The Ctp type IVb pilus locus of Agrobacterium tumefaciens directs formation of the common pili and contributes to reversible surface attachment

Abstract

Agrobacterium tumefaciens can adhere to plant tissues and abiotic surfaces and forms biofilms. Cell surface appendages called pili play an important role in adhesion and biofilm formation in diverse bacterial systems. The A. tumefaciens C58 genome sequence revealed the presence of the ctpABCDEFGHI genes (cluster of type IV pili; Atu0216 to Atu0224), homologous to tad-type pilus systems from several bacteria, including Aggregatibacter actinomycetemcomitans and Caulobacter crescentus. These systems fall into the type IVb pilus group, which can function in bacterial adhesion. Transmission electron microscopy of A. tumefaciens revealed the presence of filaments, significantly thinner than flagella and often bundled, associated with cell surfaces and shed into the external milieu. In-frame deletion mutations of all of the ctp genes, with the exception of ctpF, resulted in nonpiliated derivatives. Mutations in ctpA (a pilin homologue), ctpB, and ctpG decreased early attachment and biofilm formation. The adherence of the ctpA mutant could be restored by ectopic expression of the paralogous pilA gene. The ΔctpA ΔpilA double pilin mutant displayed a diminished biovolume and lower biofilm height than the wild type under flowing conditions. Surprisingly, however, the ctpCD, ctpE, ctpF, ctpH, and ctpI mutants formed normal biofilms and showed enhanced reversible attachment. In-frame deletion of the ctpA pilin gene in the ctpCD, ctpE, ctpF, ctpH, and ctpI mutants caused the same attachment-deficient phenotype as the ctpA single mutant. Collectively, these findings indicate that the ctp locus is involved in pilus assembly and that nonpiliated mutants, which retain the CtpA pilin, are proficient in attachment and adherence.

FIG 1

TEM of A. tumefaciens C58. Cells were negatively stained with 2% uranyl acetate and visualized by TEM. Arrow, flagellum; filled triangles, common (Ctp) pili.

FIG 2

ctp genes encode the production of common pili. (A) Gene maps of the ctp cluster and the unlinked pilA gene (Atu3514). The ctp cluster contained nine genes (Atu0216 to Atu0224). Arrows indicate promoters in the ctp cluster. (B) TEM of A. tumefaciens C58, ctp mutants, and complemented derivatives. Arrows, flagella; filled triangles, Ctp pili; open triangles, Ctp pilus bundles. Cells were negatively stained with uranyl acetate and visualized by TEM.

FIG 3

Biofilm formation by A. tumefaciens ctp deletion mutants. Static biofilms of A. tumefaciens C58 and all ctp mutants were incubated for 72 h on coverslips. All bacterial strains grown in shaking liquid culture overnight were diluted to an OD₆₀₀ of 0.05 and incubated in 12-well plates with PVC coverslips for 72 h at 28°C. After rinsing, coverslips were stained with 1% CV and the adherent biomass was quantified by measuring the absorbance of acetic acid-solubilized CV, normalized for planktonic culture growth (A₆₀₀/OD₆₀₀). Error bars show standard deviations of results of assays performed in triplicate.

FIG 4

Complementation of class I Ctp mutants. A. tumefaciens mutants were complemented by plasmid-borne, ectopically expressed constructs (P_lac-ctpA, P_lac-ctpB, and P_lac-ctpG). (A) Seventy-two-hour static biofilm assays on coverslips prepared as described in the legend to Fig. 3 and grown with 500 μM IPTG induction. (B) Short-term irreversible binding assays on coverslips with 500 μM IPTG induction. Coverslips were incubated with cell culture for 1 h and then subjected to strong washing. Total numbers of attached cells per field of view (∼3.13 × 10⁴ μm²) were determined by counting with a Nikon E800 in bright-field mode at ×100 magnification. The values are mean results of 10 fields of view, and the error bars show the standard deviations.

FIG 5

Flow cell biofilms of wild-type C58 and a ΔctpA ΔpilA double mutant. SCLM images of flow cell biofilms formed by wild-type C58 (A) and the ΔctpA ΔpilA double mutant (B) each harboring a P_tac-gfpmut3 plasmid (pJZ383) after 5 days of incubation with a Nikon A1 SCLM with a 488-nm laser for excitation and a 500- to 550-nm filter for emission. The crossed lines in each image indicate the positions of the sagittal, three-dimensional reconstructions in the side bars. The error bars show the standard errors of the means. The images were rendered with the Nikon Elements software package. The autoCOMSTAT program was used to calculate the biovolume per substratum area (C) and average biofilm height per substratum area (D). The values are averages calculated for 12 to 15 image stacks collected from three channels for each strain per time point.

FIG 6

Irreversible attachment of class II Ctp mutants: Coverslips were incubated with cell cultures for 1 h, subjected to robust rinsing, stained with af-WGA for 30 min, and then vigorously rinsed again. The total numbers of attached cells per field of view (∼3.13 × 10⁴ μm²) were determined with a Nikon E800 at ×100 magnification in bright-field mode. Enumeration of UPPs was done by determining the number of af-WGA-stained red fluorescent dots by fluorescence microscopy. The values are mean results of 10 fields of view, and the error bars show the standard deviations.

FIG 7

Comparison of reversible and stable surface attachment. Short-term binding assays were performed with A. tumefaciens C58 and ΔuppC single and ΔuppC/class II Ctp double mutants. Coverslips were incubated with cell culture for 1 h and then minimally rinsed (black bars, total numbers of reversible attached cells) or vigorously rinsed (gray bars, total numbers of irreversibly attached cells). Total numbers of attached cells per field of view (∼3.13 × 10⁴ μm²) were determined with a Nikon E800 in bright-field mode at ×100 magnification. The values are mean results of 10 fields of view, and the error bars show the standard deviations. Asterisks show that attached cells were rarely observed.

FIG 8

The ctpA mutation is epistatic to class II Ctp mutants. Static biofilm formation and stable short-term surface attachment of C58 and ΔctpA, class II, and ΔctpA/class II Ctp mutants were measured. Gray bars are the total numbers of irreversible attached cells per field of view (∼3.13 × 10⁴ μm²) from short-term binding assays viewed with a Nikon E800 at ×100 magnification in bright-field mode. The values are mean values of 10 fields of view, and the error bars show the standard deviations of the means. Black bars are quantifications of acetic acid-solubilized CV on 72-h coverslip biofilms. Adherent biomass was normalized by growth (A₆₀₀/OD₆₀₀), and error bars are standard deviations of results of assays run in triplicate.