Xylella fastidiosa Differentially Accumulates Mineral Elements in Biofilm and Planktonic Cells

Abstract

Xylella fastidiosa is a bacterial plant pathogen that infects numerous plant hosts. Disease develops when the bacterium colonizes the xylem vessels and forms a biofilm. Inductively coupled plasma optical emission spectroscopy was used to examine the mineral element content of this pathogen in biofilm and planktonic states. Significant accumulations of copper (30-fold), manganese (6-fold), zinc (5-fold), calcium (2-fold) and potassium (2-fold) in the biofilm compared to planktonic cells were observed. Other mineral elements such as sodium, magnesium and iron did not significantly differ between biofilm and planktonic cells. The distribution of mineral elements in the planktonic cells loosely mirrors the media composition; however the unique mineral element distribution in biofilm suggests specific mechanisms of accumulation from the media. A cell-to-surface attachment assay shows that addition of 50 to 100 µM Cu to standard X. fastidiosa media increases biofilm, while higher concentrations (>200 µM) slow cell growth and prevent biofilm formation. Moreover cell-to-surface attachment was blocked by specific chelation of copper. Growth of X. fastidiosa in microfluidic chambers under flow conditions showed that addition of 50 µM Cu to the media accelerated attachment and aggregation, while 400 µM prevented this process. Supplementation of standard media with Mn showed increased biofilm formation and cell-to-cell attachment. In contrast, while the biofilm accumulated Zn, supplementation to the media with this element caused inhibited growth of planktonic cells and impaired biofilm formation. Collectively these data suggest roles for these minerals in attachment and biofilm formation and therefore the virulence of this pathogen.

Response of Xylella fastidiosa to Zinc: Decreased Culturability, Increased Exopolysaccharide Production, and Formation of Resilient Biofilms under Flow Conditions

“…To our knowledge, this is the first study in which it was demonstrated that Zn alters the expression of genes involved in biosynthesis of EPS, augments EPS production, and affects culturability in a bacterium. However, the observed inhibition of growth caused by higher concentrations of Zn in the media is in agreement with previous reports (12,13,15,16). Zn tolerance/resistance are particularly significant for the X. fastidiosa life cycle, since Zn-containing fungicides (49)(50)(51)(52) and foliar Zn sprays are commonly used for fertilization (49,50,53), increasing the Zn content in the host.…”

“…Although the metal induces a large increase in the production of EPS, most of the bacterial cells remain in the planktonic fraction in batch cultures. These results suggest that Zn promotes cell-to-cell attachment (shown previously by our group [13]) while it decreases surface-cell attachment. The former may be an important factor in the Zn-induced biofilm structures observed in microfluidic chambers after reinoculation.…”

“…The same is true for EPSs, which binds cation to their negatively charged groups (71,72). The negatively charged residues in the EPS from X. fastidiosa (21,41) could chelate Zn, which was shown to accumulate Zn in biofilms (13), thus preventing accumulation of toxic levels inside the bacterial cell. A similar well-characterized case involves the response of E. amylovora to copper, where the bacterium increases EPS production upon contact with high levels of Cu (73).…”

“…Moreover, recent studies by our group show that infection with X. fastidiosa does not cause significant changes in the levels of Zn in hosts (7). Likewise, in vitro studies of the effect of Zn on X. fastidiosa have produced contradictory results: while supplementation of synthetic medium with Zn reduces the growth of X. fastidiosa (12,13), Zn concentrations in xylem vessels have been positively (14) or negatively (15,16) correlated with growth of X. fastidiosa. Application of Zn drenches to grapevines was shown to decrease X. fastidiosa populations, but the effects on disease development were inconsistent (16).…”

“…The biofilm matrix can contain EPS, proteins, and/or nucleic acids (19), and during infection, EPS protects bacteria from coming into contact with toxic molecules and can induce symptoms such as wilt in the host, as in Ralstonia solanacearum or Erwinia amylovora infections (20). In X. fastidiosa, it was demonstrated recently that EPS production is funda-mental for biofilm production, virulence, and insect transmission (21) and that X. fastidiosa biofilms accumulate high levels of Zn and other heavy metals (13). Also, in the closely related Xanthomonas spp., mutants affected in Zn homoeostasis are compromised in EPS production and virulence (22,23).…”