Exopolysaccharide biosynthesis enables mature biofilm formation on abiotic surfaces by Herbaspirillum seropedicae

Abstract

H. seropedicae associates endophytically and epiphytically with important poaceous crops and is capable of promoting their growth. The molecular mechanisms involved in plant colonization by this microrganism are not fully understood. Exopolysaccharides (EPS) are usually necessary for bacterial attachment to solid surfaces, to other bacteria, and to form biofilms. The role of H. seropedicae SmR1 exopolysaccharide in biofilm formation on both inert and plant substrates was assessed by characterization of a mutant in the espB gene which codes for a glucosyltransferase. The mutant strain was severely affected in EPS production and biofilm formation on glass wool. In contrast, the plant colonization capacity of the mutant strain was not altered when compared to the parental strain. The requirement of EPS for biofilm formation on inert surface was reinforced by the induction of eps genes in biofilms grown on glass and polypropylene. On the other hand, a strong repression of eps genes was observed in H. seropedicae cells adhered to maize roots. Our data suggest that H. seropedicae EPS is a structural component of mature biofilms, but this development stage of biofilm is not achieved during plant colonization.

Figure 1. Electrophoretic pattern of EPS isolated from H. seropedicae strains SmR1 (wild type) and EPSEB (epsB mutant).

SDS-PAGE was performed with EPS extracted by cold ethanol precipitation of the supernatant of biofilm growing bacteria in glass fiber submersed in NFbHPN medium.

Figure 2. H. seropedicae biofilm formation on glass fiber.

Light microscopy was performed with H. seropedicae SmR1 and EPSEB (epsB mutant) grown in the presence of glass fiber for 12 hours, without (A,B) and with (C,D) addition of purified wild-type EPS (100 µg.mL⁻¹). Arrows indicate attached bacteria. Asterisks indicate mature biofilm colonies. For biofilm expression analyses (E), H. seropedicae MHS-01 cells were grown for 12 h in the presence or absence of glass fiber, the free living bacteria were directly used and biofilm bacteria were recovered from glass fiber by vortex. β-galactosidase activity was determined, standardized by total protein concentration, and expressed as nmol ONP.(min.mg protein) ⁻¹± standard deviation. Different letters indicate significant differences (p<0.01, Duncan multiple range test) in epsG expression between the tested conditions.

Figure 3. Maize root colonization by H. seropedicae wild type (black bars) and epsB (gray bars) mutant strain.

Results are shown as average of Log₁₀ (number of bacteria.g⁻¹ of fresh root) ± standard deviation. d.a.i. = days after inoculation.

Figure 4. H. seropedicae strains competition for attachment on maize roots.

H. seropedicae wild type (black bars) and epsB ⁻ (gray bars) strains were inoculated on maize separately (A) or co-inoculated in a 1∶1 proportion (B), with the total of bacteria inoculated per plantlet indicated in the x axis. Results are shown as average of Log₁₀ (number of recovered attached bacteria.g⁻¹ of fresh root) ± standard deviation, CFU = colony forming units.

Figure 5. H. seropedicae attachment and epiphytic colonization of maize roots.

H. seropedicae SmR1+pHC60 (GFP- wild type) and EPSEB+pHC60 (GFP- epsB mutant) strains were inoculated on maize, and immediately after inoculation (A) or 7 days after inoculation (B), longitudinal samples of the roots were analyzed by laser scan confocal microscopy. Legends under the figures show positioning coordinates of the tridimensional images.

Figure 6. Resistance of H. seropedicae strains to chemical stress.

H. seropedicae wild type (black lines) and EPSEB (gray lines) strains were plated on solid NFbHPN medium containing the compounds. Data expressed as percentage of colony forming units (CFU) in the test plates compared to the control after 24 hours of growth at 30°C.

Figure 7. Regulation of H. seropedicae epsG expression during maize colonization.

For maize colonization expression analyses, 10⁸ H. seropedicae MHS-01 (epsG::lacZ) cells were inoculated in the hydroponic system. After 24 hours, the cells from the hydroponic medium were collected by centrifugation. The cells attached to roots or to polypropylene spheres (PP) were removed by vortex and concentrated by centrifugation. For all the samples the β-galactosidase activity was determined, standardized by total protein concentration, and expressed as nmol ONP.(min.mg protein)⁻¹± standard deviation. Different letters indicate significant differences (p<0.01, Duncan multiple range test) in epsG expression between the tested conditions.