UC Berkeley

Biosurfactants are biologically produced compounds that reduce interfacial tensions due to their water- and oil- loving groups. These amphiphilic substances are widely investigated for their potential commercial exploitation, yet little evidence has been assembled for their direct roles in the environment for the bacteria that produce them. In order to better enable the investigation of biosurfactants, we developed an efficient method for biosurfactant detection that is more sensitive than the standard drop collapse assay, as well as capable of detecting surfactants that have low water solubility and would normally be overlooked. A large number of bacteria recovered from different environments were assessed for biosurfactant production using this atomized oil assay. Detectable biosurfactant production was found to be quite common amongst culturable bacteria, with 5 to 13% of all bacteria from various habitats expressing this trait. Furthermore, we deployed the atomized oil assay in two mutagenesis screens to determine the biosynthetic and regulatory pathways of biosurfactant production in Pseudomonas syringae pv. syringae B728a.

A recurring theme that emerges in this dissertation is the importance of biosurfactant production for life on a surface. Not only were biosurfactant-producing bacteria more commonly found in terrestrial surface environments such as leaves than in aqueous samples, but the bacteria that produce biosurfactants were also more likely to produce such compounds when grown on a surface compared to planktonically. Furthermore, the patterns of regulation of the biosurfactants produced by P. syringae also provide additional support for their importance at surfaces. Syringafactin production is higher in cells grown on agar plates than in broth cultures. Also, an unidentified surfactant is produced in larger quantities when P. syringae is grown on hydrated rough surfaces compared to smooth agar plates.

Examination of the control of biosurfactant production in P. syringae revealed that syringafactin is regulated by SyfR, a divergently transcribed LuxR-type regulator. SyfR is the mediator of the surface sensing response since in the absence of functional SyfR protein, the SyfR promoter is equally induced in broth and plate cultures, but when present, both the transcription of SyfR and syringafactin is increased on agar plates. A new function for this type of LuxR-type regulator was thus demonstrated. Furthermore, random mutants with altered surfactant production were identified using the atomized oil assay enabling the investigation of biosynthetic and regulatory genes required for the unidentified biosurfactant produced by P. syringae B728a. This surfactant has low water solubility and is synthesized by an acyltransferase that when expressed in trans in E. coli is sufficient for its production. Production of this surfactant is dependent on proper flagellar assembly and the compound was thus termed BRF (biosurfactant regulated by the flagella). Mutations in genes necessary for early establishment of the flagellar apparatus abolish BRF production, while mutations that stimulate higher flagellin production increase BRF production. Flagellin synthesis is up-regulated at surfaces, and BRF synthesis was co-regulated with flagellin synthesis under conditions of varying agar concentrations in culture media. The induction of BRF production was especially pronounced during growth on hydrated paper discs, where both flagellin and BRF are induced more highly than growth on agar surfaces. BRF was induced even more highly in cells grown in broth cultures, independent of levels of flagellin production. Thus BRF is not restricted to surface production, but its production on a surface appears to be regulated by flagellar surface sensing.

In addition to assembling support from environmental collections and genetic regulation that biosurfactant production is linked to life on a surface, we directly tested its importance in planta. Biosurfactant production by P. syringae B728a increased the wettability of the leaf surface. While syringafactin production provides a slight increase in the epiphytic fitness of P. syringae on the leaf surface, no contribution of BRF could be found under the conditions tested. Syringafactin appears to either increase the colonizable area of the leaf surface (water droplets with lower surface tension have increased surface area), or to increase the local density of bacteria on leaves. Purified syringafactin increased the water permeability of isolated plant cuticles, lending support that its production allows for increased nutrient access on leaves. BRF did not significantly alter the permeability of cuticles, nor do strains defective in its production have lower measurable fitness. BRF thus might function as a flagellar lubricant enabling surface motility. Combined evidence from environmental studies, investigations of genetic regulation, as well as in planta experimentation reveals that biosurfactants have multiple roles at surfaces.