Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species

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

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

<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

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

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

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

<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