doi: 10.1371/journal.pbio.3000728.
eCollection 2020 Jun.
Modulation of bacterial multicellularity via spatio-specific polysaccharide secretion
Affiliations
- PMID: 32516311
- PMCID: PMC7310880
- DOI: 10.1371/journal.pbio.3000728
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Modulation of bacterial multicellularity via spatio-specific polysaccharide secretion
PLoS Biol.
.
Abstract
The development of multicellularity is a key evolutionary transition allowing for differentiation of physiological functions across a cell population that confers survival benefits; among unicellular bacteria, this can lead to complex developmental behaviors and the formation of higher-order community structures. Herein, we demonstrate that in the social δ-proteobacterium Myxococcus xanthus, the secretion of a novel biosurfactant polysaccharide (BPS) is spatially modulated within communities, mediating swarm migration as well as the formation of multicellular swarm biofilms and fruiting bodies. BPS is a type IV pilus (T4P)-inhibited acidic polymer built of randomly acetylated β-linked tetrasaccharide repeats. Both BPS and exopolysaccharide (EPS) are produced by dedicated Wzx/Wzy-dependent polysaccharide-assembly pathways distinct from that responsible for spore-coat assembly. While EPS is preferentially produced at the lower-density swarm periphery, BPS production is favored in the higher-density swarm interior; this is consistent with the former being known to stimulate T4P retraction needed for community expansion and a function for the latter in promoting initial cell dispersal. Together, these data reveal the central role of secreted polysaccharides in the intricate behaviors coordinating bacterial multicellularity.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
(A) Schematic representation of the 3 Wzx/Wzy-dependent polysaccharide-assembly pathways in M. xanthus DZ2/DK1622. The genomic “MXAN” locus tag identifier for each respective gene (black) has been indicated below the specific protein name (white). (B) Gene conservation and synteny diagrams for EPS, MASC, and BPS clusters in M. xanthus DZ2/DK1622 compared to the evolutionarily closest genome containing a contiguous cluster (see S1 Fig). Locus tags highlighted by pale blue boxes correspond to genes such as enzymes involved in monosaccharide synthesis, modification, or incorporation into precursor repeat units of the respective polymer. White circles depict the presence of a homologous gene encoded elsewhere in the chromosome (but not syntenic with the remainder of the EPS/MASC/BPS biosynthesis cluster). BPS, biosurfactant polysaccharide; BYK, bacterial tyrosine autokinase; EPS, exopolysaccharide; IM, inner membrane; MASC, major spore coat; OM, outer membrane.
(A) Box plots of the swarm surface obtained on 0.5% agar from T4P-dependent motility after 48 hours. The lower and upper boundaries of the boxes correspond to the 25th and 75th percentiles, respectively. The median (line through center of boxplot) and mean (+) of each dataset are indicated. Lower and upper whiskers represent the 10th and 90th percentiles, respectively; data points above and below the whiskers are drawn as individual points. Asterisks denote datasets displaying statistically significant dataset differences (p < 0.05) compared with WT, as determined via 1-way ANOVA with Tukey’s multiple comparisons test. A minimum of 4 biological replicate values were obtained, each the mean of 3 technical replicates. Raw values and detailed statistical analysis are available (S1 Data). (B) EPS-, MASC-, and BPS-pathway mutant swarm physiologies. Top: T4P-dependent motility after 48 hours (scale bar: 2 mm). Bottom: Fruiting body formation after 72 hours (main panel, scale bar: 1 mm; magnified inset, scale bar: 400 μm). BPS, biosurfactant polysaccharide; EPS, exopolysaccharide; MASC, major spore coat; T4P, type IV pilus; WT, wild type.
(A) Boxplots of trypan blue dye retention to indicate the levels of surface-associated polysaccharide production in various strains relative to WT. The lower and upper boundaries of the boxes correspond to the 25th and 75th percentiles, respectively. The median (line through center of boxplot) and mean (+) of each dataset are indicated. Lower and upper whiskers represent the 10th and 90th percentiles, respectively; data points above and below the whiskers are drawn as individual points. Asterisks denote datasets displaying statistically significant differences in distributions (p < 0.05) shifted higher (**) or lower (*) than WT, as determined via Wilcoxon signed-rank test performed relative to “100.” Raw values and detailed statistical analysis are available (S2 Data). (B) Real-time clearance of hexadecane–CYE supernatant emulsions from BPS+ and BPS− strains; values are the mean of 3 biological replicates (+/− SEM). OD600 values were normalized to their first registered values, whereas registered time points are displayed at their actual occurrence. Inset: Scanning time (post-mixing) for a given cuvette (containing a culture supernatant–hexadecane emulsion) until a first absolute value for OD600 could be registered by the spectrophotometer, for samples with(out) BPS (n = 3). Asterisk (*) denotes statistically significant difference in mean value compared with pilA mutant (p = 0.0031), as determined via unpaired Student’s t test. Raw values and detailed statistical analysis are available (S2 Data). (C) Time course of normalized surface tension values (via digital-drop tensiometry) from representative submerged-culture supernatants. Surface tension values across all time points were normalized against the initial surface tension value (t = 0) for each respective strain (S3C Fig). Strains tested: WT, MASC− (ΔwzaS), BPS− MASC− (ΔwzaB ΔwzaS), EPS− MASC− (ΔwzaX ΔwzaS), EPS− BPS− MASC− (ΔwzaX ΔwzaB ΔwzaS). Inset: Slope values from biological replicate time courses (each represented by a different shape) for each strain. Slopes were calculated by fitting the time-course curves with a fourth-degree polynomial function. Raw values are available (S3 Data). (D) T4P-dependent swarm spreading in the presence of exogenous di-rhamnolipid-C14-C14 biosurfactant from Burkholderia thailandensis E264. The lower and upper boundaries of the boxes correspond to the 25th and 75th percentiles, respectively. The median (line through center of boxplot) and mean (+) of each dataset are indicated. Lower and upper whiskers represent the 10th and 90th percentiles, respectively. Asterisks denote datasets displaying statistically significant differences in mean values (p < 0.05) compared with WT swarms, as determined via 1-sample t test performed relative to “100.” Raw values and detailed statistical analysis are available (S1 Data). (E) Real-time clearance of hexadecane–CYE supernatant emulsions from T4P+ and T4P− BPS-producing strains; values are the mean of 3 biological replicates (+/− SEM). OD600 values were normalized to their first registered values, whereas registered time points are displayed at their actual occurrence. Inset: Scanning time (post-mixing) for a given cuvette (containing a culture supernatant–hexadecane emulsion) until a first absolute value for OD600 could be registered by the spectrophotometer, for samples with(out) a functional T4P (n = 4). Asterisk (*) denotes statistically significant difference in mean value compared with ΔwzaX (p = 0.0265), as determined via unpaired Student’s t test. Raw values and detailed statistical analysis are available (S2 Data). BPS, biosurfactant polysaccharide; CYE, casitone-yeast extract; EPS, exopolysaccharide; MASC, major spore coat; OD600, optical density at 600 nm; T4P, type IV pilus; WT, wild type.
(A)
1H NMR spectra of concentrated supernatants from BPS+ ΔwzaX ΩpilA and BPS− ΔwzaB ΩpilA cultures. (B)
1H–13C HSQC spectrum of deacetylated acidic polysaccharide originally isolated from ΔwzaX ΩpilA supernatant. Analysis was performed at 27°C, 500 MHz. Resonance peak colors: black, C–H; green, C–H2. (C) Negative-mode high cone voltage (180 V) ESI-MS of deacetylated acidic polysaccharide from ΔwzaX ΩpilA supernatant. (D) Chemical structure of the BPS polymer tetrasaccharide RU. BPS, biosurfactant polysaccharide; d -ManNAc, N-acetyl-d -mannosamine; d -ManNAcA, N-acetyl-d -mannosaminuronic acid; ESI-MS, electrospray ionization mass spectrometry; HSQC, heteronuclear single quantum correlation; RU, repeating unit.
(A) EPS− (ΔwzaX) and BPS− (ΔwzaB) cells from exponentially growing cultures were mixed at the indicated ratios to a final concentration of OD600 5.0. Pure and mixed cultures were then spotted on CYE 0.5% agar and imaged after 48 hours at 32°C. (B) Swarm areas with temporal tracking of pure and mixed cultures were treated as described and imaged at 24, 48, and 72 hours. Each data point is the average of 4 biological replicates and is displayed +/− SEM. Mixed/pure cultures with statistically significant differences (p ≤ 0.05) in mean surface areas (at 72 hours relative to WT) are indicated with an asterisk (*), as determined via 1-way ANOVA followed by Dunnett’s multiple comparisons test. Raw values and detailed statistical analysis are available (S1 Data). (C) EPS− (ΔwzaX) PpilA-OMss-sfGFP and BPS− (ΔwzaB) PpilA-IMss-mCherry cells were mixed at a 1:1 ratio as in (A), spotted on agar pads, and imaged via fluorescence microscopy after 24 hours. The 2 images on the right are magnified views of the colony center and colony edge approximately indicated by the inset boxes in the “composite” image. BPS, biosurfactant polysaccharide; CYE, casitone-yeast extract; IMss, inner-membrane signal sequence; OMss, outer membrane signal sequence; OD600, optical density at 600 nm; sfGFP, superfolder green fluorescent protein; WT, wild type.
(A) Dual-labeled (PEPS-sfGFP + PBPS-mCherry) WT cells (strain EM709) from exponentially growing cultures were spotted on developmental media at a final concentration of OD600 5.0 and imaged at 48 hours. Images were scaled as described in “Material and methods.” The 2 images on the right are magnified views of the colony center and colony edge at positions approximately indicated by the inset boxes in the composite image. (B) Flow cytometry analysis of WT + PEPS-sfGFP + PBPS-mCherry cells harvested from the colony interior or periphery following incubation for 48 hours. Cells were analyzed for intensity of sfGFP and mCherry fluorescence. Results of 3 independent experiments are displayed (first experiment, blue lines; second experiment, green lines; third experiment, magenta lines). For each experiment, a total population of 300,000–500,000 events was used and statistically analyzed. Differences between fluorescence intensity at the colony center versus edges are significant (p < 0.0001) for all experiments except for the first experiment with sfGFP. Errors bars are set at 1% confidence. Signals obtained with the nonfluorescent WT strain were subtracted from the fluorescence signals of strain EM709. Raw values are available (S3 Data). BPS, biosurfactant polysaccharide; Expt., experiment; EPS, exopolysaccharide; OD600, optical density at 600 nm; sfGFP, superfolder green fluorescent protein; WT, wild type.
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