RESUMEN
The use of environmental DNA (eDNA) to monitor rare and elusive species has great potential for conservation biology. Traditional surveying methods can be time-consuming, labour-intensive, subject to error or can be invasive and potentially damaging to habitat. The Trinidad golden treefrog (Phytotriades auratus) is one such species that would benefit from such an approach. This species inhabits the giant bromeliad (Glomeropitcairnia erectiflora) on two peaks on the Caribbean island of Trinidad. Traditional survey methods for this species have required the destruction of the giant bromeliad, which is the only known habitat of this frog. Here we described the development of an eDNA PCR-based assay that uses water drawn from the water-filled phytotelmata of the giant bromeliad along with the use of a synthetic DNA positive control that can be easily amplified in the bacterium Escherichia coli. The assay can detect to a DNA concentration of 1.4ng. Sampling of 142 bromeliads using this method revealed 9% were positive for P. auratus DNA. These data suggest that eDNA methods also have great potential for revealing the presence of elusive species in arboreal habitats.
Asunto(s)
Anuros/genética , Bromelia , ADN/genética , Especies en Peligro de Extinción , Reacción en Cadena de la Polimerasa/métodos , Animales , Trinidad y TobagoRESUMEN
The formation of biofilms is an important survival strategy allowing rhizobia to live on soil particles and plant roots. Within the microcolonies of the biofilm developed by Rhizobium leguminosarum, rhizobial cells interact tightly through lateral and polar connections, forming organized and compact cell aggregates. These microcolonies are embedded in a biofilm matrix, whose main component is the acidic exopolysaccharide (EPS). Our work shows that the O-chain core region of the R. leguminosarum lipopolysaccharide (LPS) (which stretches out of the cell surface) strongly influences bacterial adhesive properties and cell-cell cohesion. Mutants defective in the O chain or O-chain core moiety developed premature microcolonies in which lateral bacterial contacts were greatly reduced. Furthermore, cell-cell interactions within the microcolonies of the LPS mutants were mediated mostly through their poles, resulting in a biofilm with an altered three-dimensional structure and increased thickness. In addition, on the root epidermis and on root hairs, O-antigen core-defective strains showed altered biofilm patterns with the typical microcolony compaction impaired. Taken together, these results indicate that the surface-exposed moiety of the LPS is crucial for proper cell-to-cell interactions and for the formation of robust biofilms on different surfaces.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Lipopolisacáridos/metabolismo , Antígenos O/metabolismo , Raíces de Plantas/microbiología , Rhizobium leguminosarum/fisiología , Lipopolisacáridos/genética , Datos de Secuencia Molecular , Antígenos O/genética , Rhizobium leguminosarum/genética , Rhizobium leguminosarum/crecimiento & desarrollo , Rhizobium leguminosarum/metabolismo , Análisis de Secuencia de ADNRESUMEN
The type I protein secretion system of Rhizobium leguminosarum bv. viciae encoded by the prsD and prsE genes is responsible for secretion of the exopolysaccharide (EPS)-glycanases PlyA and PlyB. The formation of a ring of biofilm on the surface of the glass in shaken cultures by both the prsD and prsE secretion mutants was greatly affected. Confocal laser scanning microscopy analysis of green-fluorescent-protein-labeled bacteria showed that during growth in minimal medium, R. leguminosarum wild type developed microcolonies, which progress to a characteristic three-dimensional biofilm structure. However, the prsD and prsE secretion mutants were able to form only an immature biofilm structure. A mutant disrupted in the EPS-glycanase plyB gene showed altered timing of biofilm formation, and its structure was atypical. A mutation in an essential gene for EPS synthesis (pssA) or deletion of several other pss genes involved in EPS synthesis completely abolished the ability of R. leguminosarum to develop a biofilm. Extracellular complementation studies of mixed bacterial cultures confirmed the role of the EPS and the modulation of the biofilm structure by the PrsD-PrsE secreted proteins. Protein analysis identified several additional proteins secreted by the PrsD-PrsE secretion system, and N-terminal sequencing revealed peptides homologous to the N termini of proteins from the Rap family (Rhizobium adhering proteins), which could have roles in cellular adhesion in R. leguminosarum. We propose a model for R. leguminosarum in which synthesis of the EPS leads the formation of a biofilm and several PrsD-PrsE secreted proteins are involved in different aspects of biofilm maturation, such as modulation of the EPS length or mediating attachment between bacteria.