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.     

The MotA protein of E. coli is a proton-conducting component of the flagellar motor

A number of mutants of motA, a gene necessary for flagellar rotation in E. coli, were isolated and characterized. Many mutations were dominant, owing to competition between functional and nonfunctional MotA for a limited number of sites on the flagellar motor. A new class of mutant was discovered in which flagellar torque is normal at low speeds but reduced at high speeds. Hydrogen isotope effects on these mutants indicate that MotA catalyzes proton transfer. We confirmed an earlier observation that overproduction of MotA leads to accumulation of the protein in the cytoplasmic membrane and to significant decreases in growth rate. When nonfunctional mutant variants of MotA were overproduced instead, they accumulated in the cytoplasmic membrane, but growth was not impaired. These results also suggest that MotA conducts protons. This was confirmed by measuring the proton permeabilities of vesicles containing wild-type or mutant MotA proteins.     

Co-overproduction and localization of the Escherichia coli motility proteins motA and motB.

The motility genes motA and motB of Escherichia coli were placed under control of the Serratia marcescens trp promoter. After induction with beta-indoleacrylic acid, the levels of MotA and MotB rose over about a 3-h period, reaching plateau levels approximately 50-fold higher than wild-type levels. Both overproduced proteins inserted into the cytoplasmic membrane. Growth and motility were essentially normal, suggesting that although the motor is a proton-conducting device, MotA and MotB together do not constitute a major proton leak. Derivative plasmids which maintained an intact version of motB but had the motA coding region deleted in various ways were constructed. With these, the levels of MotB were much lower, reaching a peak within 30 min after induction and declining thereafter; pulse-chase measurements indicated that a contributing factor was MotB degradation. The low levels of MotB occurred even with an in-frame internal deletion of motA, whose translational initiation and termination sites were intact, suggesting that it is the MotA protein, rather than the process of MotA synthesis, that is important for MotB stability. Termination at the usual site of overlap with the start of motB (ATGA) was not an absolute requirement for MotB synthesis but did result in higher rates of synthesis than when translation of motA information terminated prematurely. Even in the total absence of MotA, the MotB that was synthesized was found exclusively in the cytoplasmic membrane fraction. In wild-type cells, MotA was estimated by immunoprecipitation to be in about fourfold excess over MotB; a previous estimate of 600 +/- 250 copies of MotA per cell then yielded an estimate of 150 +/- 70 copies of MotB per cell.     

Ion-coupling determinants of Na+-driven and H+-driven flagellar motors

The bacterial flagellar motor is a tiny molecular machine that uses a transmembrane flux of H(+) or Na(+) ions to drive flagellar rotation. In proton-driven motors, the membrane proteins MotA and MotB interact via their transmembrane regions to form a proton channel. The sodium-driven motors that power the polar flagellum of Vibrio species contain homologs of MotA and MotB, called PomA and PomB. They require the unique proteins MotX and MotY. In this study, we investigated how ion selectivity is determined in proton and sodium motors. We found that Escherichia coli MotA/B restore motility in DeltapomAB Vibrio alginolyticus. Most hypermotile segregants isolated from this weakly motile strain contain mutations in motB. We constructed proteins in which segments of MotB were fused to complementary portions of PomB. A chimera joining the N terminus of PomB to the periplasmic C terminus of MotB (PotB7(E)) functioned with PomA as the stator of a sodium motor, with or without MotX/Y. This stator (PomA/PotB7(E)) supported sodium-driven motility in motA or motB E.coli cells, and the swimming speed was even higher than with the original stator of E.coli MotA/B. We conclude that the cytoplasmic and transmembrane domains of PomA/B are sufficient for sodium-driven motility. However, MotA expressed with a B subunit containing the N terminus of MotB fused to the periplasmic domain of PomB (MomB7(E)) supported sodium-driven motility in a MotX/Y-dependent fashion. Thus, although the periplasmic domain of PomB is not necessary for sodium-driven motility in a PomA/B motor, it can convert a MotA/B proton motor into a sodium motor.     

Requirements for conversion of the Na(+)-driven flagellar motor of Vibrio cholerae to the H(+)-driven motor of Escherichia coli

Bacterial flagella are powered by a motor that converts a transmembrane electrochemical potential of either H(+) or Na(+) into mechanical work. In Escherichia coli, the MotA and MotB proteins form the stator and function in proton translocation, whereas the FliG protein is located on the rotor and is involved in flagellar assembly and torque generation. The sodium-driven polar flagella of Vibrio species contain homologs of MotA and MotB, called PomA and PomB, and also contain two other membrane proteins called MotX and MotY, which are essential for motor rotation and that might also function in ion conduction. Deletions in pomA, pomB, motX, or motY in Vibrio cholerae resulted in a nonmotile phenotype, whereas deletion of fliG gave a nonflagellate phenotype. fliG genes on plasmids complemented fliG-null strains of the parent species but not fliG-null strains of the other species. FliG-null strains were complemented by chimeric FliG proteins in which the C-terminal domain came from the other species, however, implying that the C-terminal part of FliG can function in conjunction with the ion-translocating components of either species. A V. cholerae strain deleted of pomA, pomB, motX, and motY became weakly motile when the E. coli motA and motB genes were introduced on a plasmid. Like E. coli, but unlike wild-type V. cholerae, motility of some V. cholerae strains containing the hybrid motor was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone under neutral as well as alkaline conditions but not by the sodium motor-specific inhibitor phenamil. We conclude that the E. coli proton motor components MotA and MotB can function in place of the motor proteins of V. cholerae and that the hybrid motors are driven by the proton motive force.     

Regulated underexpression of the FliM protein of Escherichia coli and evidence for a location in the flagellar motor distinct from the MotA/MotB torque generators.

The FliM protein of Escherichia coli is essential for the assembly and function of flagella. Here, we report the effects of controlled low-level expression of FliM in a fliM null strain. Disruption of the fliM gene abolishes flagellation. Underexpression of FliM causes cells to produce comparatively few flagella, and most flagella built are defective, producing subnormal average torque and fluctuating rapidly in speed. The results imply that in a normal flagellar motor, multiple molecules of FliM are present and can function independently to some degree. The speed fluctuations indicate that stable operation requires most, possibly all, of the normal complement of FliM. Thus, the FliM subunits are not as fully independent as the motility proteins MotA and MotB characterized in earlier work, suggesting that FliM occupies a location in the motor distinct from the MotA/MotB torque generators. Several mutations in fliM previously reported to cause flagellar paralysis in Salmonella typhimurium (H. Sockett, S. Yamaguchi, M. Kihara, V.M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992) were made and characterized in E. coli. These mutations did not cause flagellar paralysis in E. coli; their phenotypes were more complex and suggest that FliM is not directly involved in torque generation.     

Hybrid Motor with H+- and Na+-Driven Components Can Rotate Vibrio Polar Flagella by Using Sodium Ions

The bacterial flagellar motor is a molecular machine that converts ion flux across the membrane into flagellar rotation. The coupling ion is either a proton or a sodium ion. The polar flagellar motor of the marine bacterium Vibrio alginolyticus is driven by sodium ions, and the four protein components, PomA, PomB, MotX, and MotY, are essential for motor function. Among them, PomA and PomB are similar to MotA and MotB of the proton-driven motors, respectively. PomA shows greatest similarity to MotA of the photosynthetic bacterium Rhodobacter sphaeroides. MotA is composed of 253 amino acids, the same length as PomA, and 40% of its residues are identical to those of PomA. R. sphaeroides MotB has high similarity only to the transmembrane region of PomB. To examine whether the R. sphaeroides motor genes can function in place of the pomA and pomB genes of V. alginolyticus, we constructed plasmids including both motA and motB or motA alone and transformed them into missense and null pomA-paralyzed mutants of V. alginolyticus. The transformants from both strains showed restored motility, although the swimming speeds were low. On the other hand, pomB mutants were not restored to motility by any plasmid containing motA and/or motB. Next, we tested which ions (proton or sodium) coupled to the hybrid motor function. The motor did not work in sodium-free buffer and was inhibited by phenamil and amiloride, sodium motor-specific inhibitors, but not by a protonophore. Thus, we conclude that the proton motor component, MotA, of R. sphaeroides can generate torque by coupling with the sodium ion flux in place of PomA of V. alginolyticus.     

Genetic mapping of tyramine oxidase and arylsulfatase genes and their regulation in intergeneric hybrids of enteric bacteria.

The genes for arylsulfatase (atsA) and tyramine oxidase (tynA) have been mapped in Klebsiella aerogenes by P1 transduction. They are linked to gdhD and trp in the order atsA-tynA-gdhD-trp-pyrF. Complementation analysis using F' episomes from Escherichia coli suggested an analogous location of these genes in E. coli, although arylsulfatase activity was not detected in E. coli. P1 phage and F' episomes were used to create intergeneric hybrid strains of enteric bacteria by transfer of the ats and tyn genes between K. aerogenes, E. coli, and Salmonella typhimurium. Intergeneric transduction of the tynK gene from K. aerogenes to an E. coli restrictionless strain was one to two orders less frequent than that of the leuK gene. The tyramine oxidase of E. coli and S. typhimurium in regulatory activity resemble very closely the enzyme of K. aerogenes. The atsE gene from E. coli was expressed, and latent arylsulfatase protein was formed in K. aerogenes and S typhimurium. The results of tyramine oxidase and arylsulfatase synthesis in intergeneric hybrids of enteric bacteria suggest that the system for regulation of enzyme synthesis is conserved more than the structure or function of enzyme protein during evolution.     

Expression and Localization of Escherichia coli Alkaline Phosphatase Synthesized in Salmonella typhimurium Cytoplasm

The Escherichia coli structural gene for alkaline phosphatase was inserted into Salmonella typhimurium by episomal transfer in order to determine whether this enzyme would continue to be localized to the periplasmic space of the bacterium even though it was formed in a cell that does not synthesize alkaline phosphatase. The S. typhimurium heterogenote synthesized alkaline phosphatase under conditions identical to that observed with E. coli. This enzyme appeared to be identical to that synthesized by E. coli, and was quantitatively released from the bacterial cell by spheroplast formation with lysozyme. These results showed that localization is not a property unique to the E. coli cell and suggested that, in E. coli, enzyme location is related to the structure of the protein. Formation of alkaline phosphatase in the S. typhimurium heterogenote was repressed in cells growing in a medium with excess inorganic phosphate, even though only one of the three regulatory genes for this enzyme is on the episome. Thus, S. typhimurium can supply the products of the other two regulatory genes essential for repression even though this bacterium seems to lack the structural gene for alkaline phosphatase.     

araB Gene and nucleotide sequence of the araC gene of Erwinia carotovora.

The araB and araC genes of Erwinia carotovora were expressed in Escherichia coli and Salmonella typhimurium. The araB and araC genes in E. coli, E. carotovora, and S. typhimurium were transcribed in divergent directions. In E. carotovora, the araB and araC genes were separated by 3.5 kilobase pairs, whereas in E. coli and S. typhimurium they were separated by 147 base pairs. The nucleotide sequence of the E. carotovora araC gene was determined. The predicted sequence of AraC protein of E. carotovora was 18 and 29 amino acids longer than that of AraC protein of E. coli and S. typhimurium, respectively. The DNA sequence of the araC gene of E. carotovora was 58% homologous to that of E. coli and 59% homologous to that of S. typhimurium, with respect to the common region they share. The predicted amino acid sequence of AraC protein was 57% homologous to that of E. coli and 58% homologous to that of S. typhimurium. The 5' noncoding regions of the araB and araC genes of E. carotovora had little homology to either of the other two species.     

Chromosomal location and expression of the structural gene for major outer membrane protein Ia of Escherichia coli K-12 and of the homologous gene of Salmonella typhimurium.

The gene determining the structure of a major outer membrane protein of Escherichia coli, protein Ia, has been located between serC and pyrD, at the min 21 region of the linkage map. This is based on the isolation and characterization of E. coli-Salmonella typhimurium intergeneric hybrids as well as analyses of a mutation (ompF2) affecting the formation of protein Ia. When the serC region of the S. typhimurium chromosome was transduced by phage P1 into E. coli, two classes of transductants were obtained; one produced protein Ia like the parental strain of E. coli, whereas the other produced not protein Ia but a pair of outer membrane proteins structurally related to 35K protein, one of the major outer membrane proteins of S. typhimurium. Furthermore, a strain of S. typhimurium harboring an F' plasmid which carries the ompF region of the E. coli chromosome was found to produce a protein indistinguishable from protein Ia, beside the outer membrane proteins characteristic to the parental Salmonella strain. These results suggest that the structural genes for protein Ia (E. coli) and for 35K protein (S. typhimurium) are homologous to each other and are located at the ompF region of the respective chromosome. The bearing of these findings on the genetic control of protein Ia formation is discussed.     

Two outer membrane transport systems for vitamin B12 in Salmonella typhimurium.

The involvement of an outer membrane transport component for vitamin B12 uptake in Salmonella typhimurium, analogous to the btuB product in Escherichia coli, was investigated. Mutants of S. typhimurium selected for resistance to bacteriophage BF23 carried mutations at the btuB locus (butBS) (formerly called bfe, at the analogous map position as the E. coli homolog) and were defective in high-affinity vitamin B12 uptake. The cloned E. coli btuB gene (btuBE) hybridized to S. typhimurium genomic DNA and restored vitamin B12 transport activity to S. typhimurium btuBS mutants. An Mr-60,000 protein in the S. typhimurium outer membrane was repressed by growth with vitamin B12 and was eliminated in a btuBS mutant. The btuBS product thus appears to play the same role in vitamin B12 transport by S. typhimurium as does the E. coli btuBE product. A second vitamin B12 transport system that is not present in E. coli was found by cloning a fragment of S. typhimurium DNA that complemented btuB mutants for vitamin B12 utilization. In addition to this plasmid with a 6-kilobase insert of S. typhimurium DNA, vitamin B12 utilization by E. coli btuB strains required the btuC and btuD products, necessary for transport across the cytoplasmic membrane, but not the btuE or tonB product. The plasmid conferred low levels of vitamin B12-binding and energy-dependent transport activity but not susceptibility to phage BF23 or utilization of dicyanocobinamide. The cloned S. typhimurium DNA encoding this new transport system did not hybridize to the btuBE gene or to E. coli chromosomal DNA and therefore does not carry the S. typhimurium btuBS locus. Increased production of an Mr -84,000 polypeptide associated with the outer membrane was seen. The new locus appears to be carried on the large plasmid in most S. typhimurium strains. Thus S. typhimurium possesses both high- and low-affinity systems for uptake of cobalamins across the outer membrane.     

Characterization of the Salmonella typhimurium phoE gene and development of Salmonella-specific DNA probes.

In Escherichia coli K-12, the phoE gene, encoding a phosphate-limitation-inducible outer membrane pore protein (PhoE), is closely linked to the genes proA and proB. When the corresponding fragment of the Salmonella typhimurium chromosome was transferred to E. coli K-12 using an RP4::miniMu plasmid, pULB113, no production of S. typhimurium PhoE could be detected. Nevertheless, DNA hybridization studies revealed that the corresponding plasmid did contain S. typhimurium phoE. Production of S. typhimurium PhoE in E. coli was detected only after subcloning the gene in a multicopy vector. Nucleotide (nt) sequence analysis showed extensive homology of S. typhimurium phoE to the E. coli gene and suggested possible explanations for the low expression of S. typhimurium phoE in E. coli. In addition, the sequence information was used to develop Salmonella-specific DNA probes. Two oligodeoxyribonucleotides were synthesized based on nt sequences encoding the fifth and eighth cell-surface-exposed regions of PhoE. When used in polymerase chain reactions, these probes turned out to be specific, i.e., no crossreactions occurred with the non-Salmonella strains, whereas 132 out of 133 tested Salmonella strains were recognized.