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Escherichia coli chemoreceptors can sense changes in temperature for thermotaxis. Here we found that the aerotaxis transducer Aer, a homolog of chemoreceptors lacking a periplasmic domain, mediates thermoresponses. We propose that thermosensing by the chemoreceptors is a general attribute of their highly conserved cytoplasmic domain (or their less conserved transmembrane domain). doi: 10.1128/JB.01219-09

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.

[Na.sub.v]BP, found in alkaliphilic Bacillus pseudofirmus OF4, is a member of the bacterial voltage-gated Na+ channel superfamily. The alkaliphile requires [Na.sub.v]BP for normal chemotaxis responses and for optimal pH homeostasis during a shift to alkaline conditions at suboptimally low [Na.sup.+] concentrations. We hypothesized that interaction of [Na.sub.v]BP with one or more other proteins in vivo, specifically methyl-accepting chemotaxis proteins (MCPs), is involved in activation of the channel under the pH conditions that exist in the extremophile and could underpin its role in chemotaxis; MCPs transduce chemotactic signals and generally localize to cell poles of rod-shaped cells. Here, immunofluorescence microscopy and fluorescent protein fusion studies showed that an alkaliphile protein (designated McpX) that cross-reacts with antibodies raised against Bacillus subtilis McpB co-localizes with [Na.sub.v]BP at the cell poles of B. pseudofirmus OF4. In a mutant in which [Na.sub.v]BP-encoding ncbA is deleted, the content of McpX was close to the wild-type level but McpX was significantly delocalized. A mutant of B. pseudofirmus OF4 was constructed in which cheAW expression was disrupted to assess whether this mutation impaired polar localization of McpX, as expected from studies in Escherichia coli and Salmonella, and, if so, whether [Na.sub.v]BP would be similarly affected. Polar localization of both McpX and [Na.sub.v]BP was decreased in the cheAW mutant. The results suggest interactions between McpX and [Na.sub.v]BP that affect their co-localization. The inverse chemotaxis phenotype of ncbA mutants may result in part from MCP delocalization.

The FimA fimbriae of Porphyromonas gingivalis, the causative agent of periodontitis, have been implicated in various aspects of pathogenicity, such as colonization, adhesion and aggregation. In this study, the four open reading frames (ORF1, ORF2, ORF3 and ORF4) downstream of the fimbrilin gene (fimA) in strain ATCC 33277 were examined. ORF2, ORF3 and ORF4 were demonstrated to encode minor components of the fimbriae and were therefore renamed fimC, fimD and fimE, respectively. Immunoblotting analyses revealed that inactivation of either fimC or fimD by an ermF-ermAM insertion, but not inactivation of ORF1, was accompanied by concomitant loss of the products from the downstream genes, raising the possibility that fimC, fimD and time constitute a transcription unit. The fimE mutant produced FimC and FimD, but fimbriae purified from it contained neither protein, suggesting that FimE is required for the assembly of FimC and FimD onto the fimbrilin (FimA) fibre. The fimC, fimD and time mutants lost autoaggregation abilities. Fimbriae purified from these three mutants showed attenuated binding activities to glyceraldehyde-3-phosphate dehydrogenase of Streptococcus oralis and to two extracellular matrix proteins, fibronectin and type I collagen. These results suggest that FimE, as well as FimC and FireD, play critical roles in the adhesive activities of the mature FimA fimbriae in P. gingivalis.

The P ring of the bacterial flagellar motor consists of multiple copies of FlgI, a periplasmic protein. The intramolecular disulfide bond in FlgI has previously been reported to be essential for P-ring assembly in Escherichia coli, because the P ring was not assembled in a dsbB strain that was defective for disulfide bond formation in periplasmic proteins. We, however, found that the two Cys residues of FlgI are not conserved in other bacterial species. We then assessed the role of this intramolecular disulfide bond in FlgI. A Cys-eliminated FlgI derivative formed a P ring that complemented the flagellation defect of our [delta]flgI strain when it was overproduced, suggesting that disulfide bond formation in FlgI is not absolutely required for P-ring assembly. The levels of the mature forms of the FlgI derivatives were significantly lower than that of wild-type FlgI, although the precursor protein levels were unchanged. Moreover, the FlgI derivatives were more susceptible to degradation than wild-type FlgI. Overproduction of FlgI suppressed the motility defect of [delta]dsbB cells. Additionally, the low level of FlgI observed in the [delta]dsbB strain increased in the presence of L-cystine, an oxidative agent. We propose that intramolecular disulfide bond formation facilitates the rapid folding of the FlgI monomer to protect against degradation in the periplasmic space, thereby allowing its efficient self-assembly into the P ring.

Vibrio cholerae has three sets of chemotaxis (Che) proteins, including three histidine kinases (CheA) and four response regulators (CheY) that are encoded by three che gene clusters. We deleted the cheY genes individually or in combination and found that only the cheY3 deletion impaired chemotaxis, reinforcing the previous conclusion that che cluster II is involved in chemotaxis. However, this does not exclude the involvement of the other clusters in chemotaxis. In other bacteria, phospho-CheY binds directly to the flagellar motor to modulate its rotation, and CheY overexpression, even without CheA, causes extremely biased swimming behavior. We reasoned that a V. cholerae CheY homolog, if it directly controls flagellar rotation, should also induce extreme swimming behavior when overproduced. This was the case for CheY3 (che cluster II). However, no other CheY homolog, including the putative CheY (CheY0) protein encoded outside the che clusters, affected swimming, demonstrating that these CheY homologs cannot act directly on the flagellar motor. CheY4 very slightly enhanced the spreading of an Escherichia coli cheZ mutant in semisolid agar, raising the possibility that it can affect chemotaxis by removing a phosphoryl group from CheY3. We also found that V. cholerae CheY3 and E. coli CheY are only partially exchangeable. Mutagenic analyses suggested that this may come from coevolution of the interacting pair of proteins, CheY and the motor protein FliM. Taken together, it is likely that the principal roles of che clusters I and III as well as cheY0 are to control functions other than chemotaxis.

In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.

Research has been conducted on the receptor methylation in Escherichia coli chemotaxis. The role of this receptor in adaptation has been investigated and the details are reported.

Research has been conducted on the Vibrio cholerae genome. Results indicate that one of the genes present in these genomes is important for the chemotaxis under standard conditions.

Research has been conducted on PomA which is the Vibrio alginolyticus sodium-driven polar-flagellar motor component. Experiments with cysteine substitutions in the PomA periplasmic region have been carried out.

Results suggest that cysteine substitution for basic or polar amino acid residues in the Tcp chemoreceptor indicate that arginine, lysine, and tyrosine are involved in the recognition of citrate.

Research has been conducted on the Vibrio alginolyticus sodium-driven motor. The results of the cysteine-scanning mutagenesis carried out to characterize periplasmic loop regions are presented.

Research has been conducted on the Rhodobacter sphaeroides. Investigation on its ability to function in place of the pomA and pomB genes of Vibrio alginolyticus is discussed.

The proteins PomA, PomB, MotX and MotY appear to be implicated in force generation of the sodium-driven polar flagella of Vibrio alginolyticus. PomA and PomB appear to be related and to produce a sodium channel. Through the use of antipeptide antibodies against PomA and PomB, immunoprecipitation was performed to determine whether these proteins form a complex. The in vivo stabilities of PomA and PomB were also investigated. Results showed that PomA and PomB functionally interact with each other.

Random mutagenesis of the pomA gene encoding a putative channel component of the Na+-driven polar flagellar motor of Vibrio alginolyticus was conducted to identify residues responsible for the function of PomA. Thirty-seven non-motile mutants were isolated and 26 independent mutations in the pomA gene were identified. Results demonstrate that the mutant proteins that exhibit dominance are expressed and inserted into the motor but do not function properly.

Vibrio cholerae is a very motile bacterium which has a single polar flagellum as a locomotion organelle. The genes that are highly homologous to the critical genes of the Na+-driven polar flagellar motor of Vibrio alginolyticus were observed in the database of V. cholerae. The energy source for this flagellar motor was examined. The Na+ dependence and the sensitivity to the Na+ motor-specific inhibitor of the motility of the V. cholerae strains were studied. Findings showed that the polar flagellar motor of V. cholerae is driven by an Na+ motive force.

Escherichia coli and Salmonella typhimurium can sense chemoeffectors in the environment and either migrate to or move away from the stimulus. A key element in effector identification and response generation is chemotactic adaptation. Adaptation in Escherichia coli and Salmonella typhimurium is dependent on the pentapeptide comprising the carboxyl terminus of chemoreceptors. Methylation of selected residues in this region enable these organisms to adapt.

A study was conducted on the cloning of a DNA fragment which complemented a polar flagellum-defective (pof) mutation of Vibrio alginolyticus. The fragment had two complete and two partial open reading frames. Deletion analysis indicates that ORF2 complements the pof mutation. Moreover, the findings suggest that RpoN plays a role in the expression of polar flagellar genes.

A study was conducted on the thermosensing function of mutant aspartate chemoreceptors by replacing methyl-accepting sites with alanine. The results indicate that the negative charge at the methylation sites is a factor in determining thermosensor phenotypes although the size and shape of the side chain may also be important factors. Moreover, there was clear differentiation in the warm, cold and null thermosensor phenotypes.

The vibrio alginolyticus polar flagellum has fast rotation using a motor driven by sodium ions. MotX and motY are genes identified in marine bacteria vibrio parahaemolyticus and v. alginolyticus, for the sodium-driven flagellar motor. Additional sodium motor genes pomA and pomB were identified in V. alginolyticus, with similarities to MotA and MotB, particularly in the transmembrane regions.

Swarm-deficient Vibrio alginolyticus mutants were isolated from a Laf-defective mutant were analyzed to characterize the mechanisms that modulates flagellar length during the flagellar protein assembly. Analysis of Pof-defective strains indicated the presence of three groups of mutants that were designated as chemotaxis-defective, flagellation-defective and motility-defective strains. The wild-type and mutant strains also exhibited swimming speeds that were dependent on sodium concentration.

Vibrio alginolyticus has two types of flagellar systems, polar flagella and lateral flagella. Upon treatment with phenol, lateral-flagella-defective (Pof+ Laf-) mutants exhibited a tumbling state which is characterized by frequent forward and backward movements. A tethered-cell method revealed that this motion was due to the frequent changing of direction of motion of the flagellar motor. Treatment of cells with attractant serine abolished the tumbling motion and cells swam smoothly. In contrast, phenol treatment had no effect on polar-flagella-defective mutants, suggesting that lateral flagella can adapt to repellent stimulus.

Mutations in the C-terminal of the Salmonella typhimurium fliK gene produce the polyhook phenotype in which there is no hook assembly termination and filament assembly initiation. The defect in filament assembly is removed by extragenic suppressors in the flaB gene. Small deletions in the FliK N-terminal and central regions produce normal hook length control and wild-type swarming. Larger deletions result in loss of hook length control and poor swarming. FliK probably measures the hook length and at a specific length signals FlhB to export flagellin instead of hook proteins.

The flagellar hook length control protein FliK of Salmonella typhimurium and Escherichia coli have molecular masses of 41,748 and 39,246 Da respectively, and are fairly hydrophilic in nature. The proteins have a C-terminal and a central domain that have a high level of proline and glutamine amino acids, and a N-terminal. The proteins are 50% identical and the C-terminal region is the most conserved. Amino acid sequence analysis shows that the FliK protein is unlikely to be the molecular-ruler for determining the hook length.

The motY gene encoding a component of the sodium-driven polar flagellar motor in Vibrio alginolyticus was cloned. Nucleotide sequence analysis indicated that the gene encodes a 293-amino-acid polypeptide with a single putative transmembrane segment that is 94% identical to the MotY of V. parahaemolyticus. Their C-terminal domains were similar to the C-terminal domains of many peptidoglycan-interacting proteins. Findings were consistent with the hypothesis that MotYis a component of the force-generating unit.

The mutant receptor Tsr-T156C retains serine and repellent-sensing abilities when Cys in Escherichia coli chemoreceptor Tsr is substituted by Thr-156, but loses its serine-sensing abilities when treated in vivo with sulfhydryl modifying reagents like N-ethylmaleimide (NEM). The NEM is bound to Tsr-T156C and the binding decreases in serine's presence. Chemical modification of the Tsr ligand-binding site provides a basis for purification and further helps in vivo and vitro investigations of the chemoreceptor protein.

Application of osmotic shock to the bacterial cells of Vibrio alginolyticus results in the elimination of DNase from the periplasmic region, leading to a 10-fold rise in bacterial cell transformation during electroporation. Washing of the cells with 5-10 mM Mg2+ contributes to the 10-fold increase in transformation. Optimum DNA transformation is seen during the early stationary phase entry of the cells and in the presence of 5.0 to 7. 5 kV per cm electric field strength during electroporation.

The Salmonella typhimurium FliI protein was examined genetically and biochemically. Site-directed mutagenesis of important residues in the F1 beta subunit resulted in loss of flagellation, indicating that FliI is an ATPase involved in flagellar assembly. The FliI protein was overexpressed and purified. It demonstrated ATP binding but not hydrolysis. The number of copies of FliI in wild-type cells was estimated to be about 1,500. Moreover, the various flagellar proteins (FliI, FlhA and FliP) and their homologs are components of a superfamily of protein export systems.

The cloning and sequencing of an Escherichia coli gene encoding an alpha-amylase were reported. A homologous gene was found to exist in Salmonella typhimurium. Minicell experiments identified the product of the E. coli gene, named amyA, as a 56-kDa protein, and the sequence of AmyA showed that it is a cytoplasmic enzyme. Moreover, the results established that AmyA is an alpha-amylase and that the linear alpha-glucan amylose is the most effective substrate.

The flagellar gene systems in Escherichia coli and Salmonella typhimurium are arranged in three major clusters, the flagellar regions I, II and III. Analysis of region III showed that there is a new region between fliD and fliE, most of which can be deleted without visible effects on flagellar activity. However, further analyses showed that this region also contains two genes which appear to be involved in flagellation. Therefore, region III has been subdivided into regions IIIa containing fliABCD and IIIb, containing fliEFGHIJKLMNOPQR. The two newly described genes have been designated fliS and fliT.

Escherichia coli and Salmonella typhimurium flagellar systems are encoded by at least 40 genes presently grouped into regions I, II, IIIa, and IIIb. One of the genes, fliE, which lies at the start of region IIIb, is a monocistronic operon oriented divergently from the large fliF operon. DNA sequencing showed that its product, FliE, is part of the flagellar hook-basal body. Nine subunits of the FliE proteins was estimated to be part of one basal body. Its functional role remains to be elucidated.