RESUMEN
Methylobacterium organophilum CZ-2 synthesized polyhydroxyalkanoates (PHAs) under nitrogen limitation with CH4 as carbon source and when either citrate or propionate was added as cosubstrates. The highest PHAs content (yPHA) in closed flasks was obtained in the CH4-citrate and CH4-propionate experiments attaining values of 0.82 and 0.68, respectively. M. organophilum CZ-2 cultivated in bioreactors with citrate and continuous CH4 addition yielded a final PHAs concentration of 143 gm(-3) containing hydroxybutyrate (HB), hydroxyvalerate (HV) and hydroxyoctanoate (HO), in a 55:35:10 ratio, with, yPHA of 0.88 and a CH4 elimination capacity (EC) of 20 gm(-3) h(-1). With propionate, the yPHA was 0.3 and the EC around 8 gm(-3) h(-1). From 1H and 13C NMR experiments it was found that the polymer produced with CH4-citrate contained six different monomers: 3HB, 3HV, 4HV, 4-hydroxyheptanoate (4HH), 3HO and 4HO, showing the great versatility of this PHAs producing bacterium.
Asunto(s)
Ácido Cítrico/metabolismo , Metano/metabolismo , Methylobacterium/metabolismo , Polihidroxialcanoatos/biosíntesis , Polihidroxialcanoatos/aislamiento & purificación , Propionatos/metabolismo , Methylobacterium/clasificación , Especificidad de la EspecieRESUMEN
Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage.
Asunto(s)
Biodiversidad , Methylobacterium/fisiología , Paenibacillus/fisiología , Raíces de Plantas/microbiología , Solanum tuberosum/crecimiento & desarrollo , Solanum tuberosum/microbiología , Simbiosis , Recuento de Colonia Microbiana , Dermatoglifia del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Genotipo , Methylobacterium/clasificación , Methylobacterium/crecimiento & desarrollo , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Paenibacillus/clasificación , Paenibacillus/crecimiento & desarrollo , Análisis de Secuencia de ADN , Solanum tuberosum/genéticaRESUMEN
The rhizosphere is an ecosystem exploited by a variety of organisms involved in plant health and environmental sustainability. Abiotic factors influence microorganism-plant interactions, but the microbial community is also affected by expression of heterologous genes from host plants. In the present work, we assessed the community shifts of Alphaproteobacteria phylogenetically related to the Rhizobiales order (Rhizobiales-like community) in rhizoplane and rhizosphere soils of wild-type and transgenic eucalyptus. A greenhouse experiment was performed and the bacterial communities associated with two wild-type (WT17 and WT18) and four transgenic (TR-9, TR-15, TR-22, and TR-23) eucalyptus plant lines were evaluated. The culture-independent approach consisted of the quantification, by real-time polymerase chain reaction (PCR), of a targeted subset of Alphaproteobacteria and the assessment of its diversity using PCR-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Real-time quantification revealed a lesser density of the targeted community in TR-9 and TR-15 plants and diversity analysis by principal components analysis, based on PCR-DGGE, revealed differences between bacterial communities, not only between transgenic and nontransgenic plants, but also among wild-type plants. The comparison between clone libraries obtained from the transgenic plant TR-15 and wild-type WT17 revealed distinct bacterial communities associated with these plants. In addition, a culturable approach was used to quantify the Methylobacterium spp. in the samples where the identification of isolates, based on 16S rRNA gene sequences, showed similarities to the species Methylobacterium nodulans, Methylobacterium isbiliense, Methylobacterium variable, Methylobacterium fujisawaense, and Methylobacterium radiotolerans. Colonies classified into this genus were not isolated from the rhizosphere but brought in culture from rhizoplane samples, except for one line of the transgenic plants (TR-15). In general, the data suggested that, in most cases, shifts in bacterial communities due to cultivation of transgenic plants are similar to those observed when different wild-type cultivars are compared, although shifts directly correlated to transgenic plant cultivation may be found.
Asunto(s)
Alphaproteobacteria/aislamiento & purificación , Eucalyptus/microbiología , Methylobacterium/aislamiento & purificación , Raíces de Plantas/microbiología , Plantas Modificadas Genéticamente/microbiología , Microbiología del Suelo , Alphaproteobacteria/clasificación , Alphaproteobacteria/genética , ADN Bacteriano/análisis , Ecosistema , Electroforesis/métodos , Biblioteca de Genes , Genes de ARNr , Variación Genética , Methylobacterium/clasificación , Methylobacterium/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genéticaRESUMEN
Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis disease in sweet orange. There is evidence that X. fastidiosa interacts with endophytic bacteria present in the xylem of sweet orange, and that these interactions, particularly with Methylobacterium mesophilicum, may affect disease progress. However, these interactions cannot be evaluated in detail until efficient methods for detection and enumeration of these bacteria in planta are developed. We have previously developed standard and quantitative PCR-based assays specific for X. fastidiosa using the LightCycler system [Li, W.B., Pria Jr., L.P.M.W.D., X. Qin, and J.S. Hartung, 2003. Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings. Phytopathology 93:953-958.], and now report the development of both standard and quantitative PCR assays for M. mesophilicum. The assays are specific for M. mesophilicum and do not amplify DNA from other species of Methylobacterium or other bacteria commonly associated with citrus or plant tissue. Other bacteria tested included Curtobacterium flaccumfaciens, Pantoea agglomerans, Enterobacter cloacae, Bacillus sp., X. fastidiosa, Xanthomonas axonopodis pv. citri, and Candidatus Liberibacter asiaticus. We have demonstrated that with these methods we can quantitatively monitor the colonization of xylem by M. mesophilicum during the course of disease development in plants artificially inoculated with both bacteria.