Role for µ-opioid receptor in antidepressant effects of δ-opioid receptor agonist KNT-127.

Previous pharmacological data have shown the possible existence of functional interactions between µ- (MOP), κ- (KOP), and δ-opioid receptors (DOP) in pain and mood disorders. We previously reported that MOP knockout (KO) mice exhibit a lower stress response compared with wildtype (WT) mice. Moreover, DOP agonists have been shown to exert antidepressant-like effects in numerous animal models. In the present study, the tail suspension test (TST) and forced swim test (FST) were used to examine the roles of MOP and DOP in behavioral despair. MOP-KO mice and WT mice were treated with KNT-127 (10 mg/kg), a selective DOP agonist. The results indicated a significant decrease in immobility time in the KNT-127 group compared with the saline group in all genotypes in both tests. In the saline groups, immobility time significantly decreased in MOP-KO mice compared with WT mice in both tests. In female MOP-KO mice, KNT-127 significantly decreased immobility time in the TST compared with WT mice. In male MOP-KO mice, however, no genotypic differences were found in the TST after either KNT-127 or saline treatment. Thus, at least in the FST and TST, the activation of DOP and absence of MOP had additive effects in reducing measures of behavioral despair, suggesting that effects on this behavior by DOP activation occur independently of MOP.

Structure of the cytoplasmic domain of FlhA and implication for flagellar type III protein export.

FlhA is the largest integral membrane component of the flagellar type III protein export apparatus of Salmonella and is composed of an N-terminal transmembrane domain (FlhA(TM)) and a C-terminal cytoplasmic domain (FlhA(C)). FlhA(C) is thought to form a platform of the export gate for the soluble components to bind to for efficient delivery of export substrates to the gate. Here, we report a structure of FlhA(C) at 2.8 A resolution. FlhA(C) consists of four subdomains (A(C)D1, A(C)D2, A(C)D3 and A(C)D4) and a linker connecting FlhA(C) to FlhA(TM). The sites of temperature-sensitive (ts) mutations that impair protein export are distributed to all four domains, with half of them at subdomain interfaces. Analyses of the ts mutations and four suppressor mutations to the G368C ts mutation suggested that FlhA(C) changes its conformation for its function. Molecular dynamics simulation demonstrated an open-close motion with a 5-10 ns oscillation in the distance between A(C)D2 and A(C)D4. These results along with further mutation analyses suggest that a dynamic domain motion of FlhA(C) is essential for protein export.

Role of the C-terminal cytoplasmic domain of FlhA in bacterial flagellar type III protein export.

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhA(C)) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, which has a mutation in the sequence encoding FlhA(C). The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhA(C) consists of at least three subdomains, a flexible linker, FlhA(CN), and FlhA(CC), and that FlhA(CN) becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhA(C) not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure.

Crystallization and preliminary X-ray analysis of FliJ, a cytoplasmic component of the flagellar type III protein-export apparatus from Salmonella sp.

The axial component proteins of the bacterial flagellum are synthesized in the cytoplasm and then translocated into the central channel of the flagellum by the flagellar type III protein-export apparatus for self-assembly at the distal growing end of the flagellum. FliJ is an essential cytoplasmic component of the export apparatus. In this study, Salmonella FliJ with an extra three residues (glycine, serine and histidine) attached to the N-terminus as the remainder of a His tag (GSH-FliJ) was purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion technique using PEG 300 as a precipitant. GSH-FliJ crystals grew in the hexagonal space group P6(1)22 or P6(5)22. While the native crystals diffracted to 3.3 A resolution, the diffraction resolution limit of mercury derivatives was extended to 2.1 A. Anomalous and isomorphous difference Patterson maps of the mercury-derivative crystal showed significant peaks in their Harker sections, indicating the usefulness of the derivative data for structure determination.

Structural Insights into the Substrate Specificity Switch Mechanism of the Type III Protein Export Apparatus.

Bacteria use a type III protein export apparatus for construction of the flagellum, which consists of the basal body, the hook, and the filament. FlhA forms a homo-nonamer through its C-terminal cytoplasmic domains (FlhAC) and ensures the strict order of flagellar assembly. FlhAC goes through dynamic domain motions during protein export, but it remains unknown how it occurs. Here, we report that the FlhA(G368C) mutation biases FlhAC toward a closed form, thereby reducing the binding affinity of FlhAC for flagellar export chaperones in complex with their cognate filament-type substrates. The G368C mutations also restrict the conformational flexibility of a linker region of FlhA (FlhAL), suppressing FlhAC ring formation. We propose that interactions of FlhAL with its neighboring subunit converts FlhAC in the ring from a closed conformation to an open one, allowing the chaperon/substrate complexes to bind to the FlhAC ring to form the filament at the hook tip.

Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism.

Symbiobacterium thermophilum is an uncultivable bacterium isolated from compost that depends on microbial commensalism. The 16S ribosomal DNA-based phylogeny suggests that this bacterium belongs to an unknown taxon in the Gram-positive bacterial cluster. Here, we describe the 3.57 Mb genome sequence of S.thermophilum. The genome consists of 3338 protein-coding sequences, out of which 2082 have functional assignments. Despite the high G + C content (68.7%), the genome is closest to that of Firmicutes, a phylum consisting of low G + C Gram-positive bacteria. This provides evidence for the presence of an undefined category in the Gram-positive bacterial group. The presence of both spo and related genes and microscopic observation indicate that S.thermophilum is the first high G + C organism that forms endospores. The S.thermophilum genome is also characterized by the widespread insertion of class C group II introns, which are oriented in the same direction as chromosomal replication. The genome has many membrane transporters, a number of which are involved in the uptake of peptides and amino acids. The genes involved in primary metabolism are largely identified, except those that code several biosynthetic enzymes and carbonic anhydrase. The organism also has a variety of respiratory systems including Nap nitrate reductase, which has been found only in Gram-negative bacteria. Overall, these features suggest that S.thermophilum is adaptable to and thus lives in various environments, such that its growth requirement could be a substance or a physiological condition that is generally available in the natural environment rather than a highly specific substance that is present only in a limited niche. The genomic information from S.thermophilum offers new insights into microbial diversity and evolutionary sciences, and provides a framework for characterizing the molecular basis underlying microbial commensalism.

Functional defect and restoration of temperature-sensitive mutants of FlhA, a subunit of the flagellar protein export apparatus.

The flagellar axial component proteins are exported to the distal end of the growing flagellum for self-assembly by the flagellar type III export apparatus. FlhA is a key membrane protein of the export apparatus, and its C-terminal cytoplasmic domain (FlhA(C)) is a part of an assembly platform for the three soluble export components, FliH, FliI, and FliJ, as well as export substrates and chaperone-substrate complexes. FlhA(C) is composed of a flexible linker region and four compact domains (A(C)D1-A(C)D4). At 42 °C, a temperature-sensitive (TS) G368C mutation in FlhA(C) blocks the export process after the FliH-FliI-FliJ-substrate complex binds to the assembly platform, but it remains unknown how it does so. In this study, we analyzed a TS mutant variant, FlhA(C)(G368C), and its pseudorevertant variants FlhA(C)(G368C/L359F), FlhA(C)(G368C/G364R), FlhA(C)(G368C/R370S), and FlhA(C)(G368C/P550S) using far-ultraviolet circular dichroism. Whereas the denaturation of the wild-type FlhA(C) occurs in a single step, FlhA(C)(G368C) and its pseudorevertant variants showed thermal transitions, at least, in two steps. The first transition of FlhA(C)(G368C) can further be divided into reversible and following irreversible transitions, which correspond to the denaturation of A(C)D2 and A(C)D1, respectively. We show the relation between the reversible transition and the TS defect in the exporting function of FlhA(C)(G368C) and that the loss of function is caused by denaturation of A(C)D2. We suggest that A(C)D2 is directly involved in the translocation of export substrates.