Genetic evidence for a switching and energy-transducing complex in the flagellar motor of Salmonella typhimurium.

The flaAII.2, flaQ, and flaN genes of Salmonella typhimurium are important for assembly, rotation, and counterclockwise-clockwise switching of the flagellar motor. Paralyzed and nonchemotactic mutants were subjected to selection pressure for partial acquisition of motility and chemotaxis, and the suppressor mutations of the resulting pseudorevertants were mapped and isolated. Many of the intergenic suppressor mutations were in one of the other two genes. Others were in genes for cytoplasmic components of the chemotaxis system, notably cheY and cheZ; one of the mutations was found in the cheA gene and one in a motility gene, motB. Suppression among the three fla genes was allele specific, and many of the pseudorevertants were either cold sensitive or heat sensitive. We conclude that the FlaAII.2, FlaQ, and FlaN proteins form a complex which determines the rotational sense, either counterclockwise or clockwise, of the motor and also participates in the conversion of proton energy into mechanical work of rotation. This switch complex is probably mounted to the base of the flagellar basal body and, via binding of the CheY and CheZ proteins, receives sensory information and uses it to control flagellar operation.     

Transductional analysis of the flagellar genes in Pseudomonas aeruginosa.

Complementation in bacteriophage E79 tv-l-mediated transduction and the phenotypic properties of the flagellar genes in Pseudomonas aeruginosa PAO were investigated by using 195 flagellar mutants of this organism. A total of 15 fla. 1 mot, and 2 che cistrons were identified. At least 5 fla cistrons (fla V to flaZ) and one mot cistron resided in one region, and at least 10 fla cistrons (flaA to flaJ) and two che cistrons (cheA and cheB) resided in another. The flaC mutants exhibited cistron-specific leakiness on motility agar plates. The flaE cistron may be the structural gene for the component protein of the flagellar filament. The cheA mutations, which resulted in pleiotropic phenotypes for flagellar formation, motility, and taxis, belonged to the same complementation group as the flaF mutations; that is, we inferred that cheA and flaF are synonymous.     

Mutant MotB proteins in Escherichia coli.

The MotB protein of Escherichia coli is an essential component in each of eight torque generators in the flagellar rotary motor. Based on its membrane topology, it has been suggested that MotB might be a linker that fastens the torque-generating machinery to the cell wall. Here, we report the isolation and characterization of a number of motB mutants. As found previously for motA, many alleles of motB were dominant, as expected if MotB is a component of the motor. In other respects, however, the motB mutants differed from the motA mutants. Most of the mutations mapped to a hydrophilic, periplasmic domain of the protein, and nothing comparable to the slow-swimming alleles of motA, which show normal torque when tethered, was found. Some motB mutants retained partial function, but when tethered they produced subnormal torque, indicating that their motors contained only one or two functional torque generators. These results support the hypothesis that MotB is a linker.     

Nucleotide sequence of the Escherichia coli motB gene and site-limited incorporation of its product into the cytoplasmic membrane.

The motB gene product of Escherichia coli is an integral membrane protein required for rotation of the flagellar motor. We have determined the nucleotide sequence of the motB region and find that it contains an open reading frame of 924 nucleotides which we ascribe to the motB gene. The predicted amino acid sequence of the gene product is 308 residues long and indicates an amphipathic protein with one major hydrophobic region, about 22 residues long, near the N terminus. There is no consensus signal sequence. We postulate that the protein has a short N-terminal region in the cytoplasm, an anchoring region in the membrane consisting of two spanning segments, and a large cytoplasmic C-terminal domain. By placing motB under control of the tryptophan operon promoter of Serratia marcescens, we have succeeded in overproducing the MotB protein. Under these conditions, the majority of MotB was found in the cytoplasm, indicating that the membrane has a limited capacity to incorporate the protein. We conclude that insertion of MotB into the membrane requires the presence of other more hydrophobic components, possibly including the MotA protein or other components of the flagellar motor. The results further reinforce the concept that the total flagellar motor consists of more than just the basal body.     

An extreme clockwise switch bias mutation in fliG of Salmonella typhimurium and its suppression by slow-motile mutations in motA and motB.

Pseudorevertants (second-site suppressor mutants) were isolated from a set of parental mutants of Salmonella with defects in the flagellar switch genes fliG and fliM. Most of the suppressing mutations lay in flagellar region IIIb of the chromosome. One fliG mutant, SJW2811, gave rise to a large number of suppressor mutations in the motility genes motA and motB, which are in flagellar region II. SJW2811, which has a three-amino-acid deletion (delta Pro-Ala-Ala) at positions 169 to 171 of FliG, had an extreme clockwise motor bias that produced inverse smooth swimming (i.e., swimming by means of clockwise rotation of a hydrodynamically induced right-handed helical bundle), and formed Mot(-)-like colonies on semisolid medium. Unlike previously reported inverse-swimming mutants, it did not show a chemotactic response to serine, and it remained inverse even in a delta che background; thus, its switch is locked in the clockwise state. The location of the mutation further underscores the conclusion from a previous study of spontaneous missense mutants (V. M. Irikura, M. Kihara, S. Yamaguchi, H. Sockett, and R. M. Macnab, J. Bacteriol. 175:802-810, 1993) that a relatively localized region in the central part of the FliG sequence is critically important for switching. All of the second-site mutations in motA and motB caused some impairment of motility, both in the pseudorevertants and in a wild-type fliG background. The mechanism of suppression of the fliG mutation by the mot mutations is complex, involving destabilization of the right-handed flagellar bundle as a result of reduced motor speed. The mutations in the MotA and MotB sequences were clustered to a considerable degree as follows: in transmembrane helices 3 and 4 of MotA and the sole transmembrane helix of MotB, at helix-membrane interfaces, in the cytoplasmic domains of MotA, and in the vicinity of the peptidoglycan binding region of the periplasmic domain of MotB. The potential importance of Lys28 and Asp33 of the MotB sequence for proton delivery to the site of torque generation is discussed.     

Three genes of a motility operon and their role in flagellar rotary speed variation in Rhizobium meliloti.

The peritrichous flagella of Rhizobium meliloti rotate only clockwise and control directional changes of swimming cells by modulating flagellar rotary speed. Using Tn5 insertions, we have identified and sequenced a motility (mot) operon containing three genes, motB, motC, and motD, that are translationally coupled. The motB gene (and an unlinked motA) has been assigned by similarity to the Escherichia coli and Bacillus subtilis homologs, whereas motC and motD are new and without known precedents in other bacteria. In-frame deletions introduced in motB, motC, or motD each result in paralysis. MotD function was fully restored by complementation with the wild-type motD gene. By contrast, deletions in motB or motC required the native combination of motB and motC in trans for restoring normal flagellar rotation, whereas complementation with motB or motC alone led to uncoordinated (jiggly) swimming. Similarly, a motB-motC gene fusion and a Tn5 insertion intervening between motB and motC resulted in jiggly swimming as a consequence of large fluctuations in flagellar rotary speed. We conclude that MotC biosynthesis requires coordinate expression of motB and motC and balanced amounts of the two gene products. The MotC polypeptide contains an N-terminal signal sequence for export, and Western blots have confirmed its location in the periplasm of the R. meliloti cell. A working model suggests that interactions between MotB and MotC at the periplasmic surface of the motor control the energy flux or the energy coupling that drives flagellar rotation.     

Analysis of the motA flagellar motor gene from Rhodobacter sphaeroides a bacterium with a unidirectional, stop-start flagellum

Rhodobacter sphaeroides swims by unidirectional rotation of a single medial flagellum, re-orienting randomly by Brownian motion when flagellar rotation tops and restarts. Previously we identified a mutant with a paralysed flagellum, which was complemented by a Rhodobacter gene that had homology to motB of Escherichia coli , a bacterium with bidirectional flagella. In the current work, interposon mutagenesis upstream of the Rhodobacter motB gene gave rise to another paralysed mutant, RED5. DNA sequence analysis of this upstream region showed one open reading frame, the predicted polypeptide sequence of which shows homology to the MotA protein of E. coli . MotA is thought to be a proton 'pore' involved in converting proton-motive force into flagellar rotation. Several potential proton-binding amino acids were conserved between putative membrane-spanning regions of R. sphaeroides and E. coli MotA sequences, along with a highly charged cytoplasmic linker region. Complementation studies with mutant RED5 showed the presence of an active promoter upstream from motA which was found to be necessary for expression of both motA and motB , Examination of the upstream DNA sequence showed only one putative promoter-like sequence which resembled a σ⁵⁴- type promoter, including a potential enhancer binding site. The overall similarities between the R. sphaeroides MotA protein and those from other bacteria suggest that, despite the novel unidirectional rotation of he R. sphaeroides flagellum, the function of the MotA protein is similar to that in bacteria with bidirectional flagella.     

Bacteriophage lambda mutants (lambdatp) that overproduce repressor.

Lambda tp mutants, selected for their ability to form turbid plaques on lon hosts, overproduce repressor. The tp1 and tp2 mutations have been located within (or adjacent to) the cIII gene. The tp1 mutation reduced late gene expression, as measured by endolysin synthesis (in the absence of functional cI repressor) and progeny phage yield. The tp4 mutation was mapped in the cY-cII region, and complementation tests indicated that tp4 affects the diffusible product of the cII gene. The tp4 mutation also reduced progeny production, but did not markedly affect endolysin synthesis.     

Roles of cheY and cheZ gene products in controlling flagellar rotation in bacterial chemotaxis of Escherichia coli.

To understand output control in bacterial chemotaxis, we varied the levels of expression of cellular cheY and cheZ genes and found that the overproduction of the corresponding proteins affected Escherichia coli swimming behavior. In the absence of other signal-transducing gene products, CheY overproduction made free-swimming cells tumble more frequently. A plot of the fraction of the population that are tumbling versus the CheY concentration was hyperbolic, with half of the population tumbling at 30 microM (25,000 copies per cell) CheY monomers in the cytosol. Overproduction of aspartate receptor (Tar) by 30-fold had a negligible effect on CheY-induced tumbling, so Tar does not sequester CheY. CheZ overproduction decreased tumbling in all tumbling mutants except certain flaAII(cheC) mutants. In the absence of other chemotaxis gene products, CheZ overproduction inhibited CheY-induced tumbling. Models for CheY as a tumbling signal and CheZ as a smooth-swimming signal to control flagellar rotation are discussed.     

flaAII (motC, cheV) of Salmonella typhimurium is a structural gene involved in energization and switching of the flagellar motor.

The flaAII gene of Salmonella typhimurium has also been termed motC and cheV, because defective alleles may give rise to a nonflagellate, paralyzed, or nonchemotactic phenotype. We isolated a temperature-sensitive motility mutant (MY1) and have found that the mutation occurs in the flaAII gene. In temperature-jump experiments, MY1 could be converted from highly motile to paralyzed within 0.5 s, demonstrating that flaAII is a structural gene whose product is immediately essential for motor rotation. The mutant, although chemotactic at permissive temperatures (less than 36 degrees C), had a higher clockwise rotational bias than did the wild type; it can therefore be regarded simultaneously as motC(Ts) and cheV (tumbly). The only previously reported S. typhimurium cheV mutant was smooth-swimming. A shift toward counterclockwise bias accompanied loss of rotational speed in the restrictive temperature range. This result, by analogy with known proton motive force effects on motor switching, further indicates a central role of the flaAII (motC, cheV) protein in the energy transduction and switching process. Since there is no evidence associating it with the isolable entity known as the basal body, it may reside at the cytoplasmic face of the flagellar motor.     

Chemotactic mechanism of Salmonella typhimurium: preliminary mapping and characterization of mutants.

Some new, generally nonchemotactic mutants of Salmonella typhimurium were isolated and they, together with previously isolated mutants and some from other investigators, were mapped. Most of the mutants were classified in nine complementation groups, which are probably individual genes. Of these, five map at the end of the flagella region and appear in the order motB-(cheWcheP)-cheX-cheQ-cheR-flaC. Two of the mutations, cheU and cheV, map in the flaQ and flaAII genes, respectively. The remaining genes, cheS and cheT, have not yet been mapped. Most of the mutants are phenotypically smoothly swimming, but some are constantly tumbling. Two of the groups show dominant behavior as recipients in genetic crosses; the rest are recessive. The mutants vary in their responses to stimuli but, since their responses to all chemoeffectors are abnormal, the central processing, rather than individual, receptors must be impaired. The two mutations that coincide with genes for flagella probably involve the locus of the final delivery of sensing signal to the flagella.     

The complex flagellar torque generator of Pseudomonas aeruginosa

Flagella act as semirigid helical propellers that are powered by reversible rotary motors. Two membrane proteins, MotA and MotB, function as a complex that acts as the stator and generates the torque that drives rotation. The genome sequence of Pseudomonas aeruginosa PAO1 contains dual sets of motA and motB genes, PA1460-PA1461 (motAB) and PA4954-PA4953 (motCD), as well as another gene, motY (PA3526), which is known to be required for motor function in some bacteria. Here, we show that these five genes contribute to motility. Loss of function of either motAB-like locus was dispensable for translocation in aqueous environments. However, swimming could be entirely eliminated by introduction of combinations of mutations in the two motAB-encoding regions. Mutation of both genes encoding the MotA homologs or MotB homologs was sufficient to abolish motility. Mutants carrying double mutations in nonequivalent genes (i.e., motA motD or motB motC) retained motility, indicating that noncognate components can function together. motY appears to be required for motAB function. The combination of motY and motCD mutations rendered the cells nonmotile. Loss of function of motAB, motY, or motAB motY produced similar phenotypes; although the swimming speed was only reduced to approximately 85% of the wild-type speed, translocation in semisolid motility agar and swarming on the surface of solidified agar were severely impeded. Thus, the flagellar motor of P. aeruginosa represents a more complex configuration than the configuration that has been studied in other bacteria, and it enables efficient movement under different circumstances.     

cheA, cheB, and cheC genes of Escherichia coli and their role in chemotaxis.

Motile but generally nonchemotatic (che) mutants of Escherichia coli were isolated by a simple screening method. A total of 172 independent mutants were examined, and four genes were defined on the basis of mapping and complemenvestigated by determining their null phenotypes with nonsense or bacteriophage Mu-induced mutations. The cheA and cheB products were essential in producing changes of swimming direction and flagellar rotation. The checC product appeared to be an essential component of the flagellum; however, specific mutational alterations of this component allowed flagellar assembly but prevented directional changes in swimming. Since some cheB mutants changed directions incessantly, this gene product may also serve to control the direction of flagellar rotation in response to chemoreceptor signals. Thus most or all of the common elements in the signalling process were involved in the generation and regulation of changes in the direction of flagellar rotation.     

MotD of Sinorhizobium meliloti and related alpha-proteobacteria is the flagellar-hook-length regulator and therefore reassigned as FliK

The flagella of the soil bacterium Sinorhizobium meliloti differ from the enterobacterial paradigm in the complex filament structure and modulation of the flagellar rotary speed. The mode of motility control in S. meliloti has a molecular corollary in two novel periplasmic motility proteins, MotC and MotE, that are present in addition to the ubiquitous MotA/MotB energizing proton channel. A fifth motility gene is located in the mot operon downstream of the motB and motC genes. Its gene product was originally designated MotD, a cytoplasmic motility protein having an unknown function. We report here reassignment of MotD as FliK, the regulator of flagellar hook length. The FliK gene is one of the few flagellar genes not annotated in the contiguous flagellar regulon of S. meliloti. Characteristic for its class, the 475-residue FliK protein contains a conserved, compactly folded Flg hook domain in its carboxy-terminal region. Deletion of fliK leads to formation of prolonged flagellar hooks (polyhooks) with missing filament structures. Extragenic suppressor mutations all mapped in the cytoplasmic region of the transmembrane export protein FlhB and restored assembly of a flagellar filament, and thus motility, in the presence of polyhooks. The structural properties of FliK are consistent with its function as a substrate specificity switch of the flagellar export apparatus for switching from rod/hook-type substrates to filament-type substrates.     

Synthesis of mot and che gene products of Escherichia coli programmed by hybrid ColE1 plasmids in minicells.

Hybrid Escherichia coli ColE1 plasmids carrying the genes for motility (mot) and chemotaxis (che) were transferred to a minicell-producing strain. The mot and che genes on the hybrid plasmid directed protein synthesis in minicells. Polypeptides synthesized in minicells were identical to the products of the motA, motB, cheA, cheW, cheM, cheX, cheB, cheY, and cheZ genes previously identified by using hybrid lambda and ultraviolet-irradiated host cells (Silverman and Simon, J. Bacteriol. 130:1317-1325, 1977), thus confirming these gene product assignments. The products of some che genes (cheA and cheM) appeared as more than one band on polyacrylamide gel electrophoresis, but analysis of partial peptide digests of these polypeptides suggested that the multiple forms were coded for by a single gene. Measurement of the physical length of the hybrid plasmids allowed an estimate of the amount of coding capacity of the cloned deoxyribonucleic acid, which was devoted to the synthesis of the mot and che gene products. These estimates were also consistent with the hypothesis that the multiple polypeptides corresponding to cheA and cheM were the products of single genes.     

CHE-3, a cytosolic dynein heavy chain, is required for sensory cilia structure and function in Caenorhabditis elegans

Forward genetic screens using novel assays of nematode chemotaxis to soluble compounds identified three independent transposon-insertion mutations in the gene encoding the Caenorhabditis elegans dynein heavy chain (DHC) 1b isoform. These disruptions were mapped and cloned using a newly developed PCR-based transposon display. The mutations were demonstrated to be allelic to the che-3 genetic locus. This isoform of dynein shows temporally and spatially restricted expression in ciliated sensory neurons, and mutants show progressive developmental defects of the chemosensory cilia. These results are consistent with a role for this motor protein in the process of intraflagellar transport; DHC 1b acts in concert with a number of other proteins to establish and maintain the structural integrity of the ciliated sensory endings in C. elegans.     

Suppressor analysis of the MotB(D33E) mutation to probe bacterial flagellar motor dynamics coupled with proton translocation

MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow with motor rotation. Asp-33 of Salmonella enterica serovar Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, the mechanism of energy coupling remains unknown. Here, we carried out genetic and motility analysis of a slowly motile motB(D33E) mutant and its pseudorevertants. We first confirmed that the poor motility of the motB(D33E) mutant is due to neither protein instability, mislocalization, nor impaired interaction with MotA. We isolated 17 pseudorevertants and identified the suppressor mutations in the transmembrane helices TM2 and TM3 of MotA and in TM and the periplasmic domain of MotB. The stall torque produced by the motB(D33E) mutant motor was about half of the wild-type level, while those for the pseudorevertants were recovered nearly to the wild-type levels. However, the high-speed rotations of the motors under low-load conditions were still significantly impaired, suggesting that the rate of proton translocation is still severely limited at high speed. These results suggest that the second-site mutations recover a torque generation step involving stator-rotor interactions coupled with protonation/deprotonation of Glu-33 but not maximum proton conductivity.