The nucleoid as a scaffold for the assembly of bacterial signaling complexes

Abstract

The FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays.

Fig 1. FrzCD-GFP colocalizes with the nucleoid in M. xanthus.

(A) Micrographs of M. xanthus cells carrying a GFP or a mCherry fusion and stained with the DNA DAPI stain. The genetic backgrounds of the M. xanthus strains are indicated on the left. The white arrow indicates the cell whose fluorescence profiles and correlation coefficients between the DAPI and GFP localization are shown in (B) and (C), respectively. Scale bars correspond to 1μm. (B) GFP or mCherry (green or red) and DAPI fluorescence (blue) profiles with the fluorescence intensity (arbitrary units) represented on the y axis and the cell length positions with -1 and +1 indicating the poles, on the x axis. (C) Correlation coefficients between the DAPI and GFP or mCherry localization. R² values > 0,5 indicate significant correlations. (D) Box plots indicate the medians of the correlation coefficients (R²) from 10 cells (from one biological replicate) of each of the indicated strains. (E) Micrographs of a M. xanthus parB conditional mutant carrying frzCD-gfp or frzE-mCherry and DAPI stained. Micrographs were obtained upon 18h depletion of ParB. The genetic backgrounds of the M. xanthus strains are indicated on the left. Scale bars correspond to 1μm.

Fig 2. FrzCD-GFP colocalizes with the nucleoid in E. coli.

(A) Micrographs of E.coli cells carrying a GFP fusion on a plasmid and stained with the DNA DAPI stain. The genetic fusions are indicated on the left. Cells carrying frzCD-gfp were also treated with 10μg/ml cephalexin to visualize FrzCD-GFP colocalization with the multiple nucleoids of undivided cells. The white arrows indicate the cells whose fluorescence profiles are shown in (B). Cells surrounded by white boxes are taken from separate original micrographs. Scale bars correspond to 1μm. (B) GFP (green) and DAPI fluorescence (blue) profiles with the fluorescence intensity (arbitrary units) represented on the y axis and the cell length positions with -1 and +1 indicating the poles, on the x axis. (C) Correlation coefficients between the DAPI and GFP localization. R² values > 0,5 indicate significant correlations. (D) Box plots indicate the medians of the correlation coefficients (R²) from 10 cells (from one biological replicate) of each of the indicated strains.

Fig 3. FrzCD directly interacts with the DNA in vitro.

(A) Electrophoretic mobility shift assays (EMSA) on 1% agarose gels stained with ethidium bromide and developed at the UV light. The indicated concentrations of purified 6His-FrzCD were incubated with a 801 bp DNA fragment. (B) Schematic representation of the FrzCD protein domains. (C-D) The indicated increasing concentrations of 6His-FrzCD^Δ131–417 (C) and 6His- FrzCD^Δ1–130 (D) were used in EMSA assays with a 801 bp DNA fragment. (E) Average binding curves and duplicates in degraded colors of each immobilized FrzCD construct 6His- FrzCD, 6His- FrzCD^Δ131–417 or 6His- FrzCD^Δ7–27, with a 474 bp DNA fragment at a concentration of 38nM. (F) “Sliding window” representation indicating the protein charge of the first FrzCD N-terminal region at the different positions and obtained with 10, 20 and 30 residue windows (blu, green and red, respectively). (G) Increasing concentrations of 6His-FrzCD^Δ7–27 were used in EMSA assays. On the first lane of each gel, 500 ng of the 2-Log DNA ladder (0.1–10 kb, NEB) have been loaded. Data in panel (A, C, D, and G) are representative of three independent experiments.

Fig 4. The organization of FrzCD clusters depends on cluster intensity and mobility.

(A) A representative fluorescence 1 second time-lapse (left panel) and the corresponding kymograph (middle panel) of a frzCD-gfp cell (top panel). Big and small arrows indicate large and small clusters, respectively. The right panel represents the trajectories of each cluster (same color codes as on the top panel). Scale bars correspond to 1μm. (B) Cluster displacement (r) from the mean position at each given time (t). L represents the cell length. The color code corresponding to the logarithm of the ratio r/L indicates that the amplitude of the cluster displacement never exceeds 5% of the cell length. (C) Box plots indicate the distribution of the Mean Square Displacements at the different lag times; the mean of each lag value is indicate by the black dots. (D) Box plots indicate a significant decrease of the median confinement for clusters of low fluorescence intensity compared to high intensity clusters. For panels B, C and D 1039 clusters from 297 cells (two biological replicates) were analyzed. (E) The box plots and the violin plots show the measured confinements of frzCD-gfp strains blocked in the ON (frzCD_E168A-G169A-gfp, hyper-reversing) and OFF (frzCD_E202A-E203A-gfp, hypo-reversing) states. 130 and 150 clusters were analyzed for the ON and OFF states, respectively. (F). Average numbers of clusters with standard deviations (black dots and bars, respectively) for different nucleoid sizes are shown. Green dots represent measurements for individual cells. Grey zones represent the variances. 2564 clusters from two biological replicates were analyzed. (G) 909 cells were ordered according to their cell length (pixels, grey) and for each cell GFP and DAPI fluorescence are represented as green and blue dots, respectively, at their corresponding cell position. 0 is the cell center.

Fig 5. Frz cluster formation generates signal sensitivity in turn important for social behaviors.

(A) The average reversal frequencies, calculated by scoring FrzS-YFP pole-to-pole oscillations are shown as a function of the IAA concentration for wild type (black), frzCD^Δ6–130 (red) and ΔfrzCD (grey). Reversal frequencies values of wild type and frzCD^Δ6–130 can be fitted by the Hill equation with an interval of confidence of 95% (dashed lines). Error bars represent the standard errors of the means. Reversal frequency values for each IAA dose and each strain are the results of a biological duplicate (each being a technical triplicate). About one hundred cells for the wild type and frzCD^Δ1–130 strains and fifty for the ΔfrzCD strain were analyzed (refer to S3 Table for the exact number of analyzed cells for each strain and IAA doses used in this experiment). (B) Colony expansion of wild type, frzCD^Δ6–130 and ΔfrzCD cells. Error bars represent the standard deviations of the means from three biological replicates. (C) The same strains were analyzed in E. coli predation assays.