V. cholerae occurs in freshwater and marine environments in two states, the planktonic state and/or attached to a surface in a biofilm. One type of multilayer biofilm of V. cholera, consisting of multiple cell layers, depends on synthesis of an extracellular matrix comprised of the VPS exopolysaccharide and numerous VPS-associated proteins, all of which are encoded within VPS island of the V. cholerae genome. Various environmental cues, including quorum sensing autoinducers regulate transcription of genes on VPS island. Recent studies have shown that certain sugars such as glucose and mannose play key role in biofilm formation of V. cholerae. Transport of these sugars, including mannitol (a sugar alcohol) is dependent on phosphoenolpyruvate (PEP) phosphotransferase system (PTS). PTS is a conserved bacterial signal transduction pathway with various cellular functions, including transport and phosphorylation of certain sugars and their derivatives, detection of quorum sensing molecules and biofilm formation. The pathways of PTS and enzymes included in it are described in the introduction of this paper. Phosphorylation states of PTS depend on intracellular availability of PEP and the environmental availability of PTS-specific sugars, and therefore, they can be called as sensors of the nutritional status of the cell. The authors of this paper have already shown that mutations in PTS components of V. cholerae causes increased tendency of forming biofilms. PTS mutants showed increased transcription of mannitol-specific PTS component, compared to wild types and this PTS component was also induced under biofim activating conditions. This study investigated role of maninitol and mannitol-specific component of PTS in biofilm formation by V. cholerae.
The selected bacterial strains were cultured in mannitol containing medium. Mutants were created by using molecular methods and, wild and mutant strains were cultured and RNA of cells was isolated. This RNA was converted into cDNA by using reverse transcriptase and then this cDNA was used in microarray. The ectopic protein expression, total growth, biofilm formation and vpsL transcription, were measured.
Transcriptomic analysis revealed that compared to wild strain biofilm, 563 genes were differentially regulated in the PTS mutant biofilm. Same analysis between both wild and mutant types of planktonic state revealed differential regulation of only 218 genes. The majority of these differentially regulated genes were associated with transport or metabolism of carbohydrates such as sugars. Major differential regulation was with genes of certain (EII) components of PTS. These PTS components which were highly induced in both planktonic and biofilm cells, were previously found to be transcriptionally activated by the addition of PTS sugars, which increases transcription of biofilm genes. The authors hypothesized that abundance of PTS-sugars and mutation in PTS, both of these scenarios result in similar physiological state of activating biofilm formation in V. cholera.
Mannitol increases VPS-dependant biofilm formation and, it is dependent on mannitol transport in V. cholera. It was found that concentrations of mannitol needed to activate biofilm formation were lesser than mannitol concentrations naturally found in marine environments. Certain mannitol specific PTS component (EIIBMtl) activates biofilm formation in V. cholera. But it is dependant on phosphorylation state and, only unphosphorylated EIIBMtl activates biofilm formation. Mannitol and unphosphorylated EIIBMtl activate biofilm formation at transcriptional level.
Apart from its association with chitinaceous surfaces, very little is known about the natural habitats of V. cholerae in the marine environments. Various marine organisms use mannitol as osmoprotectant and it is also released during algal photosynthesis (e.g. brown algae). This study, with its robust molecular experiments showed that mannitol can induce V. cholera to form biofilms and additionally V. cholerae may also use it as a source of carbon to survive in the marine environment. This study highlights how complex the life cycle and/or surviving capabilities of this clinically important pathogen can be in the marine environment and how much still we need to explore about this pathogen in order to develop strategies for its effective control.
Ymele-Leki, P., Houot, L., & Watnick, P. I. (2013). Mannitol and the mannitol-specific enzyme IIB subunit activate Vibrio cholerae biofilm formation. Applied and environmental microbiology, 79(15), 4675-4683.