1% CAA was added to this media, along with NH4Cl, as nitrogen source. Spot inoculation of V. paradoxus EPS, P. aeruginosa PAO1, and Escherichia coli S17-1 on this swarming agar was performed (Fig 1). V. paradoxus EPS and P. aeruginosa PAO1 show strong swarming activity on this media, although the patterns are strikingly different. E. coli S17-1 shows no swarming, but robust growth, on this medium. Using gradient plates, we determined that glucose was not a suitable substrate

for swarming on FW based media using NH4Cl as nitrogen source (not shown). Figure 1 Variovorax paradoxus displays swarming motility. AZD5153 concentration Swarming plates with glucose and casamino acids inoculated with drops of P. aeruginosa PAO-1 (A), V. paradoxus EPS (B), or E. coli S17-1 (C). Inhibition of Swarming

with Congo Red Swarming requires the presence of flagellar activity, which is inhibited by Congo Red (CR) [40]. Supplementing plates with ≥ 50 μg/L CR had a strong inhibitory effect on the swarming phenotype (Fig 2). The colony did expand in diameter over a 48 h period under CR conditions, but at a much lower rate, consistent with simple growth based expansion. The microscopic analysis of the colony edges (Fig 3E–H) shows that the morphology of the edge differs markedly on plates containing CR. Robust growth of V. paradoxus EPS was observed under all CR Rabusertib treatment conditions (Fig 3A–D). Figure 2 Swarming of V. paradoxus EPS is inhibited in a dose dependent manner by the presence of Congo Red in the agar. Plates containing doses of Congo Red ranging from 1–1000 μg/L were incubated at 30°C either A) under ambient atmospheric humidity or B) in a humidified glass dish. Symbols in both panels:

No CR (black diamond), 1 μg/L CR (open square), 10 μg/L CR (filled triangle), 50 μg/L CR (×), 100 μg/L(*), 500 μg/L CR (open circle), 1000 μg/L (+). Swarm diameter measured in triplicate, reported as mean ± SEM. Figure 3 Humidity affects response to Congo Red swarming inhibition. A-D) gross morphology of V. paradoxus EPS on plates incubated at 30°C on media containing 0, 10,100, and 500 μg/L CR after 48 h. E-H) Edge images from the same culture conditions at 24 h. I-L) gross morphology of 48 h cultures on identical media incubated at 30°C in a humidified chamber. M-P) edge images from the humidified chamber incubated cultures at 24 h. Scale bar = 25 Orotidine 5′-phosphate decarboxylase microns. Role of a wetting agent in swarming Swarming is dependent on the presence of a wetting agent, which can be seen spreading on the plate (Fig 4A, B). Wetting agent is observed spreading well in advance of the colony on media containing inhibitory levels of CR (Fig 4B). The wetting agent is evident on plates without CR during the first 2d of growth (Fig 4A), and the wetting agent reduces the surface tension of the agar plate, as shown using a qualitative water drop collapse assay (Fig 4C). Figure 4 A wetting agent is present beyond the edge of the swarm.