Large cell groups form on alginate but not on digested alginate.

(A) Schematic representation of the setup of the microfluidic experiments. (B and C) Representative images at different time points of V. cyclitrophicus ZF270 cells growing in microfluidic chambers, described in detail by Dal Co et al.15, with (B) alginate medium or (C) digested alginate medium, both in their soluble form (not visible). Cells are false-colored according to their lineage identities based on cell segmentation and tracking over 24 hours. Cells without identified progenitors are colored in dark blue. See Supplemental Videos S1 (alginate) and S2 (digested alginate) for time-lapse videos. (D) Cell numbers within microfluidic chambers supplied with alginate (orange) are substantially higher than cell numbers within microfluidic chambers supplied with digested alginate (blue) (Logistic growth regression for alginate: R2 = 0.99, maximal number of cells = 1217-1564, k = 0.24-0.38 h-1; for digested alginate: R2 = 0.86-0.97, maximal number of cells = 47-100, k = 0.07-0.4 h-1). Circles indicate the number of cells present at a given time point in each chamber (nchambers = 7). Data for chambers with alginate originate from D’Souza et al.8. Lines are fits of a logistic growth regression line for each condition.

Transition from alginate to digested alginate triggers density-dependent dispersal of cells.

(A) Representative time-lapse images of V. cyclitrophicus ZF270 cells (phase contrast microscopy) in microfluidic growth chambers that were initially exposed to alginate and then switched to digested alginate. (B) Number of cells in different chambers over time, each chamber indicated by a unique color (n = 8). The carbon source is indicated by the colored background (orange: alginate; blue: digested alginate). See Supplemental Video 3 for a time-lapse video. (C) Positive relationship between the number of cells in the microfluidic growth chamber at the time of the switch and the fraction of cells that disperse after the switch. Each circle represents one growth chamber with colors corresponding to (B), and the line depicts a linear regression fit (R2 = 0.92, slope = 0.12).

Cells are more motile on digested alginate than alginate and show chemotaxis towards alginate.

(A and B) Spatial trajectories of cells supplied with (A) alginate or (B) digested alginate in representative microfluidic growth chambers are shown. Black points mark the starting point of each trajectory, pink points mark the end point of each trajectory, and colored lines mark the trajectories of cells. (C) Distributions of the mean single-cell swimming speeds (Nested t-test, p-value < 0.0007, t = 4.803, df = 10, ncells = 86 vs 375 in nchambers = 5) are shown. (D) Distributions of cell displacement over the course of a trajectory (Nested t-test test, p-value < 0.0131, t = 4.39, df = 10, ncells = 86 vs 375, and nchambers = 5) are shown. In (C) and (D) the red horizontal lines indicate the mean while black lines depict the 25th and 75th quartiles of the distribution. (E) The mean fraction of motile cells in each chamber, where motile cells are defined as cells with displacement greater than 1 µm (Mann-Whitney test on the means of five growth chambers, p-value = 0.034). In C, D, and E, each chamber was considered as an independent replicate. (F) Chemotactic index (IC) quantified by In Situ Chemotaxis Assay (ISCA) (Tukey multiple comparisons of means, 95% family-wise confidence levels as error bars, p-value < 0.05, n = 3). Asterisks indicate statistically significant differences. See Supplemental Videos S4 and S5 for time-lapse videos of swimming cells.

Twelve functional gene sets are enriched in genes with increased or decreased expression in cells grown on digested alginate.

Gene set enrichment analysis (GSEA) with (A) all KEGG pathways and KEGG BRITE categories as gene sets or with (B) a custom alginate utilization, flagellar assembly, and flagellum-driven chemotaxis gene set was performed comparing the gene counts of the transcriptome of V. cyclitrophicus ZF270 cultures grown on digested alginate and alginate (six replicates each). Gene sets with a positive enrichment score were enriched with genes with higher expression in cells grown on digested alginate relative to cells grown on alginate (BH-adjusted p-value < 0.05), whereas gene sets with negative enrichment scores were significantly enriched with genes with decreased expression on digested alginate. The number in brackets indicates the number of genes with unique K number per gene set (A) and the number of genes per gene set (B) within the V. cyclitrophicus ZF270 genome.

Digested alginate increases expression of genes involved in alginate degradation, uptake and catabolism, as well as flagellar assembly and chemotaxis.

Genome-wide differential expression analysis where the log2 fold changes of gene expression on digested alginate compared to alginate is shown for (A) alginate lyases (PL6, PL7, PL15, PL17, scissors symbol), transporters (porin kdgM, symporter toaB, symporter toaC), and metabolic enzymes shunting into the Entner-Doudoroff pathway (DEHU reductase DehR, kdgK, eda), (B) genes of the flagellar locus associated with flagellar assembly and (C) adjacent chemotaxis genes. Genes displayed in (B) and (C) are part of the KEGG pathways “Bacterial motility proteins” and “Bacterial chemotaxis”. Differential expression analysis was performed to compute the Benjamini-Hochberg-adjusted Wald test p-value (”BH-adj. p-value”, text color and box outline color) and log2 fold change (box fill color) for each gene (box). For better visibility, genes that exhibited a log2 fold gene expression change greater than 1 (i.e., doubling of expression) or less than -1 (i.e., halving of expression) are designated maximum intensity of red or blue, respectively. Genes with BH-adj. p-value smaller than 0.01 were considered significantly differentially expressed. In (A), the location of the gene products was based on Figure 1 of Wargacki et al.31 with the exception of the alginate lyases (PL6, PL7, PL15, PL17) which were placed based on their signal peptides (S: extracellular, LS: membrane-embedded, none: cytosolic). In (B) and (C) the gene location and depiction were based on the KEGG pathway “Flagellar assembly” (map02040), “Bacterial chemotaxis” (map02030), and Figure 3 of Rajagopala et al.40. Genes without known cellular location were omitted here but displayed in the genomic architecture in Fig. S5. Arrow: activation; dashed arrow: modification; “flat” arrow: inhibition; OM: outer membrane; PM: periplasm; IM: inner membrane; PL: polysaccharide lyase family; kdgM: oligogalacturonate-specific outer membrane porin; toaABC: oligoalginate symporter; DEH: 4-deoxy-L-erythro-5-hexoseulose uronic acid; dehR: DEH reductase; KDG: 2-keto-3-deoxy-gluconate; kdgK: KDG kinase; KDPG: 2-keto-3-deoxy-6-phosphogluconate; eda: KDG-6-phosphate aldolase; GAP: glyceraldehyde 3-phosphate; ED: Entner-Doudoroff; ns: not significant, i.e. BH-adj. p-value > 0.01.

Bacterial growth and regulation on patches of polysaccharides.

By integrating our results with previous studies on cooperative growth on the same system, as well as results on dispersal cycles in other systems, we highlight where the specific results of this work add to this framework (bold font). When cells encounter polymer sources, the colonization of the nutrient hotspot may be aided by the basal exoenzyme production of “sentry” enzymes (”Encounter of a new carbon source”). This phase is succeeded by group formation, which enables cells to benefit from exoenzymes of neighboring cells and diffusing degradation products (”Early-stage polysaccharide breakdown”). The following phase includes cooperative extracellular degradation of the polysaccharide source, further increasing the concentration of available degradation products. These degradation products trigger the overexpression of alginate degrading, importing, and catabolizing enzymes, ensuring swift polysaccharide degradation (”Late-stage polysaccharide breakdown”). The increased pool of breakdown products also cues flagellar swimming in a subpopulation of cells and increases the expression of chemotaxis genes. Polymeric alginate acts as chemoattractant towards new polysaccharide sources. Cells and molecules are not drawn to scale. Dark red pie symbols: intracellular and extracellular polysaccharide-degrading enzymes; orange shading: a polymeric carbon source; blue shading: monomeric or oligomeric degradation products.