Transcriptional Responses of Herbaspirillum seropedicae to Environmental Phosphate Concentration

Abstract

Herbaspirillum seropedicae is a nitrogen-fixing endophytic bacterium associated with important cereal crops, which promotes plant growth, increasing their productivity. The understanding of the physiological responses of this bacterium to different concentrations of prevailing nutrients as phosphate (Pi) is scarce. In some bacteria, culture media Pi concentration modulates the levels of intracellular polyphosphate (polyP), modifying their cellular fitness. Here, global changes of H. seropedicae SmR1 were evaluated in response to environmental Pi concentrations, based on differential intracellular polyP levels. Cells grown in high-Pi medium (50 mM) maintained high polyP levels in stationary phase, while those grown in sufficient Pi medium (5 mM) degraded it. Through a RNA-seq approach, comparison of transcriptional profiles of H. seropedicae cultures revealed that 670 genes were differentially expressed between both Pi growth conditions, with 57% repressed and 43% induced in the high Pi condition. Molecular and physiological analyses revealed that aspects related to Pi metabolism, biosynthesis of flagella and chemotaxis, energy production, and polyhydroxybutyrate metabolism were induced in the high-Pi condition, while those involved in adhesion and stress response were repressed. The present study demonstrated that variations in environmental Pi concentration affect H. seropedicae traits related to survival and other important physiological characteristics. Since environmental conditions can influence the effectiveness of the plant growth-promoting bacteria, enhancement of bacterial robustness to withstand different stressful situations is an interesting challenge. The obtained data could serve not only to understand the bacterial behavior in respect to changes in rhizospheric Pi gradients but also as a base to design strategies to improve different bacterial features focusing on biotechnological and/or agricultural purposes.

Keywords: Herbaspirillum seropedicae; adhesion; phosphate; plant-microbe interaction; polyphosphate.

FIGURE 1

Cell growth and polyP levels in sufficient and high Pi media. Cells were grown at 30°C for 24 h in NFb5 or NFb50 media. Bacterial growth and polyP levels were measured at indicated times. Growth was determined by measuring the A_{600 nm} (upper panel). PolyP levels were determined using DAPI fluorescence and data were expressed in fluorescence arbitrary units (AU) (bottom panel). Data represent the mean ± SD of at least three independent experiments with a p < 0.05.

FIGURE 2

COGs classification of differentially expressed genes in NFb50 condition relative to NFb5 condition. COG functional categories are shown. Black bars indicate upregulated genes and gray bars denote downregulated genes.

FIGURE 3

Expression of Pi metabolism-related genes, AP activity, and Pi solubilization in NFb50 condition relative to NFb5 condition. (A) Fold changes of differentially expressed genes. (B) Cells of the SmR1 strain were grown at 30°C in the indicated media. At 4, 9, and 24 h, aliquots were removed and AP activities were determined according to Materials and Methods section. (C) Cells of the SmR1 strain were grown at 30°C in M1 medium for 24 h and then spotted in NBRIP plates containing the indicated Pi concentration. Pi solubilization index was calculated as described in the Materials and Methods section. Results represent the mean ± SD of three independent experiments. For each panel, different letters indicate significant differences between conditions according to Tukey’s test with a p < 0.05.

FIGURE 4

Chemotaxis and motility in NFb50 condition relative to NFb5 condition. Fold changes of differentially expressed genes related to chemotaxis (A) and motility (B), comparing both conditions. SmR1 strain cells were cultured on semisolid NFb5 and NFb50 agar plates (0.3%) and incubated for 48 h (C). In all cases, the results represent four independent experiments performed in triplicate.

FIGURE 5

H. seropedicae colonization of maize seedlings in the high and sufficient Pi conditions. H. seropedicae SmR1 cells were grown in NFb50 medium for 24 h, washed, and inoculated on maize plantlets in PM5 or PM50 (10 replicates per condition). The number of epiphytic and endophytic bacteria was determined after 5 days of infection as mentioned in Materials and Methods. Results represent the mean ± SD of three independent experiments. Different letters indicate significant differences between conditions according to Tukey’s test with a p < 0.05.

FIGURE 6

PHB levels in SmR1 cells grown in NFb50 and NFb5 condition. PHB measurements by flow cytometry in ΔphaC1 and SmR1 strains cultivated in NFb5 and NFb50 media. At the indicated times, culture aliquots were extracted, stained with Nile Red, and analyzed by flow cytometry as mentioned in the Materials and Methods section. Data are expressed as arbitrary units (AU) of fluorescence. Results represent the mean ± SD of three independent experiments with a p < 0.05.

FIGURE 7

Molecular differences of H. seropedicae cells in the high Pi condition relative to the sufficient Pi condition. The scheme shows induced (black up arrows) and repressed (white down arrows) traits in H. seropedicae SmR1.