INVESTIGADORES
ZORREGUIETA Angeles
congresos y reuniones científicas
Título:
Extracellular components that define Rhizobium leguminosarum biofilm matrix architecture
Autor/es:
ABDIAN, P; VOZZA, N; CARAMELO, J.; VON BILDERLING C.; RUSSO, D. M.; PIETRASANTA, LI; ZORREGUIETA, A
Lugar:
Viena
Reunión:
Congreso; BIOFILMS 6. International Conference on Microbial Biofilms; 2014
Institución organizadora:
xx
Resumen:
Rhizobia are soil bacteria that establish symbiotic
interactions with leguminous plants inducing the formation of nitrogen-fixing root
nodules. They are able to grow in varied environmental conditions producing
different types of biofilms. We have studied the structure and the molecular
determinants of biofilms formed by Rhizobium
leguminosarum in vitro, a process
probably more relevant to the attachment of bacterial cells to soil particles.
Using confocal microscopy (CLSM) it was observed that the typical architecture
of a mature R. leguminosarum biofilm
consists of multiple layers of cells attached to each other mostly by lateral
interactions, forming clusters that are interlaced by water channels. The main
component of the extracellular matrix is an acidic exopolysaccharide or EPS. A
flat lawn of bacteria without any architecture was observed in EPS-defective
mutants, showing that EPS provides support to the biofilm structure. Moreover R. leguminosarum produces a capsular
polysaccharide (CPS) structurally similar to EPS that seems to be involved in
surface attachment during the first stages of biofilm development.
Other components involved in R. leguminosarum biofilm are extracellular proteins, since mutations
in the type I secretion system PrsDE reduced biofilm formation. Proteins
secreted by PrsDE include several predicted calcium binding proteins and
members of the Rap (Rhizobial adhering
proteins) family, most of them of unknown function. Interestingly, the Raps
possess one or more characteristic Ra/CHDL (cadherin like-) domains that confer
carbohydrate binding ability. Among them, we have characterized RapA by
biochemical and biophysical methods, and defined its role as a unipolar calcium
binding lectin that specifically recognizes the EPS/CPS. Mutation of rapA in
R. leguminosarum bv. viciae strain A34 produced no clear adhesion
or biofilm phenotypes, but overexpression of RapA in different wild type
backgrounds produced distinctive macroscopic phenotypes, suggesting that
cellular interactions were altered. Observation of static biofilms by CLSM
showed that RapA-overproducing strains had altered cell-to-cell interactions
within the biofilms, losing intimate contact and the typical ordered structure.
By Immunofluorescence (IF) we observed that RapA was re-distributed diffusely
around RapA-overproducing cells. Moreover, RapA-overexpression had no effect on
the adhesive phenotypes of an EPS/CPS-deficient strain, reinforcing the idea
that the EPS/CPS is required for RapA to confer a particular adhesion phenotype.
We have also studied other members of tha Rap family,
the Ply glycanases that regulate the length of EPS chains in the extracellular medium.
We have observed by atomic force microscopy (AFM) that the development of an
ordered polysaccharide network is dependent on Ply secretion. Thus, Ply mutants
are unable to form a typical biofilm. A model depicting the interaction of
RapA, Ply glycanases and acidic polysaccharides (EPS and CPS) during the
progress of biofilm matrix development is presented. Supported
by CONICET and Agencia de Promoción Científica y Tecnológica-FONCYT