Characterization of the Xylella fastidiosa PD1671 gene encoding degenerate c-di-GMP GGDEF/EAL domains, and its role in the development of Pierce's disease.

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce's disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence.

Identification of an operon, Pil-Chp, that controls twitching motility and virulence in Xylella fastidiosa.

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

Twitching motility among pathogenic Xylella fastidiosa isolates and the influence of bovine serum albumin on twitching-dependent colony fringe morphology.

Fourteen Xylella fastidiosa isolates from grapevines exhibiting Pierce's disease symptoms in California, Texas, and South Carolina were examined for type IV pilus-mediated twitching motility, a phenotype previously observed in a Temecula isolate from California. All isolates except one from South Carolina (SC 19A97) exhibited colonies with a peripheral fringe on PW agar, a feature indicative of twitching motility; however, when individual cells of SC 19A97 were examined at higher magnifications twitching motility was observed. The presence and width of colony peripheral fringes were related to the amount of bovine serum albumin (BSA) present in the medium; no or low levels of BSA (0-1.8 g L(-1)) permitted development of the widest fringe, whereas higher levels (3.5-6.0 g L(-1)) severely limited, and in many instances prevented, peripheral fringe development. The growth rate of the wild-type Temecula isolate in PW broth with different concentrations of BSA was similar for all tested concentrations of BSA; however, growth was significantly reduced in medium without BSA.

Type I and type IV pili of Xylella fastidiosa affect twitching motility, biofilm formation and cell-cell aggregation.

Xylella fastidiosa, an important phytopathogenic bacterium, causes serious plant diseases including Pierce's disease of grapevine. It is reported here that type I and type IV pili of X. fastidiosa play different roles in twitching motility, biofilm formation and cell-cell aggregation. Type I pili are particularly important for biofilm formation and aggregation, whereas type IV pili are essential for motility, and also function in biofilm formation. Thirty twitching-defective mutants were generated with an EZ : : TN transposome system, and several type-IV-pilus-associated genes were identified, including fimT, pilX, pilY1, pilO and pilR. Mutations in fimT, pilX, pilO or pilR resulted in a twitch-minus phenotype, whereas the pilY1 mutant was twitching reduced. A mutation in fimA resulted in a biofilm-defective and twitching-enhanced phenotype. A fimA/pilO double mutant was twitch minus, and produced almost no visible biofilm. Transmission electron microscopy revealed that the pili, when present, were localized to one pole of the cell. Both type I and type IV pili were present in the wild-type isolate and the pilY1 mutant, whereas only type I pili were present in the twitch-minus mutants. The fimA mutant produced no type I pili. The fimA/pilO double mutant produced neither type I nor type IV pili.

Upstream migration of Xylella fastidiosa via pilus-driven twitching motility.

Xylella fastidiosa is a xylem-limited nonflagellated bacterium that causes economically important diseases of plants by developing biofilms that block xylem sap flow. How the bacterium is translocated downward in the host plant's vascular system against the direction of the transpiration stream has long been a puzzling phenomenon. Using microfabricated chambers designed to mimic some of the features of xylem vessels, we discovered that X. fastidiosa migrates via type IV-pilus-mediated twitching motility at speeds up to 5 mum min(-1) against a rapidly flowing medium (20,000 mum min(-1)). Electron microscopy revealed that there are two length classes of pili, long type IV pili (1.0 to 5.8 mum) and short type I pili (0.4 to 1.0 mum). We further demonstrated that two knockout mutants (pilB and pilQ mutants) that are deficient in type IV pili do not twitch and are inhibited from colonizing upstream vascular regions in planta. In addition, mutants with insertions in pilB or pilQ (possessing type I pili only) express enhanced biofilm formation, whereas a mutant with an insertion in fimA (possessing only type IV pili) is biofilm deficient.

Caulobacter crescentus requires RodA and MreB for stalk synthesis and prevention of ectopic pole formation.

Caulobacter crescentus cells treated with amdinocillin, an antibiotic which specifically inhibits the cell elongation transpeptidase penicillin binding protein 2 in Escherichia coli, exhibit defects in stalk elongation and morphology, indicating that stalk synthesis may be a specialized form of cell elongation. In order to investigate this possibility further, we examined the roles of two other proteins important for cell elongation, RodA and MreB. We show that, in C. crescentus, the rodA gene is essential and that RodA depletion leads to a loss of control over stalk and cell body diameter and a stalk elongation defect. In addition, we demonstrate that MreB depletion leads to a stalk elongation defect and conclude that stalk elongation is a more constrained form of cell elongation. Our results strongly suggest that MreB by itself does not determine the diameter of the cell body or stalk. Finally, we show that cells recovering from MreB depletion exhibit a strong budding and branching cell body phenotype and possess ectopic poles, as evidenced by the presence of multiple, misplaced, and sometimes highly branched stalks at the ends of these buds and branches. This phenotype is also seen to a lesser extent in cells recovering from RodA depletion and amdinocillin treatment. We conclude that MreB, RodA, and the target(s) of amdinocillin all contribute to the maintenance of cellular polarity in C. crescentus.