Figure 1.
Predicted primary structure of AtaA annotated by the daTAA program (
http://toolkit.tuebingen.mpg.de/dataa). The colored regions represent annotated domains as indicated in the box.
Figure 2.
Detection of AtaA protein and fiber by Western-blot analysis and immunoelectron microscopy.
(A) Immunodetection of AtaA using anti-AtaA antiserum against outer membrane (OM) proteins prepared from Tol 5 WT and Tol 5 T1 (T1). (B–C) Acinetobacter sp. Tol 5 observed by immunoelectron microscopy using an anti-AtaA antibody. (B) Thin peritrichate nanofibers on Tol 5 WT cells were labeled with anti-AtaA699–1014 antibody. (C) The anti-AtaA699–1014 antibody specifically binds to the thinnest, shortest nanofibers on Tol 5 cells.
Figure 3.
Detection of AtaA protein at the OM and flow cytometry analysis for the quantification of displayed AtaA on the cell surface.
(A) Immunodetection of AtaA using anti-AtaA antiserum against OM proteins prepared from Tol 5 WT, ΔataA, ΔataA transformed with the empty vector pARP3 (ΔataA, pARP3), and ΔataA complemented with ataA in pARP3 (ΔataA, pAtaA). (B) Confirmation of surface-displayed AtaA by flow cytometry. ΔataA, pAtaA (red line) shows AtaA proteins in a level similar to Tol 5 WT (blue line) on its cell surface. ΔataA (dashed line, gray fill) and ΔataA, pARP3 (black line) strains are shown as negative controls.
Figure 4.
Adherence assay of Acinetobacter sp. Tol 5, its derivatives, and Y. enterocolitica strains.
(A) Bacterial cells adhering to 48-well polystyrene (PS) plates or type I collagen-coated plates were stained with crystal violet. Strains expressing ataA (WT; ΔataA, pAtaA) adhere to both type I collagen-coated and PS surfaces, while Tol 5 strains lacking functional ataA (ΔataA; ΔataA, pARP3) cannot adhere to either surface. Y. enterocolitica expressing YadA (WA-314) specifically adheres to the collagen surface, while YadA-deficient derivatives of WA-314 (WA-C) barely adhere to both surfaces. (B) The adherence of cells stained with crystal violet in (A) was quantified by measuring the absorbance at 590 nm (A590) of an ethanol solution used to dissolve the stain. White and black bars denote adherence to the collagen surface and the PS surface, respectively. Data are expressed as mean and SEM (n = 3) values. Statistical significance, *P<0.01.
Figure 5.
Adhesion of Acinetobacter sp. Tol 5 and its derivatives to different abiotic surfaces.
Adhesion of Tol 5 and its derivatives (ΔataA; ΔataA, pARP3; ΔataA, pAtaA) to 96-well plates made of polypropylene (PP), polyvinylchloride (PVC), and glass, and to a sample cup made from stainless steel (SUS) was assessed by the same procedure as the adherence assay for a 48-well PS plate. Photographs indicate the stained cells adhering to well surfaces. Data are expressed as mean and SEM (n = 3) values. Statistical significance, *P<0.01.
Figure 6.
Autoagglutination assay of Acinetobacter sp. Tol 5, its derivatives, and Y. enterocolitica strains.
(A) Strains expressing ataA (WT; ΔataA, pAtaA) autoagglutinate and form cell clumps that settle at the bottom of test tubes. After standing for 3 h without agitation, suspensions of these strains were transparent due to the sedimentation of cells, whereas cell suspensions of strains lacking functional ataA (ΔataA; ΔataA, pARP3) remained cloudy. Yersinia strain expressing YadA (WA-314) partially formed cell clumps. Cell suspension of WA-314 strain was slightly transparent compared with the yadA-mutant strain (WA-C). (B) Autoagglutination observed in (A) was quantified by a decrease in optical density at 660 nm (OD660). Graph bars show the autoagglutination ratio (%). Data are expressed as mean and SEM (n = 3) values. Statistical significance, *P<0.001.
Figure 7.
Expression of ataA in another Acinetobacter strain, ADP1.
(A) Immunodetection of AtaA using anti-AtaA antiserum against OM proteins prepared from Acinetobacter sp. ADP1, ADP1 transformed with the empty vector pARP3 (ADP1, pARP3), and ADP1 transformed with ataA in pARP3 (ADP1, pAtaA). (B) Flow cytometry analysis of AtaA displayed on the cell surface of ADP1 and its transformants. ADP1, pAtaA (red line) shows a large number of AtaA proteins on its cell surface. ADP1 (dashed line, gray fill) and ADP1, pARP3 (black line) strains are shown as negative controls. (C) Confirmation of AtaA fibers on strains expressing ataA (ADP1, pAtaA) by immunoelectron microscopy.
Figure 8.
Adherence and autoagglutination assays of Acinetobacter sp. ADP1 and its transformants.
(A) Adherence assay of ADP1 and its transformants (ADP1; ADP1, pARP3; ADP1, pAtaA) to 48-well PS and type I collagen-coated plates, to 96-well plates made of PP, PVC, and glass, and to a sample cup made from SUS. Data are expressed as mean ± SEM (n = 3) values. Statistical significance, *P<0.01. (B) Autoagglutination assay of ADP1 and its transformants (ADP1; ADP1, pARP3; ADP1, pAtaA). Graph bars show the autoagglutination ratio (%). Data are expressed as mean ± SEM (n = 3) values. Statistical significance, *P<0.001.
Table 1.
Bacterial strains and plasmids used in this study.