A multi-scale approach of the mechanisms underlying exopolysaccharide auto-organization in the Proteus mirabilis extracellular matrix.

For decades, the origin of the concentric ring pattern of bacterial swarming colonies has puzzled microbiologists. It was hypothesized that a periodic water activity variation originates a phase transition within the extracellular matrix water H bond network, which switches on and off the exopolysaccharide auto-organization. Both rheological and infrared spectroscopy measurements respectively performed at a molecular scale and on a currently migrating colony, have given a physical insight into the mechanisms which underlie the switch between swarming and consolidation phases. Thanks to in situ and real time infrared microspectroscopy, and thanks to the brilliance of the infrared beam at SOLEIL synchrotron, here we demonstrate that Proteus mirabilis swarming is triggered by a periodic variation of water activity at the colony edge. A dynamic behavior emerges from the global properties of the multicellular entity which relies on the ability of the bacterial cells to tune exoproduct synthesis in order to undergo sharp transitions at a given water activity threshold.

Introducing the scanning air puff tonometer for biological studies.

It is getting increasingly evident that physical properties such as elastoviscoplastic properties of living materials are quite important for the process of tissue development, including regulation of genetic pathways. Measuring such properties in vivo is a complicated and challenging task. In this paper, we present an instrument, a scanning air puff tonometer, which is able to map point by point the viscoelastic properties of flat or gently curved soft materials. This instrument is an improved version of the air puff tonometer used by optometrists, with important modifications. The instrument allows one to obtain a direct insight into gradients of material properties in vivo. The instrument capabilities are demonstrated on substances with known elastoviscoplastic properties and several biological objects. On the basis of the results obtained, the role of the gradients of elastoviscoplastic properties is outlined for the process of angiogenesis, limb development, bacterial colonies expansion, etc. which is important for bridging the gaps in the theory of the tissue development and highlighting new possibilities for tissue engineering, based on a clarification of the role of physical features in developing biological material.

Isolation of a sulphated polysaccharide from a recently discovered sponge species (Celtodoryx girardae) and determination of its anti-herpetic activity.

Exopolysaccharides (EPS) were extracted from a sponge, Celtodoryx girardae recently discovered in the Golfe du Morbihan in 2000. Sponge samples were collected monthly from November 2007 to May 2008. SEC analysis of EPS samples showed that they exhibit a unique molecular weight of approximately 800 kDa. However, infrared analysis revealed that structural seasonal variations occur. EPS fractions also exhibit significant sulphate contents and were screened in vitro for a potential antiviral activity against Herpes simplex virus type 1 (HSV-1). The best result was obtained with a sample collected in January which exhibits an EC(50) of 5.9 microg/mL without cytotoxicity on the Vero cell line. Experiments carried out to elucidate the mechanism of the EPS showed that the sulphated groups of EPS interact with the glycoproteins on the surface of the virus' membrane.

Does water activity rule P. mirabilis periodic swarming? I. Biochemical and functional properties of the extracellular matrix.

The dynamics of bacterial colonies is complex in nature because it correlates the behavior of numerous individual cells in space and time and is characterized by emergent properties such as virulence or antibiotics resistance. Because there is no clear-cut evidence that periodic swarming of P. mirabilis colonies is ruled by chemical triggers responsible for cell-to-cell signaling in most of the biofilms, we propose that the observed periodicity relies on the colony's global properties. Hence, the biochemical and functional properties of the extracellular matrix (ECM) of P. mirabilis colonies were investigated. A binary exopolysaccharide mixture (1 and 300 kDa), glycinebetaine, and a phenoglycolipid were identified. Rheology, calorimetry, and water sorption experiments performed on purified EPS bring evidence that these exoproducts exhibit marked viscoelasticity, which likely relies on large scale H bond networks. Such behavior is discussed in terms of water activity because the mechanical ECM properties were found to depend on hydration.

Does water activity rule P. mirabilis periodic swarming? II. Viscoelasticity and water balance during swarming.

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.

Mapping bacterial surface population physiology in real-time: infrared spectroscopy of Proteus mirabilis swarm colonies.

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.