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Mechanosensitive channels must make a large conformational change during the transition from the closed to the open state. The crystal structure of the open form of the Escherichia coli MscS channel was recently solved and depicts a homoheptamer (1). In this study, crosslinking of site-specific cysteine substitutions demonstrates that residues up to 10 -33 Å apart in the crystal structure readily form disulfide bridges in the closed form and can also be cross-linked by a 10-Å linker. Crosslinking between adjacent subunits stabilizes the heptameric form of the channel providing biochemical evidence to support the crystal structure. The data are consistent with the published model (1) in that the membrane domain is highly flexible and that the closed to open transition may involve a significant displacement of transmembrane helices 1 and 2, possibly by as much as 30 Å. The data are also consistent with significant flexibility of the cytoplasmic domain. Mechanosensitive (MS)1 channels underpin vital sensory processes in higher organisms and are responsible for structural integrity of bacterial cells during the transition from high to low osmolarity (downshock) (2-4). In the bacterial membrane closed MS channels must maintain the cation impermeability of the membrane, which is an essential requirement for energy transduction. Open MS channels in Escherichia coli exhibit conductances of 0.3-3 nanosiemens (5), consistent with the formation of large pores in the membrane of at least an 11-Å diameter (1, 6, 7). Clearly MS channels must undergo large conformational changes in the protein structure during the transition from the closed to the open state. The recent crystal structures of the bacterial MS channels, MscL and MscS, have been achieved for the closed and open states, respectively (1, 8). Model building, coupled with biochemical analysis of single cysteine mutants, has supported molecular dynamics-based analyses of the structural transitions that may open MscL (6, 7, 9 -11). However, the two proteins are so individual that there is little from the one structure that informs the possible mechanism of the other.The MscS protein has been much less intensively studied as a result of the relatively recent discovery of the structural gene (12). Genetic and biochemical analyses established that the protein has three transmembrane (TM) helices and a large carboxyl-terminal cytoplasmic domain (13). This was confirmed by resolution to 3.8 Å of a crystal structure of the MscS channel that also showed the protein to be a homoheptamer (1). The large cytoplasmic domain was demonstrated to form a 40-Å diameter chamber perforated by eight holes, seven of which arise at the boundary between pairs of monomers and the eighth from the formation of a ␤ barrel by the carboxyl-terminal 15 residues. In the crystal structure the TM3 helix bends at Gly 113 , such that the carboxyl-terminal part of this helix lies parallel to the membrane surface. The loop between TM2 and TM3 (residues 91-95) forms an extended chain that is part of the wa...

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The effects of the bcsE gene and BcsE protein on bacterial physiology and pathogenicity in Salmonella Typhimurium and Salmonella Group C1 were investigated. It was observed that biofilm and pellicle formation did not occur in the bcsE gene mutants of wild-type strains. Besides, the ꞌrdarꞌ (red, dry, rough) biofilm morphotype in wild-type strains changed significantly in the mutants. In terms of the bcsE gene, the swimming and swarming motility in mutant strains showed a dramatic increase compared to the wild-type strains. The Salmonella bcsE gene was cloned into Escherichia coli BL21, and the his-tagged protein produced in this strain was purified to obtain polyclonal antibodies in BALB/c mice. The antibodies were showed labeled antigen specificity to the BscE protein. As a result of immunization and systemic persistence tests carried out with BALB/c mice, BscE protein was determined to trigger high levels of humoral and cellular responses (Th1 cytokine production, IgG2a/IgG1 > 1). Systemic persistence in the liver and spleen samples decreased by 99.99% and 100% in the bcsE mutant strains. Finally, invasion abilities on HT-29 epithelial cells of wild-type strains were utterly disappeared in their bcsE gene mutant strains.

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