Fig 1.
Morphological heterogeneity in P. inhibens.
(A) Phase contrast image of cells from a mid-exponential phase culture. Asterisk indicates a pre-divisional constricted cell. (B) Fluorescent membrane staining of the cells shown in A. Asterisk indicates a septum. (C) Fluorescent membrane staining shows filamentous cells with no septa. Scale bars correspond to 1 μm.
Fig 2.
Polar polysaccharide at the center of rosettes.
(A) Fluorescent overlay image of membrane stained cells (red) and Alexa 488-conjugated WGA lectin (green). Scale bar corresponds to 1 μm. (B) Quantification of lectin fluorescence intensity per cell in arbitrary units [AU]. Shown is a box plot generated with the R software [28]. (C) Percent of population in rosettes at early growth stages (2, 4, and 6 hours) or in a mid-exponential (ME) culture (see Methods). Cultures for this time course were obtained as described in Methods. Error bars indicate standard deviation of two biological replicates.
Fig 3.
Cell type distribution does not vary with culture age.
Seven different cell types were characterized by size, labeling with Alexa 488-conjugated WGA (green) and presence of a septum using membrane stain FM4-64 (red). Each cell type was quantified at 2, 4 and 6 hours of a culture in early growth stages; n > 300 cells. Scale bar corresponds to 1 μm.
Fig 4.
Kinetics of rosette formation.
(A) Rosettes form by cell encounters. Two independent aliquots from the same synchronized culture were stained with either Alexa 488-conjugated WGA (green) or Alexa 594-conjugated WGA (red) and were mixed for 30 minutes at room temperature in buffer prior to imaging. Rosettes with dual-labeled centers are shown. Images are overlay of phase contrast (gray) with green and red fluorescence channels. (B) Rosette complexity increases over time. The number of cells per rosette was quantified over time. Error bars indicate standard deviation of two biological replicates; n > 300 cells.
Fig 5.
Attachment capability to an abiotic surface increases over time.
(A) Ratio of crystal violet absorbance in arbitrary units over cell growth measured by OD600. (B) Cells of different ages were diluted to the same cell density prior to quantifying crystal violet absorbance of attached cells. Error bars indicate standard deviation of at least two biological replicates. Note: the 10-fold difference in crystal violet absorbance values between (A) and (B) is due to different incubation times during the attachment assay. See Methods for details.
Fig 6.
Self-DNA prevents attachment to an abiotic surface.
Crystal violet absorbance of attached cells was quantified before and after addition of genomic DNA originating from either P. inhibens or E. coli. “Before treatment” measurement was conducted after 2 hours of incubation in an attachment assay (see Methods) and the different treatments were then applied. Measurements after treatments were conducted one hour following the addition of water or DNA. Error bars indicate standard deviation of at least two biological replicates. Note: crystal violet absorbance values before treatment and after treatment should be compared to time points 2 and 3 hours, respectively, in Fig 5A.