In the natural environment, most of the phages that target bacteria are thought to exist in biofilm ecosystems. The purpose of this study was to gain a clearer understanding of the reactivity of these viral particles when they come into contact with bacteria embedded in biofilms. Experimentally, we quantified lactococcal c2 phage diffusion and reaction through model biofilms using in situ fluorescence correlation spectroscopy with two-photon excitation. Correlation curves for fluorescently labeled c2 phage in nonreacting Stenotrophomonas maltophilia biofilms indicated that extracellular polymeric substances did not provide significant resistance to phage penetration and diffusion, even though penetration and diffusion were sometimes restricted because of the noncontractile tail of the viral particle. Fluctuations in the fluorescence intensity of the labeled phage were detected throughout the thickness of biofilms formed by c2-sensitive and c2-resistant strains of Lactococcus lactis but could never be correlated with time, revealing that the phage was immobile. This finding confirmed that recognition binding receptors for the viral particles were present on the resistant bacterial cell wall. Taken together, our results suggest that biofilms may act as "active" phage reservoirs that can entrap and amplify viral particles and protect them from harsh environments.

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Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers The non-domesticated Bacillus subtilis strain 3610 displays, over a wide range of humidity, hyperbranched, dendritic, swarming-like migration on a minimal agar medium. At high (70 %) humidity, the laboratory strain 168 sfp + (producing surfactin) behaves very similarly, although this strain carries a frameshift mutation in swrA, which another group has shown under their conditions (which include low humidity) is essential for swarming. We reconcile these different results by demonstrating that, while swrA is essential for dendritic migration at low humidity (30-40 %), it is dispensable at high humidity. Dendritic migration (flagella-and surfactin-dependent) of strains 168 sfp + swrA and 3610 involves elongation of dendrites for several hours as a monolayer of cells in a thin fluid film. This enabled us to determine in situ the spatiotemporal pattern of expression of some key players in migration as dendrites develop, using gfp transcriptional fusions for hag (encoding flagellin), comA (regulation of surfactin synthesis) as well as eps (exopolysaccharide synthesis). Quantitative (singlecell) analysis of hag expression in situ revealed three spatially separated subpopulations or cell types: (i) networks of chains arising early in the mother colony (MC), expressing eps but not hag; (ii) largely immobile cells in dendrite stems expressing intermediate levels of hag; and (iii) a subpopulation of cells with several distinctive features, including very low comA expression but hyper-expression of hag (and flagella). These specialized cells emerge from the MC to spearhead the terminal 1 mm of dendrite tips as swirling and streaming packs, a major characteristic of swarming migration. We discuss a model for this swarming process, emphasizing the importance of population density and of the complementary roles of packs of swarmers driving dendrite extension, while non-mobile cells in the stems extend dendrites by multiplication. INTRODUCTIONThe formation of bacterial communities, such as Bacillus subtilis colonies, was shown recently, while this work was in progress, to occur through a developmental-like program, associated with the presence of different cell types (see Vlamakis et al., 2008;Lemon et al., 2008;Ló pez & Kolter, 2010). Another form of community swarming migration over a surface, apparently occurring in thin fluid films, requires flagella, a surfactant and the production of specialized swarmer cells ( , 2008). Two types of swarming-like migration by B. subtilis have been described. On Luria broth (LB) agar at low humidity and 37 u C (primarily analysed with nondomesticated strains such as 3610), migration proceeds as an expanding confluent field, headed by small groups of bacteria, apparently with little change in individual size (Kearns & Losick, 2003;Julkowska et al., 2004). In contrast, we have described the...

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Purpose Brettanomyces bruxellensis is a serious source of concern for winemakers. The production of volatile phenols by the yeast species confers to wine unpleasant sensory characteristics which are unacceptable by the consumers and inevitably provoke economic loss for the wine industry. This ubiquitous yeast is able to adapt to all winemaking steps and to withstand various environmental conditions. Moreover, the ability of B. bruxellensis to adhere and colonize inert materials can be the cause of the yeast persistence in the cellars and thus recurrent wine spoilage. We therefore investigated the surface properties, biofilm formation capacity, and the factors which may affect the attachment of the yeast cells to surfaces with eight strains representative of the genetic diversity of the species. Methods The eight strains of B. bruxellensis were isolated from different geographical and industrial fermentation origins. The cells were grown in synthetic YPD medium containing 1% (w/v) yeast extract (Difco Laboratories, Detroit), 2% (w/v) bacto peptone (Difco), and 1% (w/v) glucose. Surface physicochemical properties as electrophoretic mobility and adhesion to hydrocarbon of the cells were studied. The ability of the strains to form biofilm was quantified using a colorimetric microtiter 96-well polystyrene plate. Biochemical characteristics were examined by colorimetric methods as well as by chemical analysis. Result Our results show that the biofilm formation ability is strain-dependent and suggest a possible link between the physicochemical properties of the studied strains and their corresponding genetic group. Conclusion The capacity to detect and identify the strains of the spoilage yeast based on their biofilm formation abilities may help to develop more efficient cleaning procedures and preventing methods.

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Brettanomyces bruxellensis is a serious source of concern for winemakers. The production of volatile phenols by the yeast species confers to wine unpleasant sensory characteristics which are unacceptable by the consumers and inevitably provoke economic loss for the wine industry. This ubiquitous yeast is able to adapt to all winemaking steps and to withstand various environmental conditions. Moreover, the ability of B.bruxellensis to adhere and colonize inert materials can be the cause of the yeast persistence in the cellars and thus recurrent wine spoilage. We therefore investigated the surface properties, biofilm formation capacity and the factors which may affect the attachment of the yeast cells to surfaces with eight strains representative of the genetic diversity of the species. Our results show that the biofilm formation ability is strain-dependent and suggest a possible link between the physicochemical properties of the studied strains and their corresponding genetic group.