Bacillus velezensis Wall Teichoic Acids Are Required for Biofilm Formation and Root Colonization

Abstract

Rhizosphere colonization by plant growth-promoting rhizobacteria (PGPR) along plant roots facilitates the ability of PGPR to promote plant growth and health. Thus, an understanding of the molecular mechanisms of the root colonization process by plant-beneficial Bacillus strains is essential for the use of these strains in agriculture. Here, we observed that an sfp gene mutant of the plant growth-promoting rhizobacterium Bacillus velezensis SQR9 was unable to form normal biofilm architecture, and differential protein expression was observed by proteomic analysis. A minor wall teichoic acid (WTA) biosynthetic protein, GgaA, was decreased over 4-fold in the Δsfp mutant, and impairment of the ggaA gene postponed biofilm formation and decreased cucumber root colonization capabilities. In addition, we provide evidence that the major WTA biosynthetic enzyme GtaB is involved in both biofilm formation and root colonization. The deficiency in biofilm formation of the ΔgtaB mutant may be due to an absence of UDP-glucose, which is necessary for the synthesis of biofilm matrix exopolysaccharides (EPS). These observations provide insights into the root colonization process by a plant-beneficial Bacillus strain, which will help improve its application as a biofertilizer.IMPORTANCEBacillus velezensis is a Gram-positive plant-beneficial bacterium which is widely used in agriculture. Additionally, Bacillus spp. are some of the model organisms used in the study of biofilms, and as such, the molecular networks and regulation systems of biofilm formation are well characterized. However, the molecular processes involved in root colonization by plant-beneficial Bacillus strains remain largely unknown. Here, we showed that WTAs play important roles in the plant root colonization process. The loss of the gtaB gene affects the ability of B. velezensis SQR9 to sense plant polysaccharides, which are important environmental cues that trigger biofilm formation and colonization in the rhizosphere. This knowledge provides new insights into the Bacillus root colonization process and can help improve our understanding of plant-rhizobacterium interactions.

Keywords: Bacillus velezensis SQR9; UDP-glucose; biofilm formation; root colonization; wall teichoic acids.

FIG 1

The biofilm architecture and protein profiles were significantly different between the sfp mutant and wild-type strains. (A) The complex colony morphology and microtiter plate assay of biofilm formation by the sfp mutant and wild-type strains in MSgg medium at 24 h. (B) Biofilm matrix protein profiles were distinct between the sfp mutant and wild-type strains. SDS-PAGE gel was stained with sliver staining. Lane Marker, protein molecular weight markers in kilodaltons; lane SQR9, protein extraction from biofilm formed by wild-type SQR9; lane Δsfp, protein extraction from biofilm formed by sfp mutant.

FIG 2

The sfp mutant and wild-type strains showed different proteomic profiles by iTRAQ analysis. (A) Distribution in various functional categories of proteins altered in the sfp mutant strain. (B) Distribution of the rhizosphere proteins in the presence of various treatments. The abscissa shows the fold change in the protein ratio of two treatments (SQR9/sfp mutant), and the ordinate represents various indicated proteins.

FIG 3

Impairment of the ggaA gene postponed the formation of cellular biofilms and decreased the root colonization capability of the B. velezensis SQR9 strain. (A) Microtiter plate assay of biofilm formation by the wild-type and mutant strains. (B) OD₆₀₀ of solubilized crystal violet from the microtiter plate assay over time for the wild-type and mutant strains. Error bars indicate the standard deviations based on three different replicated experimental values. Different letters above the bars indicate significant differences (P < 0.05), and the subscript numbers differentiate the different time points. Mutant designations Δ2739, Δ9906, and Δ3020 represent mutants with deletions of the gene with the last four numbers of the GI accession number of each gene in the SQR9 genome in the NCBI database (accession no. CP006890), i.e., V529_33780 (https://www.ncbi.nlm.nih.gov/protein/631802739), V529_05450 (https://www.ncbi.nlm.nih.gov/protein/631799906), and V529_36590 (https://www.ncbi.nlm.nih.gov/protein/631803020), respectively.

FIG 4

The ggaA mutant strain is deficient in cucumber root colonization. (A) CLSM micrographs of cucumber roots colonization by GFP-tagged wild-type and ggaA mutant strains. Ck is a control which was not inoculated with GFP-tagged SQR9. (B) The populations of wild-type and ggaA mutant strains colonizing cucumber seeding roots. Error bars indicate the standard deviations from the results from three independent experiments. Different letters above the bars indicate significant differences (P < 0.01).

FIG 5

(A) Involvement of UDP-glucose in synthesis of WTAs and lipoteichoic acid. (B) Transcriptional levels of galE, ypfP, tagE, and gtaB in the ggaA mutant strain relative to those in wild-type strain SQR9. The experiments were carried out with pellicle obtained from microtiter plates when cells were grown in MSgg medium for 24 h. The B. velezensis SQR9 recA gene was used as an internal reference gene. RQ represents the relative expression level (relative quantification) compared to the wild-type strain. Bars represent standard deviations of data from three biological replicates.

FIG 6

GtaB is involved in biofilm formation and cucumber root colonization by B. velezensis SQR9. (A) The complex colony morphology and microtiter plate assay of biofilm formation by the gtaB mutant and wild-type strains at 24 h. (B) OD₆₀₀ of solubilized crystal violet from the microtiter plate assay for the wild-type and gtaB mutant strains at 24 h. (C) The populations of wild-type and gtaB mutant strains colonizing cucumber seeding roots. Error bars indicate the standard deviations from three independent experiments. Different letters above the bars indicate significant differences (P < 0.05).

FIG 7

The response to plant polysaccharides depends on the UDP-glucose pyrophosphorylase, GtaB. Top-down view of biofilm assay in which the indicated mutant cells were incubated for 24 h in the presence of UDP-glucose (UDPG), arabinogalactan (AG), pectin, or xylan in MSNc medium. Biofilm formation of wild-type and mutant cells in MSNc without supplements were used as a control (CK). Results are representative of three experiments.