Fig 1.
Ids mismatch during swarming causes large transcriptional shifts.
A. Cartoons show self versus non-self interactions as defined by the Ids state in and between cells. The post-transferred state of IdsD is depicted. Endogenous IdsD and IdsE are represented by “D” and “E”, while a non-self variant of IdsD is represented by “d.” B. A graphical representation of genes differentially regulated in consolidator cells isolated from clonal Δids swarms versus consolidator cells from clonal wild-type swarms. The X-axis corresponds to each gene on the P. mirabilis BB2000 chromosome. The Y-axis corresponds to log2-fold difference in relative transcript abundance. Genes with significantly different relative transcript abundance, defined as fold-change > log2 1.5, p < 0.05, are marked in red. All data are included. The genes idsA-F, which were deleted to construct the Δids strain, are noted. C. A graphical representation of genes differentially regulated in whole swarms of strain CCS06 versus consolidators from strain CCS01, constructed similarly to that of (B). CCS01 is the wild-type equivalent background for CCS06 and CCS02 as the ids genes are expressed from a plasmid in all strains; the strain background is Δids. Labeled genes are discussed in the text. D. Venn diagrams showing genes similarly differentially regulated in three separate swarm conditions for an Ids mismatch between cells: co-swarmed Δids, clonal CCS06, and clonal CCS02. Two biological repeats were performed.
Fig 2.
Cells experiencing Ids mismatch display transient tolerance to lethal concentrations of antibiotics.
A. Killing curves for cells of wildtype (cyan square), Δids (gray "x"), and ΔidsE (magenta circle) harvested from swarm plates and exposed to 100 μg ml-1 ampicillin. Note that the Y-axis is a logarithmic scale. Asterisks represent statistically significant difference between ΔidsE and wildtype (p = 0.02) calculated using a Mann-Whitney test. B. Survival of strains wildtype (cyan square), Δids (gray “x”), and ΔidsE (magenta circle) after swarm colonies were harvested and exposed to 60 μg ml-1 kanamycin (left), 50 μg ml-1 streptomycin (middle), and 1 μg ml-1 ciprofloxacin (right) for 12 hours. Asterisks represent statistically significant difference between ΔidsE and wildtype (p = 0.04) calculated using a Mann-Whitney test. C. Territorial exclusion does not result in long-term growth defects. Optical density at 600 nm (OD600 nm) was measured over time for liquid cultures of wildtype and the co-swarmed Δids strain. Liquid cultures were inoculated using cells isolated by FACS from co-swarm colonies where the Δids strain had been actively excluded from the swarm front.
Fig 3.
Cells affected by Ids mismatch induce an increase in ppGpp; the relA and spoT genes are necessary to induce Ids-mediated territorial exclusion.
A. Mean ppGpp absorbances from wildtype, the Δids strain, and the ΔidsE strain, calculated by integrating areas under peaks. Errors show standard deviations. Asterisks show statistically significant difference (p = 0.03) between ΔidsE and wildtype, calculated through a Mann-Whitney test. B. Swarm colony radius expansion of the ΔidsE, ΔidsEΔrelA, ΔidsEΔspoT, and ΔidsEΔrelAΔspoT strains after 16 hours of swarming. All replicates are shown. Asterisks show statistically significant difference between strains (p = 0.02) calculated through a Kruskal-Wallis test.
Fig 4.
The transcriptional shift observed in cells experiencing Ids mismatch occurs during consolidation periods between swarming.
A. A time-course graph showing mean swarm fluorescence intensity over time for two conditions: Δids carrying a chromosomal BB2000_0531 fluorescence reporter (MJT02) co-swarmed with wildtype (wildtype + MJT02, cyan squares) or co-swarmed with the Δids strain (Δids + MJT02, gray circles). Three biological repeats were performed. Fluorescence was measured over equivalent areas for each experimental condition. Error bars are standard deviations; a.u. means arbitrary units. Asterisks show statistically significant differences (p < 0.01) calculated through individual Mann-Whitney tests for each timepoint. B. Representative microscopy images phase contrast overlaid with Venus fluorescence (100x magnification) of wildtype + MJT02 (top) and Δids + MJT02 (bottom) co-swarms. Images shown are of the first active swarm phase (left, 1 hour) and the first period of consolidation (right, 2.5 hours). Scale bars = 10 μm.
Fig 5.
The Δids strain is excluded during consolidation between periods of swarming.
(A) 1:1 co-swarms of Δids-GFP and wildtype-DsRed or (B) wildtype-GFP and wildtype-DsRed were analyzed for GFP-associated and DsRed-associated fluorescence over the course of outward migration and through progression of the swarm-consolidation cycle. Each data point indicates the proportion of GFP-producing cells measured at the swarm front at a given time after swarm emergence. Periods of active swarm expansion were determined by eye and are highlighted in gray. Three biological repeats were performed, and each is shown.
Fig 6.
A model for Ids-mediated territorial exclusion.
Top: IdsD is exchanged between motile, elongated swarmer cells. Transfer from a wildtype cell (cyan) to a Δids cell (gray) is shown. Middle, wild-type cells expressing a cognate IdsE do not experience any effects from incoming IdsD and progress normally through consolidation phase, forming new swarmer cells. The Δids cells without a cognate IdsE (gray) continue to swarm normally after IdsD transfer until entry into consolidation phase. Bottom, upon differentiation into consolidation phase, unbound IdsD induces elevated ppGpp levels and profound transcriptional changes, resulting in a growth-arrested state incompatible with differentiation into new swarmer cells. The resultant phenotype exhibits secondary effects including increased antibiotic tolerance.
Table 1.
Strains used in this study.