The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 60 ؎ 1 kDa, whereas the native molecular mass was 71 ؎ 8 kDa according to size exclusion chromatography in the presence of the detergent octyl-␤-D-glucopyranoside. The monomeric enzyme contained (per mol of the 60-kDa subunit) 1.0 ؎ 0.1 mol of cobalamin, 0.6 ؎ 0.02 mol of cobalt, 7.1 ؎ 0.6 mol of iron, and 5.8 ؎ 0.5 mol of acid-labile sulfur. Purified PceA catalyzed the reductive dechlorination of tetrachloroethene and trichloroethene to cis-1,2-dichloroethene with a specific activity of 250 ؎ 12 nkat/mg of protein. In addition, several chloroethanes and tetrachloromethane caused methyl viologen oxidation in the presence of PceA. The K m values for tetrachloroethene, trichloroethene, and methyl viologen were 20.4 ؎ 3.2, 23.7 ؎ 5.2, and 47 ؎ 10 M, respectively. The PceA exhibited the highest activity at pH 8.1 and was oxygen sensitive, with a half-life of activity of 280 min upon exposure to air. Based on the almost identical N-terminal amino acid sequences of PceA of Dehalobacter restrictus, Desulfitobacterium hafniense strain TCE1 (formerly Desulfitobacterium frappieri strain TCE1), and Desulfitobacterium hafniense strain PCE-S (formerly Desulfitobacterium frappieri strain PCE-S), the pceA genes of the first two organisms were cloned and sequenced. Together with the pceA genes of Desulfitobacterium hafniense strains PCE-S and Y51, the pceA genes of Desulfitobacterium hafniense strain TCE1 and Dehalobacter restrictus form a coherent group of reductive dehalogenases with almost 100% sequence identity. Also, the pceB genes, which may code for a membrane anchor protein of PceA, and the intergenic regions of Dehalobacter restrictus and the three desulfitobacteria had identical sequences. Whereas the cprB (chlorophenol reductive dehalogenase) genes of chlorophenol-dehalorespiring bacteria are always located upstream of cprA, all pceB genes known so far are located downstream of pceA. The possible consequences of this feature for the annotation of putative reductive dehalogenase genes are discussed, as are the sequence around the iron-sulfur cluster binding motifs and the type of iron-sulfur clusters of the reductive dehalogenases of Dehalobacter restrictus and Desulfitobacterium dehalogenans identified by electron paramagnetic resonance spectroscopy.Tetrachloroethene (PCE), a frequently detected groundwater contaminant, is used by different bacteria as a terminal electron acceptor in a process called dehalorespiration (10,14,16,20,21,32,35,40,46). Six such isolates belong phylogenetically to the subphylum Firmicutes of the gram-positive bacteria, whereas other isolates are affiliated with phylogenetic groups such as the ␦ and ε subclasses of the Proteobacteria and the green no...

Wrinkled morphology is a distinctive phenotype observed in mature biofilms produced by a great number of bacteria. Here we study the formation of macroscopic structures (wrinkles and folds) observed during the maturation of Bacillus subtilis pellicles in relation to their mechanical response. We show how the mechanical buckling instability can explain their formation. By performing simple tests, we highlight the role of confining geometry and growth in determining the symmetry of wrinkles. We also experimentally demonstrate that the pellicles are soft elastic materials for small deformations induced by a tensile device. The wrinkled structures are then described by using the equations of elastic plates, which include the growth process as a simple parameter representing biomass production. This growth controls buckling instability, which triggers the formation of wrinkles. We also describe how the structure of ripples is modified when capillary effects are dominant. Finally, the experiments performed on a mutant strain indicate that the presence of an extracellular matrix is required to maintain a connective and elastic pellicle.biofilm elasticity | biofilm growth | wrinkles formation B acterial biofilms most often refer to communities that self-assemble into a cohesive extracellular matrix on solid surfaces or as pellicles floating on top of liquids. Bacteria self-organize in a collective behavior, giving a large-scale coherence to the system. Biofilms thus represent a protected life mode allowing bacteria to survive in hostile environments and from where they can disperse to colonize new niches (1, 2). A primary characteristic of biofilm formation is the production of exopolymeric substances by some cells. These substances mostly consists of exopolysaccharides (EPS), a few specific proteins, and nucleic acids (3-5), but their exact composition depends on the strain of the bacterium and the type of nutrients present in the culture medium (6, 7). When studied in a laboratory, wild-type strains of Bacillus subtilis are known to produce floating pellicles of rich and complex multiscale architectures (8, 9). The vertical structures range from the local 50-μm-scale "fruiting bodies" (10) to the extended macroscopic patterns illustrated in Fig. 1. As recently suggested in ref. 11, multiscale roughness could play a role in increasing the defense capability of B. subtilis against vapor and liquid antimicrobial agents.This study centers on the physical forces acting on biofilm and determining their morphologies at the macroscopic scale. To proceed, we restrict our experimental approach to the simplified case where macroscopic pellicles stand on rich static media. We primarily focus on the pellicles formed by the wild-type strain NCIB 3610, but present some features measured on another wildtype strain DV1 to obtain a more complete picture of existing morphologies. Of course the phenotypes are even more complex in nature given the possible multistrain and species coassembly and the diversity of settings and environments.T...

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