After inoculating the bacteria by toothpick onto large filter paper strips on an agar plate, we could observe the distance the bacteria were able to travel by removing the paper at chosen times and allowing the growth of the bacteria that had penetrated through the paper. While a ∆syfA mutant strain progressed at a rate of 0.18 cm/hr and a fleQ– mutant moved at a rate of only 0.06 cm/hr, a ∆syfA/∆brfA strain moved at a rate of only 0.14 cm/hr. All of these surfactant mutants moved much slower than the WT strain , which suggests that both syringafactin and BRF contribute to the form of motility that enables movement through porous materials. Although the surfactants are not necessary for motility through porous paper, they strongly facilitate the process. This is quite distinct from swimming motility to which neither surfactant contributes to the process, and swarming motility where one or the other surfactant are essential. Because it appeared that BrfA and flagellin determinants were expressed in a similar fashion under several different growth conditions, we tested a variety of media conditions to determine whether this coordinated response was always linked. We thus examined the transcriptional response of both fliC and brfA in cells grown in nutrient broth alone , nutrient broth amended with a variety of agar concentrations , as well as paper discs on solid plates. For all of the solidified plates and the paper, the relative levels of expression of fliC and brfA were highly similar in cells grown on a given solid surface. However, brfA was induced, while fliC was down-regulated when cells were grown in a plate containing still liquid media with no added agar. Flagellin has been previously found to be synthesized at lower levels in liquid medium compared to a similar solidified medium. 

Since flagella apparently serve as surface sensors, it appears that flagellar surface sensing might also be contributing to the regulation of brfA. It is curious, however,best strawberry planter that in liquid medium BRF production is enhanced, leading us to hypothesize that the flagella might not be the sole signal inducing surfactant production in this condition. Because it appeared that expression of fliC-encoded flagellin and brfA was unlinked in cells grown in planktonic conditions, we further evaluated whether this was true under all conditions. In other bacteria it has been noted that flagellar surface-sensing on both hard agar and softer swarming plates can be mimicked by growth in a broth in which the viscosity is increased with the addition of polyvinylpyrrolidine 360. We therefore tested the expression of brfA and fliC in still and agitated broth cultures containing 10% PVP-360. Although PVP-360 induces flagella production in some other bacteria, we found equivalent levels of expression of fliC as in unamended broth culture and presume that this reflected a similar accumulation of flagellin in P. syringae. Additionally, the addition of PVP-360 did not appear to have a large effect on brfA transcription. In agreement with earlier results, broth culture conditions reduced the transcription of fliC while increasing the expression of brfA. Futhermore, it appeared that agitation of the broth cultures further increased expression of brfA while further decreasing expression of fliC. Although we did not observe an induction of transcription of fliC upon addition of PVP-360 as has been noted in other taxa, a dramatic effect of increasing broth medium viscosity on cell shape was apparent. Curiously, cells that were grown with agitation in media containing 10% PVP-360 exhibited a hyper-elongated state similar to that associated with the swarming phenotype in other bacteria. In this culture condition the cells grew up to 20 times the length of a normal cell and appeared multinucleate. Although in most bacterial taxa this phenotype is linked with swarming motility, we found no evidence that cells of P. syringae were elongated when cultured on low agar swarming plates.

Likewise, no elongated cells were seen in cultures in non-agitated KB broth amended with 10% PVP-360, nor any other culturing conditions tested. Given that culturing of P. syringae in broth medium induced expression of brfA and production of BRF we determined whether this condition also eliminates the influence of flagellar assembly on production of this surfactant. We measured the expression of brfA in ∆syfA/fleQ-, ∆syfA/fliC-, and ∆syfA mutant backgrounds when cells were grown in broth cultures. We observed a dramatic up-regulation of GFP fluorescence in all mutant strains harboring pPbrfAgfp in shaken broth media, and the expression of brfA appeared similar in ∆syfA/fliC– compared to ∆syfA. This suggests that flagellin does not play a role in sensing the liquid environment in broth media. In contrast, although brfA expression in a ∆syfA/fleQ– strain was higher in cells cultured in broth media compared to on agar plates, its level of expression was only about as high as that of a motile strain cultured on plates. Thus, although the inability to establish the flagellar base has some role in transcriptional repression of brfA expression in broth medium, it appears that other factors play a larger role in its regulation. It remains to be seen if this regulation is operative at the level of surfactant production, or whether it only affects transcription of brfA, since supernatants of broth cultures do not exhibit water drop collapse. Given that broth culture conditions greatly affected expression of brfA, we investigated what features of such a culture influenced this regulation.However, less than 2% as many cells were produced in still broth compared to shaken broth medium after a given time of incubation. This suggested that cell density might contribute to the high levels of brfA transcription in shaken broth cultures and that expression might increase concomitantly with cell density and thus time in cultures.

We therefore compared levels of GFP fluorescence in strains harboring pPbrfA-gfp after one and two days of growth in broth medium and on plates. While similar levels of GFP fluorescence were seen at all sampling times and growth conditions in a constitutively fluorescent strain harboring pP519n-gfp, the expression of brfA apparently increased in both broth and plate cultures over time. This induction with age of culture appeared to be much greater in broth compared to plate cultures,strawberry grow pot which might reflect the preferential accumulation of a signal in broth culture. Thus, although cell density might contribute to brfA expression independently from flagella surface sensing, it appears that this effect also is conditional on growth conditions and may involve another signal. While BRF apparently aids motility both on low-agar swarming plates and on hydrated porous papers these behaviors were always observed in a ∆syfA mutant incapable of producing syringafactin. We therefore wanted to ascertain whether there was a role for BRF production in a WT background. A ∆brfA strain did not differ from the WT strain in its speed of movement through porous paper. However, this strain did differ from the WT strain in the manner in which it moved on swarming plates. The ∆brfA strain produced tendrils of cells that moved away from the point of inoculation that were much broader than the WT strain. Such apparent movement was initially as fast as that of the WT strain, but unlike the WT strain, this mutant failed to fully explore the swarming plate; even after four days, a colony of ∆brfA had not covered the agar surface, whereas the WT had fully covered the swarming plate by day 2. As a further test of the role of BRF in movement of P. syringae, we over-expressed BrfA constitutively in the WT strain and observed its swarming motility. Contrary to the broad but short tendrils of cells produced by the ∆brfA mutant, over-expression of BrfA led to the formation of very long and narrow tendrils which moved and eventually covered the plate at the same speed as the WT strain. These observations are in agreement with observations in P. aeruginosa, where the branching and avoidance of other tendrils has been proposed to be due to the repellent effect of HAAs which serves to move the swarm front forwards. Although prior research has implicated biosurfactant production in flagellar motility, as far as we are aware, this is the first report to show this as a linear process, where the stages of flagellar assembly are required for proper regulation of surfactant production. We find genetic evidence that biosurfactants are tied to flagellar motility. However, this is not the first report of flagella controlling expression of non-flagellar genes. Salmonella enterica ties the expression of some virulence factors to mid-stage flagellar assembly. 

Additionally, Frye et al. identified a number of genes that are under the control of flagellar promoters but which have no apparent effect on flagella function in S. enterica. Furthermore, expression of virulence factors in Proteus mirabilis was found to be tightly co-regulated with FliC expression. Despite these reports of co-regulation, to our knowledge there has been no previous recognition that a flagellin knockout or impairment of function would serve to further up-regulate such non-flagellar genes. Why does P. syringae co-regulate expression of a biosurfactant with flagellar synthesis? Although it is tempting to speculate that BRF could function as a virulence factor, similar to the above-mentioned bacteria, preliminary evidence shows that a strain defective in production of BRF does not have reduced virulence in planta. A more likely possibility might be that BRF is used for flagellar lubrication. In this scenario, under conditions where there is increased flagella breakage, there will also be increased production of both flagellin and BRF. Production of BRF might help lubricate the sticky surface and/or flagella to minimize breakage. Microscopic and immuno-staining approaches might be utilized in future studies to determine if such a model holds for P. syringae. While BRF may be co-regulated with flagellin because it has protective effects for flagella, it might also be interpreted as a case of regulatory piggy-backing if flagellin synthesis itself is indicative of an external condition for which biosurfactant production is beneficial. One hypothesis that is gaining experimental support is that flagella can function as surface sensors, conveying to the bacterium positional information through the inhibition of flagellar rotation. It is proposed that V. parahaemolyticus and P. mirabilis can sense surfaces by monitoring flagellar torque, whereby growth on a surface impedes flagellar rotation, which signal upregulates flagellin production. However, in these models PVP addition serves to generate a similar viscous environment that impairs flagellar rotation, and similarly leads to up-regulation of swarming genes. Why do we instead see a decrease in flagellin synthesis with PVP addition in P. syringae? One clue might involve the distinctive requirements for the swarming phenotype in P. syringae; whereas cells of V. parahaemolyticus and P. mirabilis elongate and swarm on 1.5% hard agar, P. syringae requires a moister surface in order to swarm, and it does not display the elongated cell phenotype during movement. P. syringae thus may still use resistance of flagellar rotation to gather positional information, but it might use that information to make different decisions about when to swarm and produce biosurfactant. Another emerging hypothesis that has experimental support is that flagella act as wetness sensors in bacteria such as S. enterica. Bacteria export FlgM, the FliA antisigma factor, through flagella, and it is proposed that this secretion is only possible when the exterior conditions are sufficiently moist. Thus, under wetter conditions, both flagellin and the surfactant should be produced in greater quantities. This model would support our observation that a loss of flagellin does not upregulate surfactant production in broth culture; perhaps the fully hydrated conditions present in a broth culture allow maximal export of FlgM, regardless of flagellar length. However, if FlgM is optimally secreted, it remains unclear why we do not also see an up-regulation of flagellin in broth cultures compared to drier culture conditions such as growth on agar surfaces. While flagellar function itself might logically be linked to surfactant production, it remains unclear in what way flagellar glycosylation is linked to this process. For instance, why do the glycosylation mutants, especially a mutant blocked in fgt2, which has sufficient flagella function to enable unaltered swimming motility, have an equivalent effect on flagellin and surfactant synthesis as a disruption of flagellin production itself? One hypothesis could be that glycosylation blocks FlgM export through the flagella, and without adequate glycosylation, late stage flagellar genes remain activated.