Following the analysis of the biochemical and functional properties of the P. mirabilis extra cellular matrix performed in the first part of this study, the viscoelasticity of an actively growing colony was investigated in relation to water activity. The results demonstrate that the P. mirabilis colony exhibits a marked viscoelastic character likely due to both cell rafts and exoproduct H-bond networks. Besides, the water loss by evaporation during migration has been measured, whereas the experimental determination of the water diffusion coefficient in agar has allowed us to estimate the net water influx at the agar/colony interface. These data drive us to propose that a periodic increase of the water activity at the colony's periphery, mainly due to the drastic surface to volume ratio increase associated with swarming, causes the periodic and synchronous cessation of migration through the dissociation of exoproduct networks, which in turn strongly alters the matrix viscoelasticity.

We mapped the space-time distribution of stationary and swarmer cells within a growing Proteus mirabilis colony by infrared (IR) microspectroscopy. Colony mapping was performed at different positions between the inoculum and the periphery with a discrete microscope-mounted IR sensor, while continuous monitoring at a fixed location over time used an optical fiber based IR-attenuated total reflection (ATR) sensor, or "optrode." Phenotypes within a single P. mirabilis population relied on identification of functional determinants (producing unique spectral signals) that reflect differences in macromolecular composition associated with cell differentiation. Inner swarm colony domains are spectrally homogeneous, having patterns similar to those produced by the inoculum. Outer domains composed of active swarmer cells exhibit spectra distinguishable at multiple wavelengths dominated by polysaccharides. Our real-time observations agree with and extend earlier reports indicating that motile swarmer cells are restricted to a narrow (approximately 3 mm) annulus at the colony edge. This study thus validates the use of an IR optrode for real-time and noninvasive monitoring of biofilms and other bacterial surface populations.