PMC

Scanning electron microscopy of WT and TCP⁻ mutant biofilms at 48, 72, and 96 h. The arrow points at microcolonies of the WT strain formed during the development of WT biofilms on the surface of the squid pen. TCP⁻ biofilms remained undifferentiated. Bars, 10 μm.

Mixed biofilms of WT and TCP⁻ mutant strains. (A) Phase-contrast micrograph showing the differentiated biofilms developed on the surface of a squid pen by a 50:50-mixed culture of a WT and a gfp-tagged TCP⁻ mutant strain over a 96-h period. (B) Fluorescence micrograph placed over the micrograph in panel A showing the uniform distribution of the gfp-tagged TCP⁻ mutant strain within the mixed biofilms. Bars, 10 μm.

Effect of SDS treatment on WT and TCP⁻ mutant biofilms on a squid pen. Three-dimensional side views, generated by confocal scanning laser microscopy, of SDS-treated (+ SDS) or untreated (− SDS) biofilms of the WT and TCP mutant strains of V. cholerae showing the detachment of most cells from the undifferentiated TCP⁻ biofilm after SDS treatment are presented; the structured WT biofilms remained unaffected. The substratum (squid pen) is located at the bottom of the images. Bars, 50 μm.

Colonization of squid pens by a WT and a TCP⁻ mutant strain of V. cholerae. (A) Biofilm development over a 96-h period. Biofilm biomass was estimated by measuring the fluorescence from gfp-tagged cells (expressed as relative fluorescence units [RFUs]). (B) Chitinase activity associated with biofilm cells of the WT and TCP⁻ mutant strains during the colonization of squid pens. Specific activities of units of chitinase activity (U, described as the amount of enzyme that released 1 pmol of 4MU per min under the assay conditions) per RFU are shown. (C) Immunodetection of TcpA, the TCP pilin subunit, in biofilm cells in the course of 96 h and in planktonic cells at 24 h (Plank). (D) Immunodetection of TcpA in cultures grown under TCP-inducing conditions (AKI medium) or grown for 24 h in the absence of a chitinous surface in NG2 mineral medium with lactate and NH₄Cl (L/N) or with colloidal chitin (CollCh), and in LB medium.