Article Figures & Data
Figures
- FIG 1
Transformation is growth phase-dependent in A. baumannii. (A) Schematic representation of DNA uptake machinery. Type IV pilus (TFP) components are shown in blue, DNA uptake and translocation proteins are in orange, and proteins for binding and recombination of the incoming ssDNA are in green. (B) Natural transformability of A. baumannii over time. The graph on top shows the transformation frequencies, while the graph on the bottom depicts the growth of the bacteria given in optical density at 600 nm (OD600) units. (C) Relative expression values over time for a subset of competence genes (pilA, pilQ, pilT, comEA, comEC, and dprA). (D) Transformation frequencies of the wild type (WT) and the strain encoding the PilA-FLAG translational fusion. (E and F) Immunoblotting of FLAG-tagged PilA at different time points through the bacterial growth phases (as indicated in minutes) in the pilA-FLAG strain (D) or its pilQ-negative derivative (pilA-FLAG ΔpilQ) (E). Detection of Sigma70 served as loading control. Two and three biologically independent experiments were performed for panel B and panels C to F, respectively, and the mean values (±standard deviation [SD]) are shown for all graphs. <, below detection limit. Statistical analyses were performed on log-transformed data. Statistics were based on a two-way analysis of variance (ANOVA) test with Tukey’s multiple comparisons (time points compared to 90 min, as indicated by the box in the legend) (C) or an unpaired t test with Welch’s correction (D). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- FIG 2
Design and functionality of PilA cysteine knock-in variants. (A) 3D structural model of the Neisseria gonorrhoeae major type IV pilin PilE (PDB, 2HI2 [78]), which is shown alongside Phyre2 (51) structural predictions of the major pilin PilA of pandemic V. cholerae and of PilA of A. baumannii strain A118 (this study). The conserved αβ-loops are shown in greenish yellow, and the residue chosen for the cysteine exchange (A61) is shown in red. Bottom panel: sequence alignments of N. gonorrhoeae PilE (Ng; Uniprot P02974), V. cholerae PilA (Vc; protein ID AWB74893.1 [79]), and A. baumannii PilA (Ab; strain A118 and protein ID H0N27_01510) using Clustal Omega. The αβ-loop is colored in yellow. The functional cysteine substitution in V. cholerae’s PilA is highlighted (S67 [23]). Residues tested in A. baumannii in this study are shown underlined. (B) Natural transformability of PilA cysteine knock-in variants. Bars show the average transformation frequency of three independent experiments (±SD). Statistical analyses were performed on log-transformed data using a one-way ANOVA followed by Sidak’s multiple-comparison test. Each mutant strain is compared to the WT strain. #, under detection limit in at least one experiment, in which case the detection limit was used for the calculation of the average value and statistical analyses. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (C) Pilus imaging using a thiol-reactive maleimide dye. Snapshot images of A118-pilA(A61C), A118-pilA(A61C)ΔpilT, and the parental WT strain. The ΔpilA strain served as an additional negative control. The bacteria were stained with AF-488-Mal and imaged in the phase contrast (PC) or green fluorescence (Dye) channels. A merged image of both channels is shown in the bottom row (Merge). The contrast of the merged images was adjusted for best pilus visualization. An enlargement of the marked region (dotted boxes) is shown as an inset.
- FIG 3
Type IV pilus genes are essential for transformation and surface motility. (A to C) Transformation frequencies of defined mutants (details as in Fig. 1). For complementation, the strains carried a transposon without (control, TnAraC; no gene downstream of PBAD promoter) or with (TnXXXX) the complementing gene on their chromosome and were grown in the absence or presence of 0.2% (B) or 2% (C) arabinose. For all bar plots, transformation frequencies of three independent experiments are plotted as mean values (±SD). <, below detection limit (DL); #, under DL in at least one replicate (DL used for calculation of mean value). Log-transformed data were used for statistical analysis. When no transformants were obtained, the mean of the detection limit was used for statistical analyses. (D) TFP mutants are nonmotile on solid surfaces. Surface motility of the mutants described in panel A is depicted on the y axis based on the occupied area on the motility plates. Four biological experiments with three technical replicates are shown for each strain (n = 12). Images from one experimental set are shown below the graph. Statistical analyses: panels A to C, one-way ANOVA, using Sidak’s multiple comparisons test; panel D, Brown-Forsythe and Welch ANOVA tests with unpaired t test with Welch’s correction. *, P < 0.05; ****; P < 0.0001; n.s., not significant. The strains were compared to the WT (A and D) or the most appropriate control strain (boxed strain name in panels B and C).
- FIG 4
The A. baumannii PilSR and Pil-Chp systems are required for natural transformation and surface motility. (A) Schematic representation of the PilSR and Pil-Chp systems. Left (gray): upon activation, PilS phosphorylates PilR, which promotes expression of pilA. Right (purple): PilJ promotes autophosphorylation of ChpA, which subsequently phosphorylates PilG and/or PilH, resulting in increased or decreased cAMP levels, respectively. PilG and PilH were also proposed to foster T4P extension or retraction. (B to E) Transformability of TFP regulation mutants without or with complementing constructs ± inducer, as indicated. Details as in Fig. 3. The WT and WT-TnAraC served as controls. Transformation frequencies are shown as mean value (±SD) from three independent experiments. <, below detection limit (DL). For statistical analyses, a one-way ANOVA with Sidak’s multiple comparisons test was performed on log-transformed data and the different strains’ values were compared to the WT (B) or to the most appropriate control strain (boxed name in panels C to E). #, under detection limit in at least one replicate. *, P < 0.05; ****, P < 0.0001; n.s. = not significant. (F and I) Imaging of TFP in the regulatory mutants. PilA(A61C) pilus imaging of pilT-positive or pilT-negative strains (as indicated). (F) The strains were pilR-positive or pilR-negative and carried complementing pilR or its phosphomimetic pilR(D56E) variant on a transposon, as indicated. (I) The strains were deleted for the regulatory gene that is indicated above each column. Details as in Fig. 2, with the exception that only the merged images are shown. Bar = 5 μm (enlarged images are 2× magnified). (G) Relative expression of pilA in the regulatory mutants. Average values (±SD) from two independent experiments are shown, and statistics reflect a two-way ANOVA with Tukey’s multiple comparisons test in which each strain was compared to the WT. **, P < 0.01; n.s., not significant. (H) Detection of PilA-FLAG in the different regulatory mutants. Representative images of two independent replicates. Details as in Fig. 1. (J) TFP regulatory mutants are nonmotile. The surface motility of the PilSR/ChpA system mutants is shown. Details as in Fig. 3D. The motility values of each strain were compared to those of the WT using Brown-Forsythe and Welch ANOVA tests with unpaired t test with Welch’s correction. **, P < 0.01.
Tables
- TABLE 1
Bacterial strains and plasmids used in this study
- TABLE 2
Statistics on PacBio genome sequencing data and assembly
- TABLE 3
Competence and TFP-related genes in strain A118
↵a A118 gene names are according to homologs described in references 61 and 82.
↵b Locus tags of strain A118 and automatic annotations are according to accession number CP059039 of this study.
↵c Homologs were determined using PATRIC BLAST and are based on protein sequence similarities, the positions of genes in operons, and their predicted functions. Only significant BLAST hits are shown (E value <0.01). Locus tags of strain N16961 [VC(A)XXXX] are according to accession numbers NC_002505 and NC_002506 (83). Locus tags of strain ADP1 (ACIADXXXX) are according to accession number NC_005966 (84). Locus tags of strain PAO1 (PAXXXX) are according to accession number NC_002516 (85).
