Despite its notoriety as a human pathogen, Vibrio cholerae is an aquatic microbe suited to live in freshwater, estuarine, and marine environments where biofilm formation may provide a selective advantage. Here we report characterization of biofilms formed on abiotic and biotic surfaces by two non-O1/O139 V. cholerae strains, TP and SIO, and by the O1 V. cholerae strain N16961 in addition to the isolation of 44 transposon mutants of SIO and TP impaired in biofilm formation. During the course of characterizing the mutants, 30 loci which have not previously been associated with V. cholerae biofilms were identified. These loci code for proteins which perform a wide variety of functions, including amino acid metabolism, ion transport, and gene regulation. Also, when the plankton colonization abilities of strains N16961, SIO, and TP were examined, each strain showed increased colonization of dead plankton compared with colonization of live plankton (the dinoflagellate Lingulodinium polyedrum and the copepod Tigriopus californicus). Surprisingly, most of the biofilm mutants were not impaired in plankton colonization. Only mutants impaired in motility or chemotaxis showed reduced colonization. These results indicate the presence of both conserved and variable genes which influence the surface colonization properties of different V. cholerae subspecies.The ecology of the cholera-causing bacterium, Vibrio cholerae, has been considered since the 1850s, when contaminated water sources were first implicated by John Snow as a key factor associated with its epidemiology (59). However, many of the strategies that this organism employs for survival outside the human host have come to light only recently. Perhaps one of the most important discoveries is the role that plankton have in aiding the survival of V. cholerae within aquatic environments. Seminal work by Huq et al. (25) demonstrated that the presence of copepods improved the survival of V. cholerae populations. Analogous experiments performed by Islam et al. (26,27) showed that V. cholerae survival can be enhanced via association with cyanobacteria. In both cases, surface colonization by V. cholerae was evident. The propensity of V. cholerae for epibiosis is also evident in its attachment to chironomid egg masses and vascular plants (20,56).The significance of V. cholerae higher-order assemblages can be further illustrated in two ways. First, there is a correlation between V. cholerae population levels and phytoplankton and zooplankton blooms (14, 24), as well as the physical factors which drive them (10, 36). The biofilms formed by V. cholerae during these associations may aid in survival, as biofilms have been shown to offer shelter from many different environmental stresses, including protozoan grazing (40), UV light (15), oxidants (13), and toxic metals (60). Second, filtration of drinking water through sari cloth (pore size, Ͼ20 m) is an effective method for removing V. cholerae assemblages and reducing the incidence of cholera (11).The biofilm characteristics of c...