[Protein degradation in bacteria: focus on the ClpP protease].

Proteins in the cells are born (synthesized), work, and die (decomposed). In the life of a protein, its birth is obviously important, but how it dies is equally important in living organisms. Proteases secreted into the outside of cells are used to decompose the external proteins and the degradation products are taken as the nutrients. On the other hand, there are also proteases that decompose unnecessary or harmful proteins which are generated in the cells. In eukaryotes, a large enzyme complex called the proteasome is primarily responsible for degradation of such proteins. Bacteria, which are prokaryotes, have a similar system as the proteasome. We would like to explain the bacterial degradation system of proteins or the death of proteins, which is performed by ATP-dependent protease Clp, with a particular focus on the ClpXP complex, and with an aspect as a target for antibiotics against bacteria.

Site-Directed Cross-Linking Between Bacterial Flagellar Motor Proteins In Vivo.

The bacterial flagellum employs a rotary motor embedded on the cell surface. The motor consists of the stator and rotor elements and is driven by ion influx (typically H+ or Na+) through an ion channel of the stator. Ion influx induces conformational changes in the stator, followed by changes in the interactions between the stator and rotor. The driving force to rotate the flagellum is thought to be generated by changing the stator-rotor interactions. In this chapter, we describe two methods for investigating the interactions between the stator and rotor: site-directed in vivo photo-crosslinking and site-directed in vivo cysteine disulfide crosslinking.

Function and Structure of FlaK, a Master Regulator of the Polar Flagellar Genes in Marine Vibrio.

Vibrio alginolyticus has a flagellum at the cell pole, and the fla genes, involved in its formation, are hierarchically regulated in several classes. FlaK (also called FlrA) is an ortholog of Pseudomonas aeruginosa FleQ, an AAA+ ATPase that functions as a master regulator for all later fla genes. In this study, we conducted mutational analysis of FlaK to examine its ATPase activity, ability to form a multimeric structure, and function in flagellation. We cloned flaK and confirmed that its deletion caused a nonflagellated phenotype. We substituted amino acids at the ATP binding/hydrolysis site and at the putative subunit interfaces in a multimeric structure. Mutations in these sites abolished both ATPase activity and the ability of FlaK to induce downstream flagellar gene expression. The L371E mutation, at the putative subunit interface, abolished flagellar gene expression but retained ATPase activity, suggesting that ATP hydrolysis is not sufficient for flagellar gene expression. We also found that FlhG, a negative flagellar biogenesis regulator, suppressed the ATPase activity of FlaK. The 20 FlhG C-terminal residues are critical for reducing FlaK ATPase activity. Chemical cross-linking and size exclusion chromatography revealed that FlaK mostly exists as a dimer in solution and can form multimers, independent of ATP. However, ATP induced the interaction between FlhG and FlaK to form a large complex. The in vivo effects of FlhG on FlaK, such as multimer formation and/or DNA binding, are important for gene regulation. IMPORTANCE FlaK is an NtrC-type activator of the AAA+ ATPase subfamily of σ54-dependent promoters of flagellar genes. FlhG, a MinD-like ATPase, negatively regulates the polar flagellar number by collaborating with FlhF, an FtsY-like GTPase. We found that FlaK and FlhG interact in the presence of ATP to form a large complex. Mutational analysis revealed the importance of FlaK ATPase activity in flagellar gene expression and provided a model of the Vibrio molecular mechanism that regulates the flagellar number.

Formation of multiple flagella caused by a mutation of the flagellar rotor protein FliM in Vibrio alginolyticus.

Marine bacterium Vibrio alginolyticus forms a single flagellum at a cell pole. In Vibrio, two proteins (GTPase FlhF and ATPase FlhG) regulate the number of flagella. We previously isolated the NMB155 mutant that forms multiple flagella despite the absence of mutations in flhF and flhG. Whole-genome sequencing of NMB155 identified an E9K mutation in FliM that is a component of C-ring in the flagellar rotor. Mutations in FliM result in defects in flagellar formation (fla) and flagellar rotation (che or mot); however, there are a few reports indicating that FliM mutations increase the number of flagella. Here, we determined that the E9K mutation confers the multi-flagellar phenotype and also the che phenotype. The co-expression of wild-type FliM and FliM-E9K indicated that they were competitive in regard to determining the flagellar number. The ATPase activity of FlhG has been correlated with the number of flagella. We observed that the ATPase activity of FlhG was increased by the addition of FliM but not by the addition of FliM-E9K in vitro. This indicates that FliM interacts with FlhG to increase its ATPase activity, and the E9K mutation may inhibit this interaction. FliM may control the ATPase activity of FlhG to properly regulate the number of the polar flagellum at the cell pole.

Functional analysis of the N-terminal region of Vibrio FlhG, a MinD-type ATPase in flagellar number control.

GTPase FlhF and ATPase FlhG are two key factors involved in regulating the flagellum number in Vibrio alginolyticus. FlhG is a paralogue of the Escherichia coli cell division regulator MinD and has a longer N-terminal region than MinD with a conserved DQAxxLR motif. The deletion of this N-terminal region or a Q9A mutation in the DQAxxLR motif prevents FlhG from activating the GTPase activity of FlhF in vitro and causes a multi-flagellation phenotype. The mutant FlhG proteins, especially the N-terminally deleted variant, were remarkably reduced compared to that of the wild-type protein in vivo. When the mutant FlhG was expressed at the same level as the wild-type FlhG, the number of flagella was restored to the wild-type level. Once synthesized in Vibrio cells, the N-terminal region mutation in FlhG seems not to affect the protein stability. We speculated that the flhG translation efficiency is decreased by N-terminal mutation. Our results suggest that the N-terminal region of FlhG controls the number of flagella by adjusting the FlhF activity and the amount of FlhG in vivo. We speculate that the regulation by FlhG, achieved through transcription by the master regulator FlaK, is affected by the mutations, resulting in reduced flagellar formation by FlhF.

Site-directed crosslinking identifies the stator-rotor interaction surfaces in a hybrid bacterial flagellar motor.

The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses, but have not been demonstrated biochemically. Here, we used site-directed photo- and disulfide-crosslinking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-L-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA formed a crosslink with FliG. PomA residue K89 gave the highest yield of crosslinks, suggesting that it is the PomA residue nearest to FliG. UV-induced crosslinking stopped motor rotation, and the isolated hook-basal body contained the crosslinked products. pBPA inserted to replace residues R281 or D288 in FliG formed crosslinks with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide crosslinks with cysteine inserted in place of FliG residues R281 and D288, and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA.ImportanceThe bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo- and disulfide-crosslinking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

Tree of motility - A proposed history of motility systems in the tree of life.

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility.

Characterization of PomA periplasmic loop and sodium ion entering in stator complex of sodium-driven flagellar motor.

The bacterial flagellar motor is a rotary nanomachine driven by ion flow. The flagellar stator complex, which is composed of two proteins, PomA and PomB, performs energy transduction in marine Vibrio. PomA is a four transmembrane (TM) protein and the cytoplasmic region between TM2 and TM3 (loop2-3) interacts with the rotor protein FliG to generate torque. The periplasmic regions between TM1 and TM2 (loop1-2) and TM3 and TM4 (loop3-4) are candidates to be at the entrance to the transmembrane ion channel of the stator. In this study, we purified the stator complex with cysteine replacements in the periplasmic loops and assessed the reactivity of the protein with biotin maleimide (BM). BM easily modified Cys residues in loop3-4 but hardly labelled Cys residues in loop1-2. We could not purify the plug deletion stator (ΔL stator) composed of PomBΔ41-120 and WT-PomA but could do the ΔL stator with PomA-D31C of loop1-2 or with PomB-D24N of TM. When the ion channel is closed, PomA and PomB interact strongly. When the ion channel opens, PomA interacts less tightly with PomB. The plug and loop1-2 region regulate this activation of the stator, which depends on the binding of sodium ion to the D24 residue of PomB.

Effect of sodium ions on conformations of the cytoplasmic loop of the PomA stator protein of Vibrio alginolyticus.

The sodium driven flagellar stator of Vibrio alginolyticus is a hetero-hexamer membrane complex composed of PomA and PomB, and acts as a sodium ion channel. The conformational change in the cytoplasmic region of PomA for the flagellar torque generation, which interacts directly with a rotor protein, FliG, remains a mystery. In this study, we introduced cysteine mutations into cytoplasmic charged residues of PomA, which are highly conserved and interact with FliG, to detect the conformational change by the reactivity of biotin maleimide. In vivo labelling experiments of the PomA mutants revealed that the accessibility of biotin maleimide at position of E96 was reduced with sodium ions. Such a reduction was also seen in the D24N and the plug deletion mutants of PomB, and the phenomenon was independent in the presence of FliG. This sodium ions specific reduction was also detected in Escherichia coli that produced PomA and PomB from a plasmid, but not in the purified stator complex. These results demonstrated that sodium ions cause a conformational change around the E96 residue of loop2-3 in the biological membrane.

FliH and FliI ensure efficient energy coupling of flagellar type III protein export in Salmonella.

For construction of the bacterial flagellum, flagellar proteins are exported via its specific export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane (TM) export gate complex and a cytoplasmic ATPase complex consisting of FliH, FliI, and FliJ. FlhA is a TM export gate protein and plays important roles in energy coupling of protein translocation. However, the energy coupling mechanism remains unknown. Here, we performed a cross-complementation assay to measure robustness of the energy transduction system of the export apparatus against genetic perturbations. Vibrio FlhA restored motility of a Salmonella ΔflhA mutant but not that of a ΔfliH-fliI flhB(P28T) ΔflhA mutant. The flgM mutations significantly increased flagellar gene expression levels, allowing Vibrio FlhA to exert its export activity in the ΔfliH-fliI flhB(P28T) ΔflhA mutant. Pull-down assays revealed that the binding affinities of Vibrio FlhA for FliJ and the FlgN-FlgK chaperone-substrate complex were much lower than those of Salmonella FlhA. These suggest that Vibrio FlhA requires the support of FliH and FliI to efficiently and properly interact with FliJ and the FlgN-FlgK complex. We propose that FliH and FliI ensure robust and efficient energy coupling of protein export during flagellar assembly.

Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria.

SecDF interacts with the SecYEG translocon in bacteria and enhances protein export in a proton-motive-force-dependent manner. Vibrio alginolyticus, a marine-estuarine bacterium, contains two SecDF paralogs, V.SecDF1 and V.SecDF2. Here, we show that the export-enhancing function of V.SecDF1 requires Na+ instead of H+, whereas V.SecDF2 is Na+-independent, presumably requiring H+. In accord with the cation-preference difference, V.SecDF2 was only expressed under limited Na+ concentrations whereas V.SecDF1 was constitutive. However, it is not the decreased concentration of Na+ per se that the bacterium senses to up-regulate the V.SecDF2 expression, because marked up-regulation of the V.SecDF2 synthesis was observed irrespective of Na+ concentrations under certain genetic/physiological conditions: (i) when the secDF1VA gene was deleted and (ii) whenever the Sec export machinery was inhibited. VemP (Vibrio export monitoring polypeptide), a secretory polypeptide encoded by the upstream ORF of secDF2VA, plays the primary role in this regulation by undergoing regulated translational elongation arrest, which leads to unfolding of the Shine-Dalgarno sequence for translation of secDF2VA. Genetic analysis of V. alginolyticus established that the VemP-mediated regulation of SecDF2 is essential for the survival of this marine bacterium in low-salinity environments. These results reveal that a class of marine bacteria exploits nascent-chain ribosome interactions to optimize their protein export pathways to propagate efficiently under different ionic environments that they face in their life cycles.

Effect of FliG three amino acids deletion in Vibrio polar-flagellar rotation and formation.

Most of bacteria can swim by rotating flagella bidirectionally. The C ring, located at the bottom of the flagellum and in the cytoplasmic space, consists of FliG, FliM and FliN, and has an important function in flagellar protein secretion, torque generation and rotational switch of the motor. FliG is the most important part of the C ring that interacts directly with a stator subunit. Here, we introduced a three-amino acids in-frame deletion mutation (ΔPSA) into FliG from Vibrio alginolyticus, whose corresponding mutation in Salmonella confers a switch-locked phenotype, and examined its phenotype. We found that this FliG mutant could not produce flagellar filaments in a fliG null strain but the FliG(ΔPSA) protein could localize at the cell pole as does the wild-type protein. Unexpectedly, when this mutant was expressed in a wild-type strain, cells formed flagella efficiently but the motor could not rotate. We propose that this different phenotype in Vibrio and Salmonella might be due to distinct interactions between FliG mutant and FliM in the C ring between the bacterial species.

The MinD homolog FlhG regulates the synthesis of the single polar flagellum of Vibrio alginolyticus.

FlhG, a MinD homolog and an ATPase, is known to mediate the formation of the single polar flagellum of Vibrio alginolyticus together with FlhF. FlhG and FlhF work antagonistically, with FlhF promoting flagellar assembly and FlhG inhibiting it. Here, we demonstrate that purified FlhG exhibits a low basal ATPase activity. As with MinD, the basal ATPase activity of FlhG can be activated and the D171A residue substitution enhances its ATPase activity sevenfold. FlhG-D171A localizes strongly at the cell pole and severely inhibits motility and flagellation, whereas the FlhG K31A and K36Q mutants, which are defective in ATP binding, do not localize to the poles, cannot complement a flhG mutant and lead to hyperflagellation. A strong polar localization of FlhF is observed with the K36Q mutant FlhG but not with the wild-type or D171A mutant FlhG. Unexpectedly, an Ala substitution at the catalytic residue (D60A), which abolishes ATPase activity but still allows ATP binding, only slightly affects FlhG functions. These results suggest that the ATP-dependent polar localization of FlhG is crucial for its ability to downregulate the number of polar flagella. We speculate that ATP hydrolysis by FlhG is required for the fine tuning of the regulation.

Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor.

The torque of the bacterial flagellum is generated by the rotor-stator interaction coupled with the ion flow through the channel in the stator. Anchoring the stator unit to the peptidoglycan layer with proper orientation around the rotor is believed to be essential for smooth rotation of the flagellar motor. The stator unit of the sodium-driven flagellar motor of Vibrio is composed of PomA and PomB, and is thought to be fixed to the peptidoglycan layer and the T-ring by the C-terminal periplasmic region of PomB. Here, we report the crystal structure of a C-terminal fragment of PomB (PomBC) at 2.0-Å resolution, and the structure suggests a conformational change in the N-terminal region of PomBC for anchoring the stator. On the basis of the structure, we designed double-Cys replaced mutants of PomB for in vivo disulfide cross-linking experiments and examined their motility. The motility can be controlled reproducibly by reducing reagent. The results of these experiments suggest that the N-terminal disordered region (121-153) and following the N-terminal two-thirds of α1(154-164) in PomBC changes its conformation to form a functional stator around the rotor. The cross-linking did not affect the localization of the stator nor the ion conductivity, suggesting that the conformational change occurs in the final step of the stator assembly around the rotor.

Transformation of actoHMM assembly confined in cell-sized liposome.

To construct a simple model of a cellular system equipped with motor proteins, cell-sized giant liposomes encapsulating various amounts of actoHMM, the complexes of actin filaments (F-actin) and heavy meromyosin (HMM, an actin-related molecular motor), with a depletion reagent to mimic the crowding effect of inside of living cell, were prepared. We adapted the methodology of the spontaneous transfer of water-in-oil (W/O) droplets through a phospholipid monolayer into the bulk aqueous phase and successfully prepared stable giant liposomes encapsulating the solution with a physiological salt concentration containing the desired concentrations of actoHMM, which had been almost impossible to obtain using currently adapted methodologies such as natural swelling and electro-formation on an electrode. We then examined the effect of ATP on the cytoskeleton components confined in those cell-sized liposomes, because ATP is known to drive the sliding motion for actoHMM. We added α-hemolysin, a bacterial membrane pore-forming toxin, to the bathing solution and obtained liposomes with the protein pores embedded on the bilayer membrane to allow the transfer of ATP inside the liposomes. We show that, by the ATP supply, the actoHMM bundles inside the liposomes exhibit specific changes in spatial distribution, caused by the active sliding between F-actin and HMM. Interestingly, all F-actins localized around the inner periphery of liposomes smaller than a critical size, whereas in the bulk solution and also in larger liposomes, the actin bundles formed aster-like structures under the same conditions.

Disulphide cross-linking between the stator and the bearing components in the bacterial flagellar motor.

The flagellar motor is composed of the stator and the rotor, and the interaction between the stator and the rotor at the cytoplasmic region is believed to produce mechanical force for the rotation of flagella. The periplasmic region of the stator has been proposed to play an important role in assembly around and incorporation into the motor. In this study, we provide evidence suggesting that the periplasmic region of the stator component MotB interacts with the P-ring component FlgI, which functions as a bearing for the rotor along with the L-ring protein FlgH, from a site-directed disulphide cross-linking approach. First, we prepared four FlgI and three MotB cysteine-substituted mutant proteins and co-expressed them in various combinations in Escherichia coli. We detected cross-linked combinations of FlgI G11C and MotB S248C when treated with the oxidant Cu-phenanthroline or bismaleimide cross-linkers. Furthermore, we performed Cys-scanning mutagenesis around these two residues and found additional combinations of cross-linked residues. Treatment with a protonophore CCCP significantly reduced the cross-linking efficiency between FlgI and MotB in flagellated cells, but not in non-flagellated cells. These results suggest a direct contact between MotB and FlgI upon assembly of the stator into a motor.

Tributyltin sensitivity of vacuolar-type Na(+)-transporting ATPase from Enterococcus hirae.

Tributyltin chloride (TBT), an environmental pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Some members of F-ATP synthase (F-ATPase)/vacuolar type ATPase (V-ATPase) superfamily have been identified as the molecular target of this compound. TBT inhibited the activities of H(+)-transporting or Na(+)-transporting F-ATPase as well as H(+)-transporting V-ATPase originated from various organisms. However, the sensitivity to TBT of Na(+)-transporting V-ATPase has not been investigated. We examined the effect of TBT on Na(+)-transporting V-ATPase from an eubacterium Enterococus hirae. The ATP hydrolytic activity of E. hirae V-ATPase in purified form as well as in membrane-bound form was little inhibited by less than 10 microM TBT; IC50 for TBT inhibition of purified enzyme was estimated to be about 35 microM. Active sodium transport by E. hirae cells, indicating the in vivo activity of this V-ATPase, was not inhibited by 20 microM TBT. By contrast, IC50 of H(+)-transporting V-ATPase of the vacuolar membrane vesicles from Saccharomyces cerevisiae was about 0.2 microM. E. hirae V-ATPase is thus extremely less sensitive to TBT.

The peptidoglycan-binding (PGB) domain of the Escherichia coli pal protein can also function as the PGB domain in E. coli flagellar motor protein MotB.

The bacterial flagellar stator proteins, MotA and MotB, form a complex and are thought to be anchored to the peptidoglycan by the C-terminal conserved peptidoglycan-binding (PGB) motif of MotB. To clarify the role of the C-terminal region, we performed systematic cysteine mutagenesis and constructed a chimeric MotB protein which was replaced with the peptidoglycan-associated lipoprotein Pal. Although this chimera could not restore motility to a motB strain, we were able to isolate two motile revertants. One was F172V in the Pal region and the other was P159L in the MotB region. Furthermore, we attempted to map the MotB Cys mutations in the crystal structure of Escherichia coli Pal. We found that the MotB mutations that affected motility nearly overlapped with the predicted PG-binding residues of Pal. Our results indicate that, although the functions of MotB and Pal are very different, the PGB region of Pal is interchangeable with the PGB region of MotB.

Systematic Cys mutagenesis of FlgI, the flagellar P-ring component of Escherichia coli.

The bacterial flagellar motor is embedded in the cytoplasmic membrane, and penetrates the peptidoglycan layer and the outer membrane. A ring structure of the basal body called the P ring, which is located in the peptidoglycan layer, is thought to be required for smooth rotation and to function as a bushing. In this work, we characterized 32 cysteine-substituted Escherichia coli P-ring protein FlgI variants which were designed to substitute every 10th residue in the 346 aa mature form of FlgI. Immunoblot analysis against FlgI protein revealed that the cellular amounts of five FlgI variants were significantly decreased. Swarm assays showed that almost all of the variants had nearly wild-type function, but five variants significantly reduced the motility of the cells, and one of them in particular, FlgI G21C, completely disrupted FlgI function. The five residues that impaired motility of the cells were localized in the N terminus of FlgI. To demonstrate which residue(s) of FlgI is exposed to solvent on the surface of the protein, we examined cysteine modification by using the thiol-specific reagent methoxypolyethylene glycol 5000 maleimide, and classified the FlgI Cys variants into three groups: well-, moderately and less-labelled. Interestingly, the well- and moderately labelled residues of FlgI never overlapped with the residues known to be important for protein amount or motility. From these results and multiple alignments of amino acid sequences of various FlgI proteins, the highly conserved region in the N terminus, residues 1-120, of FlgI is speculated to play important roles in the stabilization of FlgI structure and the formation of the P ring by interacting with FlgI molecules and/or other flagellar components.

Roles of the intramolecular disulfide bridge in MotX and MotY, the specific proteins for sodium-driven motors in Vibrio spp.

The proteins PomA, PomB, MotX, and MotY are essential for the motor function of Na+-driven flagella in Vibrio spp. Both MotY and MotX have the two cysteine residues (one of which is in a conserved tetrapeptide [CQLV]) that are inferred to form an intramolecular disulfide bond. The cysteine mutants of MotY prevented the formation of an intramolecular disulfide bond, which is presumably important for protein stability. Disruption of the disulfide bridge in MotX by site-directed mutagenesis resulted in increased instability, which did not, however, affect the motility of the cells. These lines of evidence suggest that the intramolecular disulfide bonds are involved in the stability of both proteins, but only MotY requires the intramolecular bridge for proper function.