The powder wash was chosen for use in all the experiments requiring BRF extract.Intriguingly, while comparing different methods for bacterial inoculation on agar plates, it was observed that production of this surfactant increased dramatically when the strain was grown on the porous surface of hydrated filter paper discs placed on agar plates. This was true both when the bacteria were directly applied with a toothpick as a single spot on the paper surface, and somewhat less so when inoculated as a larger patch from an aqueous cell suspension. A variety of additional materials other than cellulose such as cotton and polyester fabrics were tested for their stimulation of apparent surfactant production, and all induced production as long as the material was wettable. The rough surface induction of surfactant production led us to the hypothesis that the surfactant might contribute to the colonization of natural surfaces and thus prompted further investigation. Of the six mutants identified as being completely blocked in biosurfactant production three of the insertions were into the global regulatory genes gacS, ompR, and fleQ, and thus were deemed to be not specifically responsible for surfactant biosynthesis. GacS is a global regulator of secondary metabolites and extracelullar enzymes , while an OmpR homolog has recently been hypothesized to be a membrane stress sensor in P. aeruginosa , and FleQ is the initial regulatory element of flagellar biosynthesis. Of the remaining genes influencing biosurfactant production, neither Psyr_0215 which is predicted to have general base excision repair activity,arandanos planta nor Psyr_4446 which is an osmotically induced outer membrane lipoprotein, are likely candidates for contributing to surfactant synthesis.

On the other hand, a predicted acyltransferase, Psyr_3129, having 48.5% identity to rhlA and 49% identity to phaG in P. aeruginosa PAO1, seemed likely to be involved directly in surfactant biosynthesis. RhlA is responsible for production of 3-alkanoic acids , the precursor to rhamnolipids in P. aeruginosa, and is independently recognized as a biosurfactant that promotes swarming motility. PhaG is involved in polyhydroxyalkanoic acid synthesis, which is a carbon and energy storage molecule. Both enzymes divert hydroxydecanoic acids from fatty acid de novo synthesis, and exhibit similar and sometimes overlapping polymerization functions. Because the transposon insertion was in the promoter region immediately upstream of Psyr_3129, we confirmed that a knockout of this gene also blocked surfactant production by constructing a chromosomal deletion of Psyr_3129 in the ∆syfA background of P. syringae. This double mutant was also incapable of swarming ability. To ensure that disruption of brfA and not genomic changes elsewhere was responsible for abrogating biosurfactant production, we complemented this gene in trans. Expression of brfA under the control of the constitutive npt2 promoter in plasmid p519n-gfp, where gfp was replaced with brfA, proved to be lethal to P. syringae. However, when brfA was inserted into pMF54 , to form plasmid pBRF2 where brfA is driven by an IPTG-inducible trc promoter, this plasmid produced viable transformants. Curiously, when this plasmid is introduced into a ∆syfA/∆brfA double mutant, biosurfactant was produced abundantly without IPTG addition , emphasizing the leaky nature of this plasmid. Addition of IPTG did not result in surfactant production beyond that observed in uninduced cells. Thus, either BrfA synthesizes the surfactant or is essential for its expression. Significantly, rhlA from P. aeruginosa has been shown to be sufficient for HAA production in E. coli , as well as an rhlA homolog in Serratia sp. ATCC 39006 which produces an unidentified biosurfactant. 

We thus tested if our potential rhlA homolog was sufficient to confer biosurfactant production in E. coli. E. coli DH5α harboring plasmid pBRF2 produced a large amount of surfactant. It is important to note that although production of this surfactant in a ∆syfA strain of P. syringae is readily detected with the atomized oil assay, it was not detectable with other assays such as the drop collapse assay or by direct chemical detection. This suggested that either the molecule had properties such as low water solubility that prevented its detection with assays such as water drop collapse, or that it was made in relatively low amounts that are not easily detected by assays with lower sensitivity. However, a ∆syfA strain carrying pBRF2 for constitutive BrfA expression was observed to cause a drop collapse , thus we presume that low rates of production in native strains explain its lack of detection in a ∆syfA strain with a drop collapse assay. Using a modified protocol for HAA extraction , we extracted BRF from plate-grown cultures of ∆syfA. The resulting powder yielded an opaque solution in water, indicative of a surfactant with low water solubility exhibiting aggregate formation. This concentrated surfactant lowered the surface tension of water to 29 dyn/cm when measured in a pendant drop assay, confirming its potent surfactant activity. It remains to be determined what the chemical structure of BRF is, and if it is HAA. An insertion into the transcription factor fleQ, which is involved in the initiation of flagellar assembly results in a total loss of surfactant production. Disruption of flgC, a Class III flagellar assembly gene that is involved in formation of the basal body rod in P. aeruginosa , also resulted in a large reduction in the surfactant halo. The identification of these two mutants led us to hypothesize that assembly of the flagellar base structure is important for production of BRF. Surprisingly, an insertion in fliC, a Class IV structural gene encoding the actual flagellin protein, resulted in enhanced surfactant production.

Furthermore, insertions in fgt1 and fgt2, two genes involved in flagellar glycosylation that have been shown in P. syringae pv. tabaci 6605 to be important for flagellar function , both also result in up-regulation of surfactant production. This suggested that once the flagellar base is assembled and flagellin synthesis is initiated, mutations which hinder flagellar assembly or functionality serve to up-regulate the production of BRF. Curiously, even though an insertion in fgt1 only impaired flagellar swimming motility while an insertion in fgt2 did not appear to confer any flagellar impairment , these mutations both stimulated surfactant production to a similar extent as a loss of flagellin itself. We remain uncertain how these mutations lead to up regulating surfactant production. To further support our hypothesis that expression of BRF is dependent on flagellar assembly itself and not merely coincidentally with expression of certain flagellar genes,square nursery pots we constructed targeted knockouts in additional flagellar genes involved at different stages of flagellar assembly. A directed knockout mutant of fleQ was deficient in surfactant production, confirming our earlier observations of an insertional mutant of this gene. Although the initial screen did not identify any insertions in Class II genes that are important for the initial establishment of the flagellar apparatus, a directed knockout of fliF exhibited a dramatic loss of surfactant production. Furthermore, a knockout of flgD, a Class III flagellar gene in an operon downstream of flgC, resulted in a similar 3-fold reduction in the size of the BRF halo. Disruption of fliA, encoding the sigma factor responsible for initiating transcription of Class IV genes, also conferred a 3-fold reduction in surfactant production. Thus, although FliA is necessary for expression of late stage flagellar genes, it does not appear necessary for production of BRF. Because the establishment of the flagellar base appears important for production of this surfactant, we postulated that perhaps the flagellum is in some way necessary for the export of BRF. In order to test this model, we introduced plasmid pBRF2 conferring constitutive BrfA expression into a ∆syfA/fleQ– double mutant strain of P. syringae. This strain, despite lacking flagella, exhibited unaltered surfactant production , indicating that flagella are not necessary for surfactant export. Thus it appears that the flagellar assembly process most likely influences brfA at the transcriptional level.

In order to investigate the contribution of flagellar assembly to transcriptional regulation of brfA we linked a gfp reporter gene to the promoter containing region 5’ to brfA in the stable plasmid vector pPROBE-GT to produce reporter plasmid pPbrfA-gfp. We introduced pPbrfA-gfp into the different insertional mutants blocked at different stages of flagellar assembly and observed that, as was indicated by the atomized oil assay, the expression of brfA was higher in a ∆syfA/fliC– mutant compared to that in either a ∆syfA/fleQ– or ∆syfA/flgC– mutant. We also constructed reporter plasmid pPfliC-gfp in which a gfp reporter gene was fused to the promoter-containing region of fliC to provide estimates of the expression of the gene encoding flagellin, a late stage flagellar gene. Similar to what was observed for expression of brfA, the expression of fliC was greatly reduced in both a ∆syfA/fleQ– and ∆syfA/flgC background but was over-expressed relative to that in a ∆syfA background alone in a ∆syfA/fliC mutant. As far as we are aware, flagellar glycosylation has not been documented to have a feedback role in flagellin biosynthesis. Although it is intuitive that a loss of flagellin production might result in constitutive activation of the late-stage flagellar genes through FliA, it is less obvious how flagellar glycosylation mutations might be feeding back to up-regulate flagella production, especially in the case of fgt2 which does not exhibit any impairment of flagellar function. In order to investigate the feedback process, we constructed transcriptional reporters of both flgB, a class II flagellar gene, and fliE, a class III flagellar gene, in addition to the fliC reporter. Reporter plasmids pPflgB-gfp and pPfliE-gfp, respectively, were separately introduced into the original ∆syfA strain as well as a ∆syfA/fgt2– strain, so that the effect of flagellar glycosylation on the expression of the three classes of flagella genes could be observed. We clearly observed that a loss of flagellar glycosylation results in up-regulation only of the late stage flagellin gene fliC and not of fliE or flgB. Loss of glycosylation most likely affects the flagella in such a way as to encourage the export of the anti-sigma factor FlgM, either through increased flagellar breakage or increased export within the flagella, thus releasing FliA from FlgM control. To address the process by which paper surfaces up-regulate production of BRF we addressed the expression of brfA under various growth conditions. The GFP fluorescence of a WT strain carrying pPbrfA-gfp was compared between when grown on filter paper discs on agar plates and when grown directly on agar plates. While GFP fluorescence exhibited by P. syringae harboring plasmid p519n-gfp conferring constitutive GFP expression was similar in these two growth conditions, much higher GFP fluorescence was observed after growth on the porous paper in the strain carrying pPbrfA-gfp. Such apparent paper surface-induced upregulation of brfA was observed in both the WT strain as well as a ∆syfA strain. No such induction of syfA was observed when strains harboring pPsyfA-gfp were grown on paper discs , indicative that syringafactin is not similarly regulated. Because we observed both enhanced production of BRF and elevated expression of brfA in cells grown on hydrated paper discs, as well as a dependence of BRF production on flagella assembly, we hypothesized that genes for flagella for motility would be up-regulated on the paper discs coincidently with those for BRF production. To test this, we compared the GFP fluorescence of cells harboring the fliC reporter plasmid pPfliC-gfp when grown on agar plates and paper discs. As hypothesized, we observed an up-regulation of genes encoding flagellin when the strain is exploring the porous paper surface. In order to examine the necessity of flagella for movement through hydrated paper, we compared the lateral spread of a WT strain and a fleQ– mutant on paper discs. While flagellated strains quickly moved both into and along the length of the paper discs, the non-flagellated strains remained at the site of inoculation and formed colonies only on top of the paper. This requirement of motility for colonization of paper disks appears very similar to that observed for exploration of a porous ceramic surface. To better determine the relative rate of movement of different strains along paper, we increased the distance over which the bacteria were allowed to move.