Microbiology (Reading) 151:3469C3482
Microbiology (Reading) 151:3469C3482. and FlgG through its C-terminal area. A deletion analysis that divides the C terminus in two halves revealed that this interacting regions for most of the rod proteins are not redundant. Our results also show that the presence of the rod proteins FliE, FlgB, FlgC, and FlgF displace the previously reported SltF-FlgJ conversation. In addition, we observed modulation of the transglycosylase activity of SltF mediated by FlgB and FlgJ that could be relevant to coordinate rod assembly with cell wall remodeling. In summary, different mechanisms regulate the flagellar lytic transglycosylase, SltF, ensuring a timely transcription, a proper localization and a controlled enzymatic activity. IMPORTANCE Several mechanisms participate in the assembly of cell-envelope-spanning macromolecular structures. The sequential expression of substrates to be exported, selective export, and a specific order of incorporation are some of the mechanisms that stand out to drive an efficient assembly process. Here, we analyze how the structural rod proteins, the scaffold protein FlgJ and the flagellar lytic enzyme SltF, interact in an orderly fashion to assemble the flagellar rod into the periplasmic space. A complex arrangement of transient interactions directs a dedicated flagellar muramidase toward the flagellar rod. All of these interactions bring this protein to the proximity of the peptidoglycan wall while also modulating its enzymatic activity. This study suggests how a dynamic network of interactions participates in controlling SltF, a prominent component for flagellar formation. and and and instead of finding this bidomain protein a short Tianeptine version of FlgJ has been identified. This short FlgJ is usually characterized by having only the scaffolding domain name, and the cell wall hydrolase Tianeptine is usually a separate polypeptide (40); in these bacteria, the cell wall hydrolase proteins are encoded by genes located in a flagellar context (39, 41). is an alphaproteobacterium that under the standard growth conditions used in the laboratory assembles a single subpolar flagellum. In this microorganism, many of the genes encoding for the components of the flagellum have been identified, and it has been demonstrated that this expression of these genes follows a hierarchical pattern in which the early flagellar components are transcribed by the RNA polymerase associated with a sigma Rabbit polyclonal to IL1R2 54 factor (E54) and the late components are dependent on E28 (42). 54, also known as RpoN, recognizes promoters with the conserved positions GG and GC that are located C24 and C12 nucleotides upstream the transcription initiation site (43), and open complex formation requires the presence of an activator protein that binds Tianeptine approximately 150?bp upstream of the promoter sequence; this type of protein is known as an enhancer-binding protein (EBP) (44). The genome encodes four genes (to transcription dependent on E28 proceeds (42, 47). In this bacterium, the flagellum-specific soluble lytic transglycosylase (SltF) is usually encoded within the operon, and the protein is usually exported to the periplasm via the general secretion (Sec) pathway, where it interacts with FlgJ and is directed to open a gap in the PG layer (40, 48, 49). SltF has a conserved enzymatic domain name Tianeptine and a C-terminal region that is relevant for flagellar formation but not for activity (41, 49). Thus far, it has been determined that this conversation between SltF and FlgJ depends on the integrity of the C-terminal domain name of SltF (41, 49). In addition, it has been shown that SltF can interact with the rod proteins FlgB and FlgF, affecting its hydrolytic activity in opposite ways, i.e., FlgB activates, whereas FlgF inhibits, enzymatic activity (50). To further understand the role of these proteins during rod biogenesis, we performed an analysis of the interactions of wild type and three different C-terminal mutant versions of SltF with the rod proteins and examined whether these interactions were modified by the inclusion of FlgJ. A model is usually proposed in which SltF is Tianeptine usually preferentially recruited to the forming rod by FliE, FlgB, FlgC, and FlgF over FlgJ, and we postulate how the observed interactions modulate SltF activity to facilitate rod assembly. RESULTS Expression of occurs in early stages of flagellar formation. The gene that codes for SltF.