<i>H. seropedicae</i> EPS is required for biofilm formation on glass fiber.

<p><i>H. seropedicae</i> strains were grown in the presence of glass fiber and purified wild type EPS (100 µg.mL⁻¹) when indicated. After 12 hours, bacteria attached to the fiber were stained with crystal violet, washed and de-stained with absolute ethanol. The absorbance of the ethanol (550 nm) was determined and subtracted from the absorbance of the control without bacteria. Different letters indicate significant difference (p<0.001, Duncan multiple range test) between biofilm formation by the strains.</p><p><i>H. seropedicae</i> EPS is required for biofilm formation on glass fiber.</p

<i>H. seropedicae</i> strains competition for attachment on maize roots.

<p><i>H. seropedicae</i> wild type (black bars) and <i>epsB</i>⁻ (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.</p

Exopolysaccharide Biosynthesis Enables Mature Biofilm Formation on Abiotic Surfaces by <i>Herbaspirillum seropedicae</i>

<div><p><i>H. seropedicae</i> 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 <i>H. seropedicae</i> SmR1 exopolysaccharide in biofilm formation on both inert and plant substrates was assessed by characterization of a mutant in the <i>espB</i> 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 <i>eps</i> genes in biofilms grown on glass and polypropylene. On the other hand, a strong repression of <i>eps</i> genes was observed in <i>H. seropedicae</i> cells adhered to maize roots. Our data suggest that <i>H. seropedicae</i> EPS is a structural component of mature biofilms, but this development stage of biofilm is not achieved during plant colonization.</p></div

Bacterial strains and plasmids used in this study.

a<p>Ap = ampicillin; Km = kanamycin; Sm = streptomycin; Tc = tetracycline; Cm = chloramphenicol; and the superscript r = resistant.</p><p>Bacterial strains and plasmids used in this study.</p

Electrophoretic pattern of EPS isolated from <i>H. seropedicae</i> strains SmR1 (wild type) and EPSEB (<i>epsB</i> mutant).

<p>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.</p

<i>H. seropedicae</i> biofilm formation on glass fiber.

<p>Light microscopy was performed with <i>H. seropedicae</i> SmR1 and EPSEB (<i>epsB</i> 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), <i>H. seropedicae</i> 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 <i>epsG</i> expression between the tested conditions.</p

Resistance of <i>H. seropedicae</i> strains to chemical stress.

<p><i>H. seropedicae</i> 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.</p

Regulation of <i>H. seropedicae epsG</i> expression during maize colonization.

<p>For maize colonization expression analyses, 10⁸<i>H. seropedicae</i> MHS-01 (<i>epsG::lacZ</i>) 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 <i>epsG</i> expression between the tested conditions.</p

Maize Root Lectins Mediate the Interaction with <i>Herbaspirillum seropedicae</i> via N-Acetyl Glucosamine Residues of Lipopolysaccharides

<div><p><i>Herbaspirillum seropedicae</i> is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for <i>H. seropedicae</i> attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene <i>waaL</i> (codes for the O-antigen ligase) is induced during rhizosphere colonization by <i>H. seropedicae</i>. Furthermore a <i>waaL</i> mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits <i>H. seropedicae</i> attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type <i>H. seropedicae</i>, but failed to agglutinate the <i>waaL</i> mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in <i>H. seropedicae</i> attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization.</p></div

Maize root colonization by different amounts of <i>H. seropedicae</i> wild-type (black bars) and <i>waaL</i> mutant (gray bars) strains.

<p>Panel A: maize was inoculated separately with the indicated amount of each bacterial strain. Panel B: maize was inoculated with a 1∶1 mixture of both strains. The total number of bacterial cells inoculated is indicated in the x axis. Results are shown as means of log₁₀ (number of bacteria.g⁻¹ of fresh root) ± standard deviation. Different letters indicate significant difference at p<0.01 (Duncan multiple range test).</p