Although we have arbitrarily classified surfactants as either causing either a “bright” or “dark” halo in oil drops surrounding a surfactant source, it is most probable that there is a spectrum of contact angles for the oil droplet that is dictated by the expected range of change of the various tensions by various surfactants as discussed above. Similarly, while all of our obtained mutants displayed bright halos, there is a possibility that the contact angles of the oil droplets could be slightly different, especially near mutants unable to produce syringafactin. However, we have not yet found a reliable method for measuring the contact angles of the atomized oil droplets observed with our assay, and no obvious differences in droplet shape were detected during microscopic observation of the droplets. It is not clear if there is an invariant correlation between a surfactant’s hydrophilic-lipophilic balance and the shape it imparts to oil droplets on an agar surface. It is, however, tempting to speculate on the utility of this assay in predicting important characteristics of novel surfactants. HLB values are a scalar factor that reflects the degree to which a surfactant is hydrophilic or lipophilic, with a value of zero reflecting a completely lipophilic molecule, a value of 10 corresponding to a compound with equivalent hydrophobic and hydrophilic groups, and values over 10 for predominantly hydrophilic molecules.For example, common surfactants such as SDS and Tween 20 have high HLB values and are therefore best suited for emulsifying a hydrophobic substance into a water phase. On the other hand, surfactants such as Silwet® L-77 with HLB values near 10 are more suited for wetting, or spreading of a water phase over surfaces such as leaves. These surfactants with balanced water- and oil- loving groups can be very effective as spreading agents, capable of lowering the surface tension of water below 30 mN/m. Rhamnolipid, with a predicted HLB of 9.5,plastic pots for planting which can lower the surface tension of water to 28 mN/m, is a highly effective spreading agent involved in bacterial motility.
Although there is no consensus on the HLB of surfactin, it is also capable of lowering the surface tension of water to 27 mN/m, suggestive that it may also have an HLB near 10. Surfactants like Silwet® L-77 which had lower HLB values conferred bright halos in our assay. The surfactants with HLB values over 13, which are most ideal for emulsification of oil into water, did not cause the oil droplets to bead, resulting in dark halos when tested by the atomized oil assay. It is interesting that none of the biosurfactants tested conferred dark halos, suggesting that their primary roles are not as emulsifiers.It is noteworthy that the measurements of biosurfactant production using the halo method were strongly correlated with the swarming capability in mutants of P. syringae strain B728a. This suggests that the area covered by surfactants at the air/water interface as measured by our assay reflects a similar distance where swarming movement of bacteria across an aqueous agar surface is facilitated. Moreover, it is significant that drop-collapse activity was not a good indicator of the swarming ability of a strain, which raises the question of what specific properties make a surfactant a good lubricant that facilitates bacterial motility. Because the drop-collapse assay only detects surfactants that are able to greatly lower the surface tension of water, this property appears unnecessary for functions such as swarming. In addition, use of the drop-collapse assay in biological screens may cause a wide array of biologically active surfactants to be overlooked. In view of that, it is interesting that a syringafactin mutant of P. syringae strain B728a appears to produce a second surfactant that can promote swarming but not cause a drop-collapse. This is in contrast to a syringafactin mutant in P. syringae strain DC3000 which does not appear to produce this second surfactant. It is also striking that no mutants were identified in strain B728a that exhibited a total absence of surfactant halo, pointing to differential regulation of syringafactin and the remaining expressed surfactant.
Furthermore, the disruption of pmpR apparently causes the down-regulation of syringafactin while conferring up-regulation of the other surfactant, suggesting its role in regulating both surfactants. While both P. syringae strains are pathogenic to plants, strain B728a is a much better epiphyte than DC3000. Perhaps this second surfactant is particularly useful for the lifestyle of epiphytes such as strain B728a on waxy leaf surfaces. We are actively pursuing the identity and specific properties of this second surfactant. The phytotoxins syringomycin and syringopeptin have been suggested to possess surfactant activities , although preliminary results have not yet provided support for the identity of either of these surfactants as the second surfactant. It is possible that combining one of the mutations found from this screen with a syfA or syfB mutation could reveal the identity of the second surfactant Some, but not all of the genes found to regulate both biosurfactant production and swarming ability in P. syringae have homologs that influence swarming in Pseudomonas aeruginosa. Disruption of Psyr_3619, encoding an RNA helicase, conferred a similar reduction in swarming as that seen in blockage of its homolog PA2840 in P. aeruginosa. Likewise, disruption of pmpR in P. aeruginosa, a homolog of Psyr_1407, resulted in enhanced swarming in both species. It is significant that P. syringae B728a mutations were not identified in homologs of any of the many other genes found to alter swarming in P. aeruginosa despite the near completeness of the mutant library, emphasizing that the surfactants that contribute to swarming in these strains differ and/or that many factors other than biosurfactant production contribute to swarming ability. It is also noteworthy that relatively few different genes apparently contribute to biosurfactant production in P. syringae B728a. The disruptions of only 12 unique genes, identified from over 7,000 screened mutants,strawberries in a pot were found to alter biosurfactant production. Assuming random transposon insertion, we predict that we have screened a library of approximately 77% of the P. syringae B728a genes. Although we have identified many of the mutations which have an effect on measured surfactant halos, we may have missed a number of mutations which negatively affected syringafactin production but were masked by a compensatory increase in production of the second surfactant.
For life on the leaf surface, Pseudomonads have been shown to employ a variety of traits to grow and survive despite fluctuating water availability. In response to desiccation stress, Pseudomonads produce alginate in order to maintain a hydrated micro-environment. Our finding of multiple components of the AlgT regulatory pathway among mutants of strain B728a with altered biosurfactant production could suggest an intimate relationship between water availability and biosurfactant production. This potential relationship warrants further exploration of either the AlgT pathway or perhaps alginate production itself as a regulator of surfactant production. The role of biosurfactants on the leaf surface is most likely complex, and as such may likely prove to have very complex regulatory networks. The atomized oil assay has revealed a likely diversity of biosurfactants that are produced by strain B728a and their complex patterns of expression, details that would have been difficult to discern using other assays for biosurfactant production. The tools and genetic resources developed here should prove useful in further studies of the roles of surfactants in the interaction of P. syringae with plants. Biosurfactants, or biologically-produced surface active agents, have received wide attention mostly for their potential for hydrocarbon dispersion and remediation.However, a wide variety of roles for biosurfactants have been since described, from biofilm formation to inhibitory activity against pathogenic organisms, sparking a renewed interest in their discovery. Given this interest in biosurfactants, the lack of knowledge of the distribution and frequency of occurrence of surfactant production in the environment is remarkable. Comprehensive examinations of biosurfactant production are lacking, and studies that have addressed this trait in a given environment can seldom be compared with those of other habitats ; both the screening methods used, as well as pre-screening culturing conditions such as medium and incubation conditions usually vary widely between studies. In a recent report we described a high-throughput assay which utilizes the application of atomized oil droplets to rapidly detect biosurfactants produced by bacteria on the surface of agar plates. This method has advantages over other common assays such as droplet collapse assays in that it can be performed for many colonies simultaneously after limited growth, does not require sample preparation of culture supernatants, and thus is suited for high throughput screening for surfactant producing strains. Moreover, this method is capable of detecting much lower concentrations of surfactants than the drop collapse assay, and therefore in principle is capable of identifying biosurfactant producing strains that would escape detection with most other methods. However, since the atomized oil assay has not yet been tested on a broad range of environmental isolates, in this study we address whether the range of strains that it can detect includes all of those detectable by the drop collapse assay.
Furthermore, although the atomized oil assay has proven effective at detecting surfactants on agar plates, traditionally broth culture supernatants are screened for biosurfactant activity using the drop collapse assay. Depending on the properties of the surface-active compound and its biological role for the producing strain, its production may depend strongly on whether the producing cells are situated at a surface or not. Since a large difference in the transcriptomes of bacteria grown planktonically versus on surfaces have been described, with about one-third of genes differentially regulated , it seems likely that biosurfactant production itself may be strongly influenced by cell culture conditions. Surface sensing is an important cue for many species to transition to surface-associated behavior such as swarming, whereby cells move across a moist surface utilizing flagella and surfactant. Although the surface regulation of flagella has been well documented , the regulation of surfactant production by surfaces has not yet been explored and will be addressed in this report. Insight into the role of biosurfactants would benefit from a better understanding of the numerical distribution of surfactant producers in different environments. A variety of isolated reports have described collections of biosurfactant producers from aqueous environments, polluted/unpolluted soils, and even clouds, with estimates of their frequency in culturable bacterial communities ranging from less than 3 to as much as 50%, but typically around 10%. However, no encompassing model that describes the selection for such a trait has emerged from these studies, perhaps because few comparative analyses of habitats have been performed. We hypothesize that hydrophobic surfaces are habitats that would be particularly selective for bacteria that produce surface active compounds. The surface of leaves that are usually covered with wax would constitute such a habitat, although surfactant production in this habitat has seldom been investigated. In order to survive on leaf surfaces, epiphytes must be able to access limited and spatially heterogeneous nutrient supplies and endure daily fluctuations in moisture availability on a water-repellent surface. Epiphytic bacteria could potentially use biosurfactants to increase the wetability of the leaf, to enhance diffusion of nutrients across the waxy cuticle, and/or aid in motility to favorable growth sites. Despite the substantial potential role of biosurfactants on leaves, only a few studies have examined their production in the phyllosphere, all of which have focused on their possible ecological role in only specific strains and have not addressed the frequency of surfactant producers on leaf surfaces. A comprehensive examination of the phyllosphere inhabitants might reveal strains and biosurfactants not normally encountered in other habitats, and would address the hypothesis of surface enrichment of producing strains. In this study we compare the frequency of surfactant producers in the phyllosphere to those in soil and water environments. We compare the atomized oil assay with the drop collapse assay to characterize surfactants made by a collection of environmental strains, further demonstrating the usefulness of this assay in high-throughput screening and its much higher sensitivity for all types of biosurfactants encountered, many of which are hydrophobic and poorly detectable by the droplet collapse assay. We also investigate the influence of planktonic versus surface-associated culture conditions on the production of biosurfactants from our environmental isolates, and find evidence for frequent contact-dependent production of surface active compounds.