RESUMEN
Flavodoxins are small electron transfer proteins containing flavin mononucleotide (FMN) as a prosthetic group, which play an important role during oxidative stress or iron limitation. The aims of this study were the identification and characterization of flavodoxins in the model aromatic-degrader Paraburkholderia xenovorans LB400 and the analyses of their protective effects during oxidative stress induced by paraquat and H2O2. Two genes (BxeA0278 and BxeB0391) encoding flavodoxins (hereafter referred to as fldX for flavodoxin from P. xenovorans), were identified at the LB400 major and minor chromosome. Genomic context of the flavodoxin-encoding genes showed genes encoding membrane proteins, transporters, and proteins involved in redox processes and biosynthesis of macromolecules. A secondary structure prediction of both LB400 flavodoxins showed the characteristic flavodoxin structure of five ß-sheets intercalated with five α-helices. FldX1 contains a loop intercalated in the fifth ß-strand, which indicates that it belongs to the long-chain flavodoxins, whereas FldX2 is a short-chain flavodoxin. A phylogenetic analysis of 73 flavodoxins from 43 bacterial genera revealed eight clusters (I-VIII), while FldX1 and FldX2 grouped separately within a long-chain and a short-chain flavodoxin clades. FldX1 and FldX2 were overexpressed in P. xenovorans. Interestingly, the strain overexpressing the long-chain flavodoxin FldX1 (p2-fldX1) showed a faster growth in glucose than the control strain. The recombinant strain overexpressing the long-chain flavodoxin FldX1 (p2-fldx1) exposed to paraquat (20 mM) possessed lower susceptibility to growth inhibition on plates and higher survival in liquid medium than the control strain. The strains overexpressing the flavodoxins FldX1 and FldX2 showed higher survival during exposure to 1 mM paraquat (>95%) than the control strain (68%). Compared to the control strain, strains overexpressing FldX1 and FldX2 showed lower lipid peroxidation (>20%) after exposure to 1 mM paraquat and a lower protein carbonylation (~30%) after exposure to 1 mM H2O2 was observed. During exposure to paraquat, strain p2-fldx1 downregulated the katG4, hpf, trxB1 and ohr genes (> 2-fold), whereas strain p2-fldx2 upregulated the oxyR and ahpC1 genes (> 2-fold). In conclusion, the flavodoxins FldX1 and FldX2 of P. xenovorans LB400 conferred protection to cells exposed to the oxidizing agents paraquat and H2O2.
Asunto(s)
Adaptación Biológica/efectos de los fármacos , Betaproteobacteria/efectos de los fármacos , Betaproteobacteria/fisiología , Flavodoxina/genética , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo/efectos de los fármacos , Paraquat/farmacología , Secuencia de Aminoácidos , Biología Computacional/métodos , Flavodoxina/química , Flavodoxina/metabolismo , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Genómica/métodos , FilogeniaRESUMEN
Sponges harbor characteristic microbiomes derived from symbiotic relationships shaping their lifestyle and survival. Haliclona fulva is encrusting marine sponge species dwelling in coralligenous accretions or semidark caves of the Mediterranean Sea and the near Atlantic Ocean. In this work, we characterized the abundance and core microbial community composition found in specimens of H. fulva by means of electron microscopy and 16S amplicon Illumina sequencing. We provide evidence of its low microbial abundance (LMA) nature. We found that the H. fulva core microbiome is dominated by sequences belonging to the orders Nitrosomonadales and Cenarchaeales. Seventy percent of the reads assigned to these phylotypes grouped in a very small number of high-frequency operational taxonomic units, representing niche-specific species Cenarchaeum symbiosum and uncultured Betaproteobacteria HF1, a new eubacterial ribotype variant found in H. fulva. The microbial composition of H. fulva is quite distinct from those reported in sponge species of the same Haliclona genus. We also detected evidence of an excretion/capturing loop between these abundant microorganisms and planktonic microbes by analyzing shifts in seawater planktonic microbial content exposed to healthy sponge specimens maintained in aquaria. Our results suggest that horizontal transmission is very likely the main mechanism for symbionts' acquisition by H. fulva. So far, this is the first shallow water sponge species harboring such a specific and predominant assemblage composed of these eubacterial and archaeal ribotypes. Our data suggests that this symbiotic relationship is very stable over time, indicating that the identified core microbial symbionts may play key roles in the holobiont functioning.
Asunto(s)
Archaea/clasificación , Betaproteobacteria/clasificación , Haliclona/microbiología , Microbiota , Agua de Mar/microbiología , Simbiosis , Animales , Archaea/genética , Archaea/aislamiento & purificación , Archaea/fisiología , Bacterias , Betaproteobacteria/genética , Betaproteobacteria/aislamiento & purificación , Betaproteobacteria/fisiología , ADN de Archaea/análisis , ADN Bacteriano/análisis , Francia , Mar Mediterráneo , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Microbiología del AguaRESUMEN
The bacterial genus Burkholderia comprises species occupying several habitats, including a group of symbionts of leguminous plants-also called beta-rhizobia-that has been recently ascribed to the new genus Paraburkholderia We used common bean (Phaseolus vulgaris L.) plants to trap rhizobia from an undisturbed soil of the Brazilian Cerrado under the vegetation type 'Cerradão'. Genetic characterization started with the analyses of 181 isolates by BOX-PCR, where the majority revealed unique profiles, indicating high inter- and intra-species diversity. Restriction fragment length polymorphism-PCR of the 16S rRNA of representative strains of the BOX-PCR groups indicated two main clusters, and gene-sequencing analysis identified the minority (27%) as Rhizobium and the majority (73%) as Paraburkholderia Phylogenetic analyses of the 16S rRNA and housekeeping (recA and gyrB) genes positioned all strains of the second cluster in the species P. nodosa, and the phylogeny of a symbiotic gene-nodC-was in agreement with the conserved genes. All isolates were stable vis-à-vis nodulating common bean, but, in general, with a low capacity for fixing N2, although some effective strains were identified. The predominance of P. nodosa might be associated with the edaphic properties of the Cerrado biome, and might represent an important role in terms of maintenance of the ecosystem, which is characterized by acid soils with high saturation of aluminum and low N2 content.
Asunto(s)
Betaproteobacteria/fisiología , Fijación del Nitrógeno/fisiología , Phaseolus/fisiología , Brasil , ADN Bacteriano/genética , Phaseolus/microbiología , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 16S/genética , Rhizobium/genética , Análisis de Secuencia de ADN , Suelo , Microbiología del Suelo , SimbiosisRESUMEN
The aim of the present study was to identify a collection of 35 Cupriavidus isolates at the species level and to examine their capacity to nodulate and fix N(2). These isolates were previously obtained from the root nodules of two promiscuous trap species, Phaseolus vulgaris and Leucaena leucocephala, inoculated with soil samples collected near Sesbania virgata plants growing in Minas Gerais (Brazil) pastures. Phenotypic and genotypic methods applied for this study were SDS-PAGE of whole-cell proteins, and 16S rRNA and gyrB gene sequencing. To confirm the ability to nodulate and fix N(2), the presence of the nodC and nifH genes was also determined, and an experiment was carried out with two representative isolates in order to authenticate them as legume nodule symbionts. All 35 isolates belonged to the betaproteobacterium Cupriavidus necator, they possessed the nodC and nifH genes, and two representative isolates were able to nodulate five different promiscuous legume species: Mimosa caesalpiniaefolia, L. leucocephala, Macroptilium atropurpureum, P. vulgaris and Vigna unguiculata. This is the first study to demonstrate that C. necator can nodulate legume species.
Asunto(s)
Betaproteobacteria/clasificación , Betaproteobacteria/aislamiento & purificación , Cupriavidus necator/fisiología , Fabaceae/microbiología , Fijación del Nitrógeno , Simbiosis , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Betaproteobacteria/genética , Betaproteobacteria/fisiología , Brasil , Análisis por Conglomerados , Cupriavidus necator/crecimiento & desarrollo , Cupriavidus necator/metabolismo , Girasa de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Electroforesis en Gel de Poliacrilamida , Fabaceae/fisiología , Datos de Secuencia Molecular , N-Acetilglucosaminiltransferasas/genética , Oxidorreductasas/genética , Filogenia , Proteoma/análisis , ARN Ribosómico 16S/genética , Nódulos de las Raíces de las Plantas , Análisis de Secuencia de ADNRESUMEN
Bacteria isolated from Mimosa nodules in Taiwan, Papua New Guinea, Mexico and Puerto Rico were identified as belonging to either the alpha- or beta-proteobacteria. The beta-proteobacterial Burkholderia and Cupriavidus strains formed effective symbioses with the common invasive species Mimosa diplotricha, M. pigra and M. pudica, but the alpha-proteobacterial Rhizobium etli and R. tropici strains produced a range of symbiotic phenotypes from no nodulation through ineffective to effective nodulation, depending on Mimosa species. Competition studies were performed between three of the alpha-proteobacteria (R. etli TJ167, R. tropici NGR181 and UPRM8021) and two of the beta-rhizobial symbionts (Burkholderia mimosarum PAS44 and Cupriavidus taiwanensis LMG19424) for nodulation of these invasive Mimosa species. Under flooded conditions, B. mimosarum PAS44 out-competed LMG19424 and all three alpha-proteobacteria to the point of exclusion. This advantage was not explained by initial inoculum levels, rates of bacterial growth, rhizobia-rhizobia growth inhibition or individual nodulation rate. However, the competitive domination of PAS44 over LMG19424 was reduced in the presence of nitrate for all three plant hosts. The largest significant effect was for M. pudica, in which LMG19424 formed 57% of the nodules in the presence of 0.5 mM potassium nitrate. In this host, ammonium also had a similar, but lesser, effect. Comparable results were also found using an N-containing soil mixture, and environmental N levels are therefore suggested as a factor in the competitive success of the bacterial symbiont in vivo.
Asunto(s)
Alphaproteobacteria/aislamiento & purificación , Alphaproteobacteria/fisiología , Betaproteobacteria/aislamiento & purificación , Betaproteobacteria/fisiología , Mimosa/microbiología , Nitrógeno/metabolismo , Simbiosis , Alphaproteobacteria/clasificación , Alphaproteobacteria/crecimiento & desarrollo , Betaproteobacteria/clasificación , Betaproteobacteria/crecimiento & desarrollo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Genes de ARNr , México , Papúa Nueva Guinea , Filogenia , Puerto Rico , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , TaiwánRESUMEN
The bacterial leaf-spot of anthurium emerged during the 1980s, in the French West Indies and Trinidad. This new bacterial disease is presently wide spread and constitutes a serious limiting factor for commercial anthurium production. Twenty-nine strains isolated from leaf-spots of naturally infected anthurium were characterized and compared with reference strains belonging to the Comamonadaceae family, the genera Ralstonia and Burkholderia, and representative fluorescent pseudomonads. From artificial inoculations 25 out of 29 strains were pathogenic on anthurium. Biochemical and physiological tests, fatty acid analysis, DNA-DNA hybridization, 16S rRNA gene sequence analysis, DNA-16S RNA hybridization were performed. The 25 pathogenic strains on anthurium were clustered in one phenon closely related to phytopathogenic strains of the genus Acidovorax. Anthurium strains were 79-99% (deltaTm range 0.2-1.6) related to the strain CFBP 3232 and constituted a discrete DNA homology group indicating that they belong to the same species. DNA-rRNA hybridization, 16S rRNA sequence and fatty acid analysis confirmed that this new species belongs to the beta-subclass of Proteobacteria and to rRNA superfamily III, to the family of Comamonadaceae and to the genus Acidovorax. The name Acidovorax anthurii is proposed for this new phytopathogenic bacterium. The type strain has been deposited in the Collection Franaise des Bactries Phytopathognes as CFBP 3232T.