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. 2018 Dec 24;7:e42057. doi: 10.7554/eLife.42057

Figure 2. Quorum sensing controls rapid, VpsL-independent aggregation of V. cholerae in liquid.

Aggregate formation of the LCD QS-locked (LCD-locked) (A), HCD QS-locked (HCD-locked) (B), and wild-type (WT) (C) V. cholerae strains after 22 h of growth. Shown are representative cross-sections through samples (A–C). The approximate extents of individual aggregates are indicated with white outlines. Magnification: 63X; scale bar: 50 μM. (D) Quantitation of total volume fraction, the total volume of the imaged region that is occupied by aggregates (Materials and methods) within the imaged region for ΔvpsL LCD-locked, ΔvpsL HCD-locked, and ΔvpsL WT strains after 22 h of growth. Representative cross-sections through the ΔvpsL LCD-locked (E–H), ΔvpsL HCD-locked (I–L), and ΔvpsL WT (M–P) V. cholerae strains at 16, 19, 22, and 25 h. (E–P) Magnification: 10X; scale bar: 250 μm. (Q) Quantitation of aggregate volume fraction. The data for T = 22 h are the same as those shown in Figure 2D. Triangle: ΔvpsL LCD-locked, circle: ΔvpsL HCD-locked, and diamond: ΔvpsL WT. (R) Average cluster volume over time for the ΔvpsL HCD-locked (circle) and ΔvpsL WT (diamond) strains. (D,Q,R) Quantitation of mean ± standard deviation (SD; N=3 biological replicates). Mean and SD were calculated using the untransformed data, not the log-transformed data, which results in asymmetric error bars. All strains in all panels harbor the fluorescent mKO reporter constitutively expressed from the chromosome.

Figure 2—source data 1. Figure source data.
elife-42057-fig2-data1.xlsx (273.3KB, xlsx)
DOI: 10.7554/eLife.42057.014

Figure 2.

Figure 2—figure supplement 1. V. cholerae aggregates form in liquid.

Figure 2—figure supplement 1.

Aggregate formation in 24-well uncoated plates (Uncoated) or in plates coated with a hydrogel layer that reduces cell attachment (Low-attachment). (A,B) ΔvpsL HCD QS-locked (HCD-locked), (C,D) ΔvpsL LCD QS-locked (LCD-locked), and (E,F) the vpvcW240R hyper biofilm former. (C,D) We note that ΔvpsL LCD-locked strains produce ‘voids’, which are regions containing few cells; we do not understand the underlying mechanism giving rise to these features. (A–D) Images taken of cells in liquid medium. (E,F) Images taken at the surface of the plate. (A–F) All strains harbor mKO constitutively expressed from the chromosome and are representative of 3 biological replicates. Magnification: 10X; scale bar: 250 μM.
Figure 2—figure supplement 2. V. cholerae forms distinct aggregates.

Figure 2—figure supplement 2.

Representative cross-section following 22 h of growth of aggregates formed in liquid by the ΔvpsL HCD-locked V. cholerae strain that constitutively expresses fluorescent mKO, mKate2, or mTFP1. Strains were independently grown for 22 h until aggregates formed. Aggregates from each culture were mixed for 60 s and then imaged. The cells carrying the different fluorescent reporters do not mix, but rather, form distinct aggregates. Magnification: 10X; scale bar: 250 μM.
Figure 2—figure supplement 3. Known Ca2+-related genes do not contribute to V. cholerae aggregation.

Figure 2—figure supplement 3.

Quantitation of aggregate formation following 22 h of growth of the ΔvpsL HCD QS-locked (HCD-locked), ΔvpsL LCD QS-locked (LCD-locked), ΔvpsL ΔvpsN HCD-locked, ΔvpsL ΔasnB HCD-locked, and ΔvpsL ΔcarR HCD-locked strains. Homologs of VpsN and AsnB (WbfF and WbfR, respectively) contribute to O-antigen and capsule synthesis, respectively, and therefore, respectively, promote and repress Ca2+-dependent biofilm formation in V. cholerae O139 strain MO10 (Kierek and Watnick, 2003a). CarR is the response regulator of the V. cholerae CarRS two-component system that responds to extracellular Ca2+ (Bilecen and Yildiz, 2009). Error bars are mean ± standard deviation, (N=3 biological replicates). All strains harbor mKO constitutively expressed from the chromosome.
Figure 2—figure supplement 3—source data 1. Figure source data.
elife-42057-fig2-figsupp3-data1.xlsx (203.9KB, xlsx)
DOI: 10.7554/eLife.42057.007
Figure 2—figure supplement 4. Known pili genes do not contribute to V. cholerae aggregation.

Figure 2—figure supplement 4.

Quantitation of total aggregate volume fraction following 22 h of growth of the ΔvpsL HCD QS-locked (HCD-locked), ΔvpsL LCD QS-locked (LCD-locked), ΔvpsL ΔtcpA HCD-locked, ΔvpsL ΔpilA HCD-locked, and ΔvpsL ΔmshA HCD-locked V. cholerae strains. tcpA, pilA, and mshA, respectively, encode structural components of the toxin co-regulated pilus (TCP), the chitin-regulated pilus (ChiRP), and the mannose-sensitive haemagglutinin pilus (MSHA) . Error bars are mean ± standard deviation (N=3 biological replicates). All strains harbor mKO constitutively expressed from the chromosome.
Figure 2—figure supplement 4—source data 1. Figure source data.
elife-42057-fig2-figsupp4-data1.xlsx (253.6KB, xlsx)
DOI: 10.7554/eLife.42057.009
Figure 2—figure supplement 5. WT and ΔvpsL strains display similar aggregation kinetics.

Figure 2—figure supplement 5.

Aggregate formation in WT V. cholerae at 16, 19, 22, and 25 h (A-D). Images are representative cross-sections of 3 biological replicates. Magnification: 10X; scale bar: 250 μm. Strain harbors mKO constitutively expressed from the chromosome.
Figure 2—figure supplement 6. V. cholerae aggregate formation is rapid.

Figure 2—figure supplement 6.

Quantitation of total aggregate volume fraction in a ΔvpsL HCD-locked V. cholerae strain harboring mKO constitutively expressed from the chromosome. Three biological replicates were sampled every 30 min from 19 h to 21 h. Error bars are mean ± standard deviation (N=2–3 technical replicates).
Figure 2—figure supplement 6—source data 1. Figure source data.
elife-42057-fig2-figsupp6-data1.xlsx (226.5KB, xlsx)
DOI: 10.7554/eLife.42057.012
Figure 2—figure supplement 7. V. cholerae aggregate formation is non-clonal.

Figure 2—figure supplement 7.

Image of a representative cross-section through a V. cholerae aggregate following 22 h of growth. The white outline shows the approximate extent of the aggregate. The culture was inoculated at T = 0 h with a roughly equal mixture of the ΔvpsL HCD-locked strain constitutively expressing either mTFP1 or mKate2 from the chromosome. Magnification: 63X,; scale bar: 25 μM.