<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

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

<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

<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

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

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

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

Proteomic Analysis of Herbaspirillum seropedicae Cultivated in the Presence of Sugar Cane Extract

Bacterial endophytes of the genus Herbaspirillum colonize sugar cane and can promote plant growth. The molecular mechanisms that mediate plant– H. seropedicae interaction are poorly understood. In this work, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth phase in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR. A total of 16 differential spots (1 exclusively expressed, 7 absent, 5 up- and 3 down-regulated) in the presence of 5% sugar cane extract were identified; thus the host extract is able to induce and repress specific genes of H. seropedicae. The differentially expressed proteins suggest that exposure to sugar cane extract induced metabolic changes and adaptations in H. seropedicae presumably in preparation to establish interaction with the plant

Proteomic Analysis of Herbaspirillum seropedicae Cultivated in the Presence of Sugar Cane Extract

Bacterial endophytes of the genus Herbaspirillum colonize sugar cane and can promote plant growth. The molecular mechanisms that mediate plant– H. seropedicae interaction are poorly understood. In this work, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth phase in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR. A total of 16 differential spots (1 exclusively expressed, 7 absent, 5 up- and 3 down-regulated) in the presence of 5% sugar cane extract were identified; thus the host extract is able to induce and repress specific genes of H. seropedicae. The differentially expressed proteins suggest that exposure to sugar cane extract induced metabolic changes and adaptations in H. seropedicae presumably in preparation to establish interaction with the plant