phylogenetic group. Intragenic recombination was evidenced for gyrB, rpoD and fdxA, but was shown to be a weaker force than mutation in the origin of intragenetic diversity. This is the first multilocus assessment of the phylogeny and population structure of an ecologically important bacterial group involved in plant disease suppression.
Assuntos
Antifúngicos/metabolismo , Genes Bacterianos , Pseudomonas/genética , Evolução Biológica , Fungos , Desequilíbrio de Ligação , Dados de Sequência Molecular , Floroglucinol/análogos & derivados , Floroglucinol/metabolismo , Filogenia , Plantas/microbiologia , Pseudomonas/metabolismo , Análise de Sequência de DNARESUMO
Hydrogen cyanide (HCN) is a broad-spectrum antimicrobial compound involved in biological control of root diseases by many plant-associated fluorescent pseudomonads. The HCN synthase is encoded by three biosynthetic genes (hcnA, hcnB, and hcnC), but little is known about the diversity of these genes in fluorescent Pseudomonas spp. and in other bacteria. Here, the partial hcnBC sequence was determined for a worldwide collection of biocontrol fluorescent Pseudomonas spp. Phylogenies based on hcnBC and deduced protein sequences revealed four main bacterial groups, but topological incongruences were found between hcnBC and rrs-based phylogenies, suggesting past lateral transfer of hcnBC among saprophytic root-colonizing pseudomonads. Three of the four groups included isolates from different countries and host plants. Yet, these groups corresponded to distinct, ecologically-adapted populations of HCN-producing biocontrol fluorescent pseudomonads, as indicated by high hcnBC distinctness ratio values and the differences in production levels of HCN in vitro found between groups. This is in accordance with previous results on catabolic properties and biocontrol abilities of these strains. HCN synthase gene diversity may thus reflect the adaptive radiation of HCN+ biocontrol fluorescent pseudomonads. Positive correlations were found between HCN production in vitro and plant protection in the cucumber/Pythium ultimum and tomato/Fusarium oxysporum f. sp. radicis-lycopersici pathosystems.