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We previously reported that the ClpXP ATP-dependent protease specifically recognizes and degrades the flagellar master transcriptional activator complex, FlhD4C2, to negatively control flagellar biogenesis. The flagellum-related protein, FliT, is also a negative regulator of flagellar regulon by inhibiting the binding of FlhD4C2 to the promoter DNA. We have found a novel pathway of FliT inhibition of FlhD4C2 activity connected to ClpXP proteolysis. An in vitro degradation assay using purified proteins shows that FliT selectively increases ClpXP proteolysis of the FlhC subunit in the FlhD4C2 complex. FliT behaves specifically to ClpXP-dependent proteolysis of FlhC. An in vitro interaction assay detects the ternary complex of FliT-FlhD4C2-ClpX. FliT promotes the affinity of ClpX against FlhD4C2 complex, whereas FliT does not directly interact with ClpX. Thus, FliT interacts with the FlhC in FlhD4C2 complex and increases the presentation of the FlhC recognition region to ClpX. The DNA-bound form of FlhD4C2 complex is resistant to ClpXP proteolysis. We suggest that the role of FliT in negatively controlling the flagellar gene expression involves increasing free molecules of FlhD4C2 sensitive to ClpXP proteolysis by inhibiting the binding to the promoter DNA as well as enhancing the selective proteolysis of FlhC subunit by ClpXP.

The RcsC/YojN/RcsB phosphorelay system controls gene expression in response to a variety of signals, including changes in temperature, osmolarity, and overproduction of membrane proteins. Transcription of certain RcsB-activated genes, such as the capsule synthesis cps operon, requires the co-activator protein RcsA, whereas expression of other RcsB-activated genes is RcsA-independent. We have established previously that a tolB mutation induces transcription of the Salmonella UDP-glucose dehydrogenase ugd gene in an RcsA- and RcsB-dependent manner. This induction is independent of the two-component systems PhoP/PhoQ and PmrA/PmrB, which are required for ugd expression in response to low Mg2+. We now report that the RcsC/YojN/RcsB system is activated in a pmrA mutant experiencing Fe3+ and low Mg2+, resulting in expression of both cps and ugd genes. However, whereas cps transcription remained RcsA-dependent, ugd transcription became RcsA-independent but dependent on the PhoP protein. S1 mapping experiments demonstrated that RcsA-dependent and -independent transcription of the ugd gene use the same promoter. DNase footprinting analysis identified a PhoP-binding site in the ugd promoter. Yet, PhoP-mediated ugd transcription required either the RcsC/YojN/RcsB or the PmrA/PmrB systems.

At the mammalian skeletal neuromuscular junction, cycling of nicotinic ACh receptors (nAChRs) is critical for the maintenance of a high postsynaptic receptor density. However, the mechanisms that regulate nAChRs recycling in living animals remain unknown. Usingin vivotime-lapse imaging, fluorescence recovery after photobleaching, and biochemical pull down assays, we demonstrated that recycling of internalized nAChRs into fully functional and denervated synapses was promoted by both direct muscle stimulation and pharmacologically induced intracellular calcium elevations. Most of internalized nAChRs are recycled directly into synaptic sites. Chelating of intracellular calcium below resting level drastically decreased cycling of nAChRs. Furthermore we found that calcium-dependent AChR recycling is mediated by Ca2+/calmodulin-dependent kinase II (CaMKII). Inhibition of CaMKII selectively blocked recycling and caused intracellular accumulation of internalized nAChRs, whereas internalization of surface receptors remained unaffected. Electroporation of CaMKII-GFP isoforms into the sternomastoid muscle showed that muscle-specific CaMKIIβm isoform is highly expressed at the neuromuscular junction (NMJ) and precisely colocalized with nAChRs at crests of synaptic folds while the CaMKIIγ and δ isoforms are poorly expressed in synaptic sites. These results indicate that Ca2+along with CaMKII activity are critical for receptor recycling and may provide a mechanism by which the postsynaptic AChR density is maintained at the NMJin vivo.

The lipopolysaccharide (LPS) is the outermost component of the cell envelope in Gram-negative bacteria. It consists of the hydrophobic lipid A, a short non-repeating core oligosaccharide and a distal polysaccharide termed O-antigen. We report here that the PmrA/PmrB and RcsC/YojN/RcsB two-component systems of Salmonella enterica serovar Typhimurium independently promote transcription of the wzzst gene, which encodes a protein that determines the chain length of the O-antigen. We show that the regulatory proteins PmrA and RcsB footprint partially overlapping regions of the wzz st promoter stimulating transcription from the same start site. Induction of the PmrA/PmrB or RcsC/YojN/RcsB systems increased the fraction of LPS molecules containing 16-35 O-antigen subunits, leading to heightened resistance to serum. The LPS of a rcsB null mutant exhibited an altered mobility in the O-antigen subunits attached to the lipid A-core region when separated on a SDS/PAGE gel, suggesting that RcsB may regulate additional LPS genes. Inactivation of the wzzst gene eliminated the enhanced swarming behaviour exhibited by the rcsB mutant. That multiple regulatory systems control wzzst expression suggests that the Wzzst protein is required under different environmental conditions. Fil: Delgado, Monica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Mouslim, Chakib. University of Washington; Estados Unidos Fil: Groisman, Eduardo A.. University of Washington; Estados Unidos

Histidine-constitutive (His c ) strains of Salmonella typhimurium undergo cell division inhibition in the presence of high concentrations of a metabolizable carbon source. Filaments formed by His c strains show constrictions and contain evenly spaced nucleoids, suggesting a defect in septum formation. Inhibitors of penicillin-binding protein 3 (PBP3) induce a filamentation pattern identical to that of His c strains. However, the His c septation defect is caused neither by reduced PBP3 synthesis nor by reduced PBP3 activity. Gross modifications of peptidoglycan composition are also ruled out. d -Cycloserine, an inhibitor of the soluble pathway producing peptidoglycan precursors, causes phenotypic suppression of filamentation, suggesting that the septation defect of His c strains may be caused by scarcity of PBP3 substrate.

The sfiW locus of Salmonella enterica, previously identified by mutations that suppress the cell division defect of His-constitutive (His(c)) strains, corresponds to serC, the bifunctional gene for phosphoserine-oxoglutarate aminotransferase (SerC) and 2-ketoerythroic acid 4-phosphate transaminase (PdxF). SerC- mutants form small, nearly spherical cells in a wild-type (His+) background, suggesting that the SerC/PdxF product acts as a septation antagonist. Suppression of His(c) filamentation by serC mutations may be explained by loss of the anti-septation activity of SerC/PdxF. The isolation of serC alleles that have lost their biosynthetic activities but are still able to inhibit septum formation suggests that the anti-septation activity of the SerC/PdxF product is unrelated to its known roles in serine and pyridoxine biosynthesis.