Chitin utilization by the insect-transmitted bacterium Xylella fastidiosa

Abstract

Xylella fastidiosa is an insect-borne bacterium that colonizes xylem vessels of a large number of host plants, including several crops of economic importance. Chitin is a polysaccharide present in the cuticle of leafhopper vectors of X. fastidiosa and may serve as a carbon source for this bacterium. Biological assays showed that X. fastidiosa reached larger populations in the presence of chitin. Additionally, chitin induced phenotypic changes in this bacterium, notably increasing adhesiveness. Quantitative PCR assays indicated transcriptional changes in the presence of chitin, and an enzymatic assay demonstrated chitinolytic activity by X. fastidiosa. An ortholog of the chitinase A gene (chiA) was identified in the X. fastidiosa genome. The in silico analysis revealed that the open reading frame of chiA encodes a protein of 351 amino acids with an estimated molecular mass of 40 kDa. chiA is in a locus that consists of genes implicated in polysaccharide degradation. Moreover, this locus was also found in the genomes of closely related bacteria in the genus Xanthomonas, which are plant but not insect associated. X. fastidiosa degraded chitin when grown on a solid chitin-yeast extract-agar medium and grew in liquid medium with chitin as the sole carbon source; ChiA was also determined to be secreted. The gene encoding ChiA was cloned into Escherichia coli, and endochitinase activity was detected in the transformant, showing that the gene is functional and involved in chitin degradation. The results suggest that X. fastidiosa may use its vectors' foregut surface as a carbon source. In addition, chitin may trigger X. fastidiosa's gene regulation and biofilm formation within vectors. Further work is necessary to characterize the role of chitin and its utilization in X. fastidiosa.

FIG. 1.

Effect of chitin on Xylella fastidiosa. (A) Growth curve of X. fastidiosa in XFM in presence or absence of colloidal chitin. (B) Log-transformed cell numbers present as planktonic or attached as a biofilm on glass in liquid XFM. Different letters on bars indicate statistically different treatments. (C) Biofilm formation of X. fastidiosa in a shaking culture. The biofilm is stained at the broth-air interface. (D) Chitin-induced transcriptional changes in X. fastidiosa; (E) in-gel chitinase activity ([4-MU(GlcNAc)₃] cleavage) of X. fastidiosa whole-cell culture extracts.

FIG. 2.

Chitin utilization by Xylella fastidiosa. (A) Bacterial growth curve in XFM medium without carbon sources (XFM Δ) or with chitin added as the only carbon source (XFM Δ-chitin). Bars (second y axis) show the progression of chiA expression over time. (B) In-gel chitinase activity [4-MU(GlcNAc)₃ cleavage] over time for X. fastidiosa culture filtrates in liquid XFM Δ-chitin, demonstrating ChiA secretion; (C) X. fastidiosa colonies grown on chitin-agar medium. The clear zones around colonies indicate chitin degradation. (D) In-gel chitinase activity (4-MU(GlcNAc)₃ cleavage) for Escherichia coli strains grown in LB medium.

FIG. 3.

Xylella fastidiosa growth and biofilm formation on hindwings of leafhopper vectors. Cells were suspended in XFM Δ, and drops were placed on wings. Wild-type (A) and cell-cell signaling rpfF mutant (B) cells were incubated up to 10 days on wings. (C) Control medium XFM Δ without cells. The upper pictures were taken at ×25 magnification and are suspension droplets; the lower pictures were taken at ×80 magnification after removing the drop of medium and rinsing the wings with water. (D) chiA expression in the cell-cell signaling mutant rpfF compared to the wild type. Different letters on bars represent statistically different treatments.

FIG. 4.

(A) Hypothetical model for chitin utilization in X. fastidiosa. Chitin is hydrolyzed to chitobiose outside the cell by chiA and passively transported into the periplasmic space as a dimer. nahA converts the substrate into N-acetylglucosamine, which is phosphorylated and transported into the cytoplasm via an ABC transporter. (B) Scanning electron microscopy micrograph of X. fastidiosa cells colonizing the mouthparts of a leafhopper vector. The arrows indicate potential degradation of the chitinous surface at the fringe of the microcolony. The picture is an unpublished image obtained in a previous study (2).