Detection of Novel Amino Acid Polymorphisms in the East Asian CagA of Helicobacter Pylori with Full Sequencing Data.

Cytotoxin-associated gene A (CagA) is generally accepted to be the most important virulence factor of Helicobacter pylori and increases the risk of developing gastric cancer. East Asian CagA, which includes the EPIYA-D segment at the C-terminal region, has a significantly higher gastric carcinogenic rate than Western CagA including the EPIYA-C segment. Although the amino acid polymorphism surrounding the EPIYA motif in the C-terminal region has been examined in detail, limited information is currently available on the amino acid polymorphism of the N-terminal region of East Asian CagA. In the present study, we analyzed the sequencing data of East Asian CagA that we obtained previously to detect amino acid changes (AACs) in the N-terminal region of East Asian CagA. Four highly frequent AACs in the N-terminal region of East Asian CagA were detected in our datasets, two of which (V356A, Y677F) exhibited reproducible specificity using a validation dataset from the NCBI database, which are candidate AACs related to the pathogenic function of CagA. We examined whether these AACs affect the functions of CagA in silico model. The computational docking simulation model showed that binding affinity between CagA and phosphatidylserine remained unchanged in the model of mutant CagA reflecting both AAC, whereas that between CagA and α5ß1 integrin significantly increased. Based on whole genome sequencing data we herein identified novel specific AACs in the N-terminal regions of EPIYA-D that have the potential to change the function of CagA.

Mutation of alanine-422 in KaiC leads to a low amplitude of rhythm in the reconstituted cyanobacterial circadian clock.

The cyanobacterial circadian oscillator can be reconstituted by mixing the purified clock proteins KaiA, KaiB, and KaiC with ATP in vitro, leading to a 24-h oscillation of KaiC phosphorylation. The cyanobacterial mutant pr1 carrying valine instead of alanine at position 422 of KaiC (KaiC-A422V) lost the ability to shift the phase of the circadian rhythm. In this study, we analyzed KaiC-A422V to investigate the effect of this single-residue substitution on the in vitro reconstitution of KaiC oscillation. KaiC-A422V exhibited low amplitude oscillations of phosphorylation with a smaller amount of Kai complex than wild-type KaiC (KaiC-WT). Although KaiA can stimulate KaiC phosphorylation, the phosphorylation level of KaiC-A422V is much lower than that of KaiC-WT even at higher KaiA concentrations. It has been suggested that monomer shuffling of KaiC is involved in entraining the in vitro rhythm. To examine whether KaiC-A422V has the capacity for monomer shuffling, we used the difference in the amplitude of the phosphorylation rhythms between KaiC-WT and KaiC-A422V as the indicator of monomer shuffling. When KaiC-A422V and KaiC-WT were mixed, the amplitude of the phosphorylation rhythm changed according to the mixing ratio. This suggests that KaiC-A422V has a reduced ability to shuffle monomers in hexameric KaiC. In addition, the A422V mutation resulted in a change of the stability of the KaiC protein.

Synechocystis KaiC3 Displays Temperature- and KaiB-Dependent ATPase Activity and Is Important for Growth in Darkness.

Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies, and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. strain PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of nonstandard KaiC homologs in vitro KaiC3 displayed ATPase activity lower than that of the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1, and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of ΔkaiAB1C1 and reduced in the absence of kaiC3 We therefore suggest a role for adaptation to darkness for KaiC3 as well as a cross talk between the KaiC1- and KaiC3-based systems.IMPORTANCE The circadian clock influences the cyanobacterial metabolism, and deeper understanding of its regulation will be important for metabolic optimizations in the context of industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is required. Synechocystis sp. strain PCC 6803 represents a major cyanobacterial model organism and harbors phylogenetically diverged homologs of the clock proteins, which are present in various other noncyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple Synechocystis Kai proteins and characterize enzymatic activities of the nonstandard clock homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness, suggesting its involvement in the regulation of nonphotosynthetic metabolic pathways.

Pressure accelerates the circadian clock of cyanobacteria.

Although organisms are exposed to various pressure and temperature conditions, information remains limited on how pressure affects biological rhythms. This study investigated how hydrostatic pressure affects the circadian clock (KaiA, KaiB, and KaiC) of cyanobacteria. While the circadian rhythm is inherently robust to temperature change, KaiC phosphorylation cycles that were accelerated from 22 h at 1 bar to 14 h at 200 bars caused the circadian-period length to decline. This decline was caused by the pressure-induced enhancement of KaiC ATPase activity and allosteric effects. Because ATPase activity was elevated in the CI and CII domains of KaiC, while ATP hydrolysis had negative activation volumes (ΔV≠), both domains played key roles in determining the period length of the KaiC phosphorylation cycle. The thermodynamic contraction of the structure of the active site during the transition state might have positioned catalytic residues and lytic water molecules favourably to facilitate ATP hydrolysis. Internal cavities might represent sources of compaction and structural rearrangement in the active site. Overall, the data indicate that pressure differences could alter the circadian rhythms of diverse organisms with evolved thermotolerance, as long as enzymatic reactions defining period length have a specific activation volume.

Phosphorylation at Thr432 induces structural destabilization of the CII ring in the circadian oscillator KaiC.

KaiC is the central oscillator protein in the cyanobacterial circadian clock. KaiC oscillates autonomously between phosphorylated and dephosphorylated states on a 24-h cycle in vitro by mixing with KaiA and KaiB in the presence of ATP. KaiC forms a C6 -symmetrical hexamer, which is a double ring structure of homologous N-terminal and C-terminal domains termed CI and CII, respectively. Here, through the characterization of an isolated CII domain protein, CIIKaiC , we show that phosphorylation of KaiC Thr432 destabilizes the hexameric state of the CII ring to a monomeric state. The results suggest that the stable hexameric CI ring acts as a molecular bundle to hold the CII ring, which undergoes dynamic structural changes upon phosphorylation.

Structural characterization of the circadian clock protein complex composed of KaiB and KaiC by inverse contrast-matching small-angle neutron scattering.

The molecular machinery of the cyanobacterial circadian clock consists of three proteins: KaiA, KaiB, and KaiC. Through interactions among the three Kai proteins, the phosphorylation states of KaiC generate circadian oscillations in vitro in the presence of ATP. Here, we characterized the complex formation between KaiB and KaiC using a phospho-mimicking mutant of KaiC, which had an aspartate substitution at the Ser431 phosphorylation site and exhibited optimal binding to KaiB. Mass-spectrometric titration data showed that the proteins formed a complex exclusively in a 6:6 stoichiometry, indicating that KaiB bound to the KaiC hexamer with strong positive cooperativity. The inverse contrast-matching technique of small-angle neutron scattering enabled selective observation of KaiB in complex with the KaiC mutant with partial deuteration. It revealed a disk-shaped arrangement of the KaiB subunits on the outer surface of the KaiC C1 ring, which also serves as the interaction site for SasA, a histidine kinase that operates as a clock-output protein in the regulation of circadian transcription. These data suggest that cooperatively binding KaiB competes with SasA with respect to interaction with KaiC, thereby promoting the synergistic release of this clock-output protein from the circadian oscillator complex.

Conversion between two conformational states of KaiC is induced by ATP hydrolysis as a trigger for cyanobacterial circadian oscillation.

The cyanobacterial circadian oscillator can be reconstituted in vitro by mixing three clock proteins, KaiA, KaiB and KaiC, with ATP. KaiC is the only protein with circadian rhythmic activities. In the present study, we tracked the complex formation of the three Kai proteins over time using blue native (BN) polyacrylamide gel electrophoresis (PAGE), in which proteins are charged with the anionic dye Coomassie brilliant blue (CBB). KaiC was separated as three bands: the KaiABC complex, KaiC hexamer and KaiC monomer. However, no KaiC monomer was observed using gel filtration chromatography and CBB-free native PAGE. These data indicate two conformational states of KaiC hexamer and show that the ground-state KaiC (gs-KaiC) is stable and competent-state KaiC (cs-KaiC) is labile and degraded into monomers by the binding of CBB. Repeated conversions from gs-KaiC to cs-KaiC were observed over 24 h using an in vitro reconstitution system. Phosphorylation of KaiC promoted the conversion from gs-KaiC to cs-KaiC. KaiA sustained the gs-KaiC state, and KaiB bound only cs-KaiC. An E77Q/E78Q-KaiC variant that lacked N-terminal ATPase activity remained in the gs-KaiC state. Taken together, ATP hydrolysis induces the formation of cs-KaiC and promotes the binding of KaiB, which is a trigger for circadian oscillations.