Lipopolysaccharide O-chain core region required for cellular cohesion and compaction of in vitro and root biofilms developed by Rhizobium leguminosarum

Abstract

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.

FIG 1

(A) Diagram of the R. leguminosarum strain 3841 locus organization involved in LPS biosynthesis. The location of each gene on the 3841 chromosome (or pRL9 plasmid) is indicated by numbers relating to the genome sequence. The positions of the Tn5 insertions in mutants are shown by black (A950 and A951) or white (B772) arrows. The lower bold lines indicate the amplified products used to generate the complementation plasmids. In the δ-lps locus, cpaA encodes an LPS-associated cation exporter, lpcB encodes a CMP-Kdo transferase, and lpcA encodes a galactosyl transferase. In the pRL9-borne β locus, lpsB2 encodes a hypothetical O-antigen biosynthesis-related protein, lpsB1 encodes a putative galactosyl transferase protein, and lpsD encodes a putative O-antigen ligase. (B) SDS-12% PAGE-silver periodate oxidation (left) or immunoblot (right) analysis of the LPS extracted from A34; 3841; lpcA, lpcB, and lpsD mutants; and complemented strains. O chains were detected by immunoblotting using the specific monoclonal antibody MAC 114 or MAC57, which recognizes the O-chain LPS from A34 or 3841, respectively. LPS I and LPS II components of the LPS are indicated.

FIG 2

Autoaggregation assays. The sedimentation profiles of liquid suspensions of R. leguminosarum strain A34 (A) or 3841 (B) derivative strains in TY medium are shown. Each point corresponds to average of replicated samples from two independent experiments.

FIG 3

Rhizobial adhesion to a hydrophobic abiotic surface. R. leguminosarum A34 or 3841 derivative strains were grown in polystyrene multiwell plates in static Y-mannitol minimal medium for 3 days at 28°C, and bacterial attachment was quantified by crystal violet staining. Horizontal values correspond to average of six replicate samples in at least two different experiments. ***, P < 0.0001. One-way analysis of variance was performed using GraphPad Prism 5 software.

FIG 4

Cellular interactions and biofilms formed by R. leguminosarum A34 derivative strains. CLSM images are horizontal (x-axis) projections of optical sections showing bacterial attachment at day 1 and the biofilms formed at day 4 in chambered glass cover slides (×1,000 magnification) by A34, the isogenic lpcA LPS mutant, and the complemented lpcA pFC222 strains. The insets are zooms (3×). Size bars indicate 2 μm.

FIG 5

Cellular interactions and biofilms formed by R. leguminosarum 3841 derivative strains. CLSM images show bacterial attachment at day 1 and biofilms formed at day 4 in chambered glass cover slides by 3841, the isogenic lpcB and lpsD LPS mutants, and the complemented lpcB pFC222 and lpsD pFC224 strains after 1 and 4 days (×1,000 magnification). The insets are zooms (3×). Size bars indicate 2 μm.

FIG 6

Rhizobial biofilm formation on pea roots. (A) Five-day-old GFP-labeled biofilm formed by the WT strains and the LPS derivative mutants. Note the compact microcolony patches formed by the A34 and 3841 WT strains and the ramified or star-like microcolonies scattered on the root epidermis developed by the mutants. 6×-zoom images (right) show the detail of a root-attached bacterial aggregate. Magnifications: ×400 (left) and ×2,400 (right). (B) CLSM images of bacterial aggregates associated to root hairs. White arrows indicate bacterial clumps associated to root hairs developed by the LPS mutants. Magnification: ×400. CLSM images are horizontal (x-axis) projections of representative images of five independent experiments. Size bars indicate 10 μm.