Architecture of the Bacterial Flagellar Distal Rod and Hook of Salmonella

The bacterial flagellum is a large molecular complex composed of thousands of protein subunits for motility. The filamentous part of the flagellum, which is called the axial structure, consists of the filament, the hook, and the rods, with other minor components-the cap protein and the hook associated proteins. They share a common basic architecture of subunit arrangement, but each part shows quite distinct mechanical properties to achieve its specific function. The distal rod and the hook are helical assemblies of a single protein, FlgG and FlgE, respectively. They show a significant sequence similarity but have distinct mechanical characteristics. The rod is a rigid, straight cylinder, whereas the hook is a curved tube with high bending flexibility. Here, we report a structural model of the rod constructed by using the crystal structure of a core fragment of FlgG with a density map obtained previously by electron cryomicroscopy. Our structural model suggests that a segment called L-stretch plays a key role in achieving the distinct mechanical properties of the rod using a structurally similar component protein to that of the hook

<i>Salmonella</i> Flagellum

Flagella-driven motility contributes to effective bacterial invasion. The bacterial flagellum of Salmonella enterica is a rotary motor powered by an electrochemical potential difference of protons across the cytoplasmic membrane. The flagellum is composed of several basal body rings and an axial structure consisting of the rod as a drive shaft, the hook as a universal joint and the filament as a helical propeller. The assembly of the axial structure begins with the rod, followed by the hook and finally the filament. A type III protein export apparatus is located at the flagellar base and transports flagellar axial proteins from the cytoplasm to the distal end of the growing flagellar structure where their assembly occurs. The protein export apparatus coordinates flagellar gene expression with assembly, allowing the hierarchy of flagellar gene expression to exactly parallel the flagellar assembly process. The basal body can accommodate a dozen stator complexes around a rotor ring complex in a load-dependent manner. Each stator unit conducts protons and pushes the rotor. In this book chapter, we will summarize our current understanding of the structure and function of the Salmonella flagellum

Structural basis of enzyme activity regulation by the propeptide of l-lysine α-oxidase precursor from Trichoderma viride

Harmuful proteins are usually synthesized as inactive precursors and are activated by proteolytic processing. l-Amino acid oxidase (LAAO) is a flavoenzyme that catalyzes the oxidative deamination of l-amino acid to produce a 2-oxo acid with ammonia and highly toxic hydrogen peroxide and, therefore, is expressed as a precursor. The LAAO precursor shows significant variation in size and the cleavage pattern for activation. However, the molecular mechanism of how the propeptide suppresses the enzyme activity remains unclear except for deaminating/decarboxylating Pseudomonasl-phenylalanine oxidase (PAO), which has a short N-terminal propeptide composed of 14 residues. Here we show the inactivation mechanism of the l-lysine oxidase (LysOX) precursor (prLysOX), which has a long N-terminal propeptide composed of 77 residues, based on the crystal structure at 1.97 Å resolution. The propeptide of prLysOX indirectly changes the active site structure to inhibit the enzyme activity. prLysOX retains weak enzymatic activity with strict specificity for l-lysine and shows raised activity in acidic conditions. The structures of prLysOX crystals that soaked in a solution with various concentrations of l-lysine have revealed that prLysOX can adopt two conformations; one is the inhibitory form, and the other is very similar to mature LysOX. The propeptide region of the latter form is disordered, and l-lysine is bound to the latter form. These results indicate that prLysOX uses a different strategy from PAO to suppress the enzyme activity and suggest that prLysOX can be activated quickly in response to the environmental change without proteolytic processing

PorA, a conserved C-terminal domain-containing protein, impacts the PorXY-SigP signaling of the type IX secretion system

Porphyromonas gingivalis, a periodontal pathogen, translocates many virulence factors including the cysteine proteases referred to as gingipains to the cell surface via the type IX secretion system (T9SS). Expression of the T9SS component proteins is regulated by the tandem signaling of the PorXY two-component system and the ECF sigma factor SigP. However, the details of this regulatory pathway are still unknown. We found that one of the T9SS conserved C-terminal domain-containing proteins, PGN_0123, which we have designated PorA, is involved in regulating expression of genes encoding T9SS structural proteins and that PorA can be translocated onto the cell surface without the T9SS translocation machinery. X-ray crystallography revealed that PorA has a domain similar to the mannose-binding domain of Escherichia coli FimH, the tip protein of Type 1 pilus. Mutations in the cytoplasmic domain of the sensor kinase PorY conferred phenotypic recovery on the ΔporA mutant. The SigP sigma factor, which is activated by the PorXY two-component system, markedly decreased in the ΔporA mutant. These results strongly support a potential role for PorA in relaying a signal from the cell surface to the PorXY-SigP signaling pathway

Purification, Characterization and Crystal Structure of Isoamylase from Thermophilic Bacteria Rhodothermus marinus

The isoamylase gene from Rhodothermus marinus was cloned into and expressed in Escherichia coli Top 10. As a result of characterization of purified R. marinus isoamylase. the enzyme had an optimum pH of 4.0 and optimum temperature of 70℃. Thermal inactivation studies of the purified R. marinus isoamylase revealed the enzymatic activity to be uninfluenced after one hour incubation at 60℃. These results suggest that R. marinus isoamylase has high thermostability. The crystallization and crystal structure analysis of R. marinus isoamylase was performed. The three-dimensional structure at 1.9Å resolution was determined in complex with the panose. R. marinus isoamylase is composed of three domains N, A and C, and, has a (β/α)8-barrel in domain A. The secondary structural alignments of the R. marinus isoamylase and P. amyloderamosa isoamylase was carried out. They have the four active-site consensus regions characteristic of the α-amylase family. And the essential residue of the α-amylase family (D359, E395, and D467) was conserved in these enzymes. R. marinus isoamylase has shorter loops than P. amyloderamosa isoamylase. And R. marinus isoamylase had no Ca2+ binding site. These results are thought to be factors of thermostability of R. marinus isoamylase.Rhodothermus marinus 由来イソアミラーゼ遺伝子を組み込んだプラスミド pBX２を使用し，大腸菌 Top10株を形質転換し，16時間の前培養，24時間の本培養後，菌体破砕し，得られた無細胞抽出液を熱処理（80℃，10 min），50ｵ硫安分画，陰イオン交換カラムクロマトグラフィー（DEAEﾝトヨパール），ハイドロキシアパタイトカラムクロマトグラフィーに供して本酵素の精製を行った．本精製酵素の性質検討を行った結果，本酵素の最適反応温度は70℃，pH４であり，また本酵素は60℃で１時間処理しても活性が低下することが無く，Pseudomonas amyloderamosa 由来イソアミラーゼよりも高い耐熱性を有することが判明した．本酵素の結晶化・X線結晶構造解析を行った結果，本酵素は P. amyloderamosa 由来イソアミラーゼと同様Ｎドメイン・ＡドメインＣドメインの３つのドメインから構成されており，活性残基（Ｄ359，Ｅ395，Ｄ467）など活性中心付近のアミノ酸残基も P. amyloderamosa 由来イソアミラーゼと同様，高度に保存されていた．本酵素の熱安定性が P. amyloderamosa 由来イソアミラーゼよりも高 い要因として，P. amyloderamosa 由来イソアミラーゼよりもループの長さが全体的に短いことと，カルシウムイオン結合サイトの欠如が挙げられた．今後さらに構造解析を進めることにより，本酵素の熱安定性機構，反応 機構など更なる知見が得られることが期待される

Analysis of the Change in Dominant Phytoplankton Species in Unstratified Lake Oshima-Ohnuma Estimated by a Bottle Incubation Experiment

1996年5?7月に、渡島大沼において、優占植物プランクトンの細胞密度を測定し、さらにボトル培養実験によりそれらの成長速度を見積り、春から夏への植物プランクトン優占種変化に関わる要因を明らかにすることを試みた。4、5月にAsterionella gracillimaが優占種となったが、その成長速度は5月には比較的低く、その後、細胞密度が低下し、この主な原因は栄養塩の不足であることが示唆された。6月後半からMelosira gramulataの成長速度が増加し、優占種となった。M.gramulataは水深6mでも成長速度が高いことから、深層で生存できることと、湖が成層を形成しないことで再懸濁により有光層に存在できる状況が同種の成長に適していることが考えられた

Glycine insertion makes yellow fluorescent protein sensitive to hydrostatic pressure

Fluorescent protein-based indicators for intracellular environment conditions such as pH and ion concentrations are commonly used to study the status and dynamics of living cells. Despite being an important factor in many biological processes, the development of an indicator for the physicochemical state of water, such as pressure, viscosity and temperature, however, has been neglected. We here found a novel mutation that dramatically enhances the pressure dependency of the yellow fluorescent protein (YFP) by inserting several glycines into it. The crystal structure of the mutant showed that the tyrosine near the chromophore flipped toward the outside of the β-can structure, resulting in the entry of a few water molecules near the chromophore. In response to changes in hydrostatic pressure, a spectrum shift and an intensity change of the fluorescence were observed. By measuring the fluorescence of the YFP mutant, we succeeded in measuring the intracellular pressure change in living cell. This study shows a new strategy of design to engineer fluorescent protein indicators to sense hydrostatic pressure

Structural Insight into the Rotational Switching Mechanism of the Bacterial Flagellar Motor

Structural analysis of a clockwise-biased rotation mutant of the bacterial flagellar rotor protein FliG provides a new model for the arrangement of FliG subunits in the motor, and novel insights into rotation switching