Therefore, to evaluate changes in the surface and cell morphology we performed Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) analyses of stationary-phase bacteria. the absence of MapB did not lead to an extensive alteration in OMP abundance, but to a reduction in the relative amounts of a protein subset, including proteins from the Omp25/31 family. Electron microscopy revealed that ?cells exhibit multiple anomalies in cell morphology, indicating that the absence of the TamB homologue in severely affects cell division. Finally, ?cells were impaired in macrophage infection and showed an attenuated virulence phenotype in the mouse model. Collectively, our results indicate that GSK 1210151A (I-BET151) the role of TamB homologue is not restricted to participating in the translocation of autotransporters across the OM but that it is essential for OM stability and protein composition and that it is involved in cell envelope biogenesis, a process that is inherently coordinated with cell division. Introduction Bacteria of the genus are gram-negative bacteria, responsible for brucellosis, a disease characterized by chronic infections, abortions and infertility in animals, and chronic fatigue in humans1. invades and replicates in a variety of host cells such as macrophages, trophoblasts, dendritic and epithelial cells, within a characteristic compartment (brucellosome) derived from the endoplasmic reticulum2. The bacterial cell envelope is the major point of interaction between intracellular pathogens and the host and, therefore, the molecules that are part of or are built within it have fundamental roles in the success of infection. The cell envelope, and in particular the outer membrane (OM), exhibits unique characteristics that make these pathogens resistant to most of the host bactericidal agents. Additionally, several evidences indicate that the envelope promotes evasion from innate immunity and is crucial to avoid intracellular destruction3. The cell envelope has been GSK 1210151A (I-BET151) subject of numerous studies due to its central role in infection success. OM forms very stable bilayers4,5 as several outer membrane proteins Rabbit Polyclonal to p90 RSK (OMPs) maintain hydrophobic interactions with other OM components and/or contain hydrophilic domains that allow their binding to the peptidoglycan5,6. In fact, it was proposed that the interaction of the peptidoglycan with the OM results in a higher OM stiffness in than in other gram-negative bacteria4,7. contains an unconventional non-endotoxic lipopolysaccharide (LPS) that confers resistance to host antimicrobials5,8. Both the lipid A and the of and and (a gammaproteobacterium), (phylum) and (a spirochaete) may have a more general role in the OM assembly22C25. While performed an analysis to identify autotransporters the BR0049 gene from 1330 came out as a possible adhesin with a low similarity to autotransporters. Probably this was due to the abundance of -helix strands that are predicted along almost the entire protein and also to a -sheet structure found in the very C-terminus of BR0049 and its orthologues from GSK 1210151A (I-BET151) other studies and recent findings on the evolution of the novel TAM machinery21 indicated that BR0049 and its orthologues from other encode proteins that are phylogenetically related to members of the TamB family. Evidence presented in this work shows that BR0049 (a TamB homologue) plays roles that go beyond that of participating in autotransporter assembly. We propose that BR0049 is additionally involved in cell envelope biogenesis, in a process that is inherently coordinated with cellular division and that is crucial for cellular integrity. Results BR0049 is a distant homologue of TamB The gene annotated as BR0049 in the 1330 genome encodes a predicted protein of 1515 amino acids. However, a careful alignment analysis of its upstream DNA region and that of its orthologues in other species ( 99% aminoacid sequence identity). Outside GSK 1210151A (I-BET151) spp., its closest homologues with an 80C84% sequence identity are the orthologues of the genus (another member of the family). In other such as rhizobia, the homologues are similar in size and share 30C50% amino acid sequence identity. Database searches using the DELTA-BLAST program were able to detect phylogenetically related proteins in such as TamB from and would also be part of the TAM system21. Similar to TamB from TamB. An additional TamB protein feature was found in BR0049* as a -sheet secondary structure is predicted by the Jpred 4 protein secondary structure server in most of the protein except for an -helical small region of about 46 amino acids (1459C1505 amino acid region) (Fig.?1A), starting at the same relative position from the C-terminal end of TamB from TAM orthologues. (A) Scheme of the predicted BR0048 and MapB domain organization. (B) MapB-3xFLAG (MapB-3xF) expression along the growth curve. Samples were harvested at the indicated optical densities (at 600?nm) of the exponential (EP) or the stationary (SP) phases. Equivalent total proteins obtained from whole-cell lysates were analyzed by Western blot using a monoclonal.