Furthermore our studies have shown blockade of SST using antisense oligonucleotide leads to the loss of neurons

For most swarming bacteria, the spreading properties of this high-population liquid swarm layer are enhanced by production of a surfactant by the bacteria. Swarming can also occur without a surfactant; Salmonella enterica, for example, swarms with the aid of an osmotic agent that is not a surfactant. P. aeruginosa swarming is aided by its production of the glycolipid surfactant di-rhamnose-b-hydroxyalkanoyl-b-hydroxyalkanoate. Expression of the rhamnolipid biosynthetic operon is initiated in a population-dependent manner described as quorum sensing. P. aeruginosa utilizes two acylhomoserine signals to regulate different sub-sets of genes. These two AHLs have specific affinity to two LuxR-homolog transcriptional regulators, LasR and RhlR; this regulation occurs in series where a fully-induced Las-system initiates activation of the Rhl-system. Quorum sensing controls rhamnolipid production through the Rhl-system where the RhlR activates expression of the rhlA and rhlB genes only when sufficient butyryl homoserine lactone is present. RhlA converts b-hydroxydecanoyl-ACP to halo-alkanoic acid and RhlB is a rhamnosyltransferase that converts HAA to monorhamnolipid. These di-rhamnolipid precursors act similarly to di-rhamnolipid in aiding swarming; HAAs, mono-rhamnolipid, and di-rhamnolipid all act as surfactants to lower surface tension. Tremblay et al., however, have shown that these molecules have distinctive chemotactic and diffusive properties. Di-rhamnolipid acts as an attractant while the precursor HAAs can act as repellants to P. aeruginosa. There also appear to be growth conditions where rhamnolipid is not required, but still aids, P. aeruginosa swarming. Therefore, while rhamnolipid synthesis is well-described and the ability to improve surface spreading of P. aeruginosa is known, the spatial and temporal actions of rhamnolipid and its precursors on surfaces are less defined. Most images of P. aeruginosa swarms show cell spreading in fractal or tendril patterns; development of these tendrils requires rhamnolipid. However, others have shown that P. aeruginosa also swarms as an expanding circle. Differences in these varied reports for P. aeruginosa swarming seem to be explained by a variety of factors including strain effects, media composition, and surface hardness. We became interested in the influence of surface hardness upon swarming and tendril formation. Higher agar or ‘‘hard’’ surfaces are known to limit swarming as do overdried plates. We found that varying the concentration of the agar within a small range greatly altered swarming patterns and overall spreading. We hypothesized that surface properties affect rhamnolipid production, which subsequently affects swarming. We provide evidence that hard agar surfaces limit the initiation of quorum sensing and rhamnolipid production in very close proximity to the advancing edge of swarming cells, which is sufficient to dominate the resultant swarm phenotype. Adding exogenous AHL signals or increasing substrate carbon did not alter wild-type swarming. Because we were unable to artificially stimulate tendril formation on hard agar, this suggests that quorum sensing on surfaces is not solely population dependent. Since swarm tendrils are due to rhamnolipid, we questioned if a lack of tendrils was due to insufficient rhamnolipid production. Adapting the methylene blue rhamnolipid plate assay utilized in several studies, we observed that wild-type qualitatively produces similar rhamnolipid amounts on both soft and hard agar. The zones of clearing that indicated production of surfactant were not differentiable between agar types. Hard agar did not prevent rhamnolipid production. These methylene blue indicator plates did not, however, provide insight into actual rhamnolipid production levels during swarming; the CTAB component of these plate assays is toxic to bacteria and swarming of P. aeruginosa was impaired on these plates. While the potential to produce rhamnolipid may be equal on these two agar surfaces, this particular assay provides little information of how P. aeruginosa behaves under more optimal conditions. We then examined expression of a rhamnolipid gene fluorescent reporter to gauge potential differences in rhamnolipid synthesis in situ during swarming. The reporter construct utilizes the promoter region of rhlA fused to green fluorescent protein. We observed that P. aeruginosa induction of PrhlA::gfp is greater at the advancing edge of soft agar swarms compared to those grown on hard agar. After 40 hours, the formation of swarm tendrils corresponded to fluorescence of PrhlA::gfp in very close proximity to the advancing edge of swarming bacteria on soft agar. On hard agar, however, when no tendrils formed, the fluorescence detected near the swarm edge was barely above background and 376less than the expression observed near the swarm edge on soft agar. These rhlA expression differences were observed only at the swarm edge; fluorescence toward the center of swarms was bright and indistinguishable on all agar types. The overall bacterial population showed no evidence of limited rhamnolipid production-most of the hard agar-grown colony area is bright with PrhlA::gfp and produced rhamnolipid on methylene blue indicator plates.