Subcellular localization of type IV pili regulates bacterial multicellular development

Abstract

In mammals, subcellular protein localization of factors like planar cell polarity proteins is a key driver of the multicellular organization of tissues. Bacteria also form organized multicellular communities, but these patterns are largely thought to emerge from regulation of whole-cell processes like growth, motility, cell shape, and differentiation. Here we show that a unique intracellular patterning of appendages known as type IV pili (T4P) can drive multicellular development of complex bacterial communities. Specifically, dynamic T4P appendages localize in a line along the long axis of the cell in the bacterium Acinetobacter baylyi. This long-axis localization is regulated by a functionally divergent chemosensory Pil-Chp system, and an atypical T4P protein homologue (FimV) bridges Pil-Chp signaling and T4P positioning. We further demonstrate through modeling and empirical approaches that subcellular T4P localization controls how individual cells interact with one another, independently of T4P dynamics, with different patterns of localization giving rise to distinct multicellular architectures. Our results reveal how subcellular patterning of single cells regulates the development of multicellular bacterial communities.

Fig. 1. Acinetobacter baylyi localizes its T4P in a line along the long axis of the cell dependent on the Pil-Chp pathway.

a Representative time-lapse images of A. baylyi extending and retracting fluorescently labeled T4P with background fluorescence subtracted. Blue arrows indicate direction of T4P movement. Scale bar, 1 µm. b Schematic of the Pil-Chp components found in A. baylyi with analogous flagellar chemotaxis protein names in parentheses. Deletions of components colored gold cause dispersed T4P localization while deletion of components in gray have no effect on localization of T4P. IM, inner membrane. c Quantification of the percentage of cells in a population with dispersed T4P. Cells with dispersed T4P were defined as cells that had T4P on multiple sides of the cell body. Each data point represents an independent, biological replicate and bar graphs indicate the mean ± SD. For each biological replicate, a minimum of 70 total cells were assessed. Statistical comparisons were made using One-Way ANOVA followed by Dunnett’s multiple comparisons test comparing log-transformed values from mutant strains to the ∆pilT parent: ns, not significant; **P < 0.01; ***P < 0.001. Exact measurements and P values are reported in the Source Data file. d Representative images of A. baylyi Pil-Chp mutants displaying dispersed or linearly localized T4P with background fluorescence subtracted. Zoomed-in images of representative single cells from each strain are outlined in dashed boxes and shown below. Scale bars, 2 µm. Genotypes of each strain used in each figure panel are outlined in Supplementary Table 1.

Fig. 2. The Pil-Chp pathway in A. baylyi has evolutionarily diverged in function to control T4P localization.

a Representative image of the parent strain containing a T4P machinery fluorescent mCherry fusion to the outer membrane secretin protein, PilQ, with background fluorescence subtracted. b Representative images of PilQ-mCherry localization in different Pil-Chp mutants with background fluorescence subtracted. c Natural transformation assays of indicated strains. Each data point represents a biological replicate and bar graphs indicate the mean ± SD. The transformation frequency of the ∆comP strain was below the limit of detection, indicated by LD. d Quantification of the percentage of cells in a population making T4P based on the presence or absence of fluorescently labeled T4P. Each data point represents an independent, biological replicate and bar graphs indicate the mean ± SD. For each biological replicate, a minimum of 70 total cells were assessed. Scale bars, 2 µm. Statistical comparisons were made using One-Way ANOVA followed by Dunnett’s multiple comparisons test comparing log-transformed values from mutant strains to the parent: ns, not significant; ****P < 0.0001. Exact measurements and P values are reported in the Source Data file. Genotypes of each strain used in each figure panel are outlined in Supplementary Table 1.

Fig. 3. The Pil-Chp pathway controls T4P localization through the positioning of a divergent FimV homologue.

a Representative image of a ∆fimV ∆pilT mutant displaying dispersed T4P labeled with AF488-maleimide dye and with background fluorescence subtracted. b Representative images of indicated protein fusion strains with background fluorescence subtracted. White outlines are cell body outlines from phase contrast images. c Quantification of fluorescence phenotypes depicted in b. Data are compiled from two independent biological replicates, and a minimum of 100 total cells were assessed for each strain. d Schematic of the proposed molecular pathways connecting the Pil-Chp system to T4P localization. Gray arrow indicates the dependence of FimV linear localization on the Pil-Chp pathway while black arrows indicate the recruitment of FimL/PilG and T4P machinery proteins to FimV. Deletions of components colored gold cause dispersed T4P localization while deletion of components in gray have no effect on localization of T4P machines. IM, inner membrane. Scale bars, 2 µm. Genotypes of each strain used in each figure panel are outlined in Supplementary Table 1.

Fig. 4. T4P localization controls multicellular interactions in A. baylyi.

a Representative images of simulated data for modeled cellular objects with different patterns of adhesin localization. b Quantification of multicellular aggregate sizes from simulated or experimental data collected for indicated strains. Each data point represents a simulation (n = 5) or biological (n = 3) replicate and bar graphs indicate the mean ± SD. Statistical comparisons were made using two-tailed Welch’s t-test: *P < 0.05; ***P < 0.001; ****P < 0.0001. Exact measurements and P values are reported in the Source Data file. c Representative images of multicellular aggregates from experimental data for indicated strains in a ∆pilT background. Scale bar, 2 µm. d Representative confocal microscopy images collected for indicated strains. Green areas are labeled T4P and red areas are cell bodies expressing cytoplasmic mRuby3. Insets show a magnified top-down view of cells in the white square on each panel. Genotypes of each strain used in each figure panel are outlined in Supplementary Table 1.