PubMed Health⌕ Search

Biomedical subjects

R M Macnab

Publications and source records attributed to R M Macnab.

At least 91 records · Page 5Linked to original sources
PubMed1986

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.

Amino Acid Sequence↗
PubMed1986

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.

Chemotaxis↗
PubMed1985

Purification and characterization of the flagellar hook-basal body complex of Salmonella typhimurium.

The hook-basal body complex of Salmonella typhimurium, a major component of its flagellar apparatus, was subjected to detailed analysis by electron microscopy and gel electrophoresis. The study was facilitated by the development of an improved protocol for isolation of the complexes in high yield and purity. Nine proteins were identified with the structure. These proteins had apparent molecular weights of 65,000 (65K), 60K, 42K, 38K, 32K, 30K, 27K, 16K, and 14K. Small but reproducible shifts in the apparent molecular weights of specific proteins from conditionally nonflagellate mutants indicated the following gene-polypeptide correspondences: flaFV, 42K; flaFVI, 32K; flaFVII, 30K; flaFIX, 38K; flaAII.1, 65K. Several new morphological features of hook-basal body complexes were recognized, including a clawlike structure on the cytoplasm-proximal M ring and additional material at the cytoplasmic face of the M ring. Based on this study and the work of others, we suggest that the morphological features of the hook-basal body complex correspond to the following proteins: hook-filament junction, 60K; hook, 42K; rod, 30K and 32K; L ring and outer cylinder wall, 27K; P ring, 38K; S ring, unknown; M ring 65K.

Bacterial Outer Membrane Proteins↗
PubMed1984

Gene sequence and predicted amino acid sequence of the motA protein, a membrane-associated protein required for flagellar rotation in Escherichia coli.

The motA and motB gene products of Escherichia coli are integral membrane proteins necessary for flagellar rotation. We determined the DNA sequence of the region containing the motA gene and its promoter. Within this sequence, there is an open reading frame of 885 nucleotides, which with high probability (98% confidence level) meets criteria for a coding sequence. The 295-residue amino acid translation product had a molecular weight of 31,974, in good agreement with the value determined experimentally by gel electrophoresis. The amino acid sequence, which was quite hydrophobic, was subjected to a theoretical analysis designed to predict membrane-spanning alpha-helical segments of integral membrane proteins; four such hydrophobic helices were predicted by this treatment. Additional amphipathic helices may also be present. A remarkable feature of the sequence is the existence of two segments of high uncompensated charge density, one positive and the other negative. Possible organization of the protein in the membrane is discussed. Asymmetry in the amino acid composition of translated DNA sequences was used to distinguish between two possible initiation codons. The use of this method as a criterion for authentication of coding regions is described briefly in an Appendix.

Amino Acid Sequence↗
PubMed1983

Asynchronous switching of flagellar motors on a single bacterial cell.

Salmonella possesses several flagella, each capable of counterclockwise and clockwise rotation. Counterclockwise rotation produces swimming, clockwise rotation produces tumbling. Switching between senses occurs stochastically. The rotational sense of individual flagella on a single cell could be monitored under special conditions (partially de-energized cells of cheC and cheZ mutants). Switching was totally asynchronous, indicating that the stochastic process operates at the level of the individual organelle. Coordinated rotation in the flagellar bundle during swimming may therefore derive simply from a high counterclockwise probability enhanced by mechanical interactions, and not from a synchronizing switch mechanism. Different flagella on a given cell had different switching probabilities, on a time scale (greater than 2 min) spanning many switching events. This heterogeneity may reflect permanent structural differences, or slow fluctuations in some regulatory process.

Bacterial Proteins↗
PubMed1983

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.

Energy Transfer↗
PubMed1982

Effects of pH and repellent tactic stimuli on protein methylation levels in Escherichia coli.

Intracellular pH (pH(int)) and extracellular pH (pH(ext)) of Escherichia coli were measured at 12-s time resolution by (31)P-nuclear magnetic resonance: a sudden neutral-to-acid shift in pH(ext) (e.g., from 7.0 to 5.6) caused a transient failure of homeostasis, with pH(int) decreasing by about 0.4 unit in ca. 30 s and then returning to its original value (ca. 7.5) over a period of several minutes. Membrane proton conductance was estimated to be 20 pmol s(-1) cm(-2) pH unit(-1). Addition of the membrane-permeant weak acid benzoate at constant pH(ext) also caused a lowering of pH(int); at high concentrations it generated an inverted transmembrane pH gradient (DeltapH). The buffering capacity of the cells was estimated by such experiments to be ca. 50 mM per pH unit. Effects of pH-related stimuli on the methyl-accepting chemotaxis proteins (MCPs) were examined: the steady-state methylation of MCP I was found to decrease when pH(int) was lowered by weak acid addition or when pH(ext) was lowered. The extent of demethylation in the latter case was too great to be explained by imperfect steady-state homeostasis; a small but reproducible undershoot in methylation level correlated with the observed short-term homeostatic failure. MCP II underwent smaller and more complex changes than MCP I, in response to pH-related stimuli. The methylation level of MCP I could not, by any condition tested, be driven below a limit of ca. 15% of the control level (unstimulated cells at pH(ext) 7.0). The weak-acid concentration needed to reach that limit was dependent on pH(ext), as would be expected on the basis of DeltapH-driven concentrative effects. The potency ranking of weak acids was the same with respect to lowering pH(int), demethylating MCP I, and causing repellent behavioral responses. The data are consistent with a model whereby MCP I and hence tactic behavior are sensitive to both pH(int) and pH(ext). Evidence is presented that pH(int) may also have a direct (non-MCP-related) effect on motor function. Comparison of methyl-(3)H- and (35)S-labeled MCP I revealed that in both unstimulated and repellent-stimulated cells the major species did not carry methyl label, yet it had an electrophoretic mobility that indicated that it was more positively charged than the unmethylated form observed in methyltransferase mutants, and it was susceptible to base hydrolysis. This suggests that a substantial fraction of MCP I molecules is methylated or otherwise modified but neither exchanges methyl label nor undergoes reverse modification by repellent stimuli.

Acetates↗
PubMed1981

pH homeostasis in Escherichia coli: measurement by 31P nuclear magnetic resonance of methylphosphonate and phosphate.

The intracellular pH of Escherichia coli cells, respiring on endogenous energy sources, was monitored continuously by 31P NMR over an extracellular pH range between 5.5 and 9. pH homeostasis was found to be good over the entire range, with the data conforming to the simple relationship intracellular pH = 7.6 + 0.1(external pH - 7.6) so that the extreme values observed for intracellular pH were 7.4 and 7.8 at external pH 5.5 and 9, respectively. As well as inorganic phosphate, we employed the pH-sensitive NMR probe methylphosphonate, which was taken up by glycerol-grown cells and was nontoxic; its pKa of 7.65 made it an ideal probe for measurement of cytoplasmic pH and alkaline external pH.

Cell Membrane↗
PubMed1981

Cytoplasmic pH mediates pH taxis and weak-acid repellent taxis of bacteria.

Bacteria migrate away from an acid pH and from a number of chemicals, including organic acids such as acetate; the basis for detection of these environmental cues has not been demonstrated. Membrane-permeant weak acids caused prolonged tumbling when added to Salmonella sp. or Escherichia coli cells at pH 5.5. Tethered Salmonella cells went from a prestimulus behavior of 14% clockwise rotation to 80% clockwise rotation when 40 mM acetate was added and remained this way for more than 30 min. A low external pH in the absence of weak acid did not markedly affect steady-state tumbling frequency. Among the weak acids tested, the rank for acidity (salicylate greater than benzoate greater than acetate greater than 5,5-dimethyl-2,4-oxazolidinedione) was the same as the rank for the ability to collapse the transmembrane pH gradient and to cause tumbling. At pH 7.0, the tumbling responses caused by the weak acids were much briefer. Indole, a non-weak-acid repellent, did not cause prolonged tumbling at low pH. Two chemotaxis mutants (a Salmonella mutant defective in the chemotaxis methylesterase and an E. coli mutant defective in the methyl-accepting protein in MCP I) showed inverse responses of enhanced counterclockwise rotation in the first 1 min after acetate addition. The latter mutant had been found previously to be defective in the sensing of gradients of extracellular pH and (at neutral pH) of acetate. We conclude (i) that taxes away from acid pH and membrane-permeant weak acids are both mediated by a pH-sensitive component located either in the cytoplasm or on the cytoplasmic side of the membrane, rather than by an external receptor (as in the case of the attractants), and (ii) that both of these taxes involve components of the chemotaxis methylation system, at least in the early phase of the response.

Acetates↗
PubMed1981

Effects of lipophilic cations on motility and other physiological properties of Bacillus subtilis.

Lipophilic cations (tetraphenylarsonium, tetraphenylphosphonium, and triphenylmethylphosphonium) caused a number of major changes in the physiology of Bacillus subtilis. Macromolecular synthesis was inhibited, adenosine 5'-triphosphate concentration increased, swimming speed was reduced, tumbling was suppressed, and the capacity to take up the cations was greatly enhanced; respiration was not significantly altered. The effects occurred at lipophilic cation concentrations in the range commonly employed for measurement of membrane potential. Neither the enhancement of cation uptake nor the motility inhibition was a consequence of alteration of membrane potential, since both effects were still seen in the presence of valinomycin, with the extent of 86Rb+ uptake indicating a constant potential. Because suppression of tumbling accompanied speed reduction, as has also been found when protonmotive force is reduced, it is likely that lipophilic cations are perturbing the process of conversion of proton energy into work, rather than simply causing structural damage.

Adenosine Triphosphate↗
PubMed1979

Chemotaxis of Salmonella typhimurium toward citrate.

Salmonella, but not Escherichia coli, was attracted to citrate, a distinction that is understandable in view of the inability of E. coli to transport tricarboxylic acids. The Salmonella response to citrate and to two previously described attractants, aspartate and malate, was mutually noncompetitive. Citrate taxis different from citrate uptake in that it did not require Na+, was constitutive, and was not repressible by glucose.

Aspartic Acid↗
PubMed1978

Inversion of a behavioral response in bacterial chemotaxis: explanation at the molecular level.

Certain cheU mutants of Salmonella show inverted chemotactic behavior, being repelled by attractants and attracted by repellents. Such a dramatic change in behavioral pattern would seem at first glance to require drastic and complex alterations in the sensory processing system. In fact, the behavior can be explained by a simple shift in the level of a response regulator and the subtle effects of this shift on flagellar function. Flagella can exist in either a left-handed or a right-handed structure depending on applied torsion. Wild-type cells swim smoothly by counterclockwise rotation of a left-handed helical bundle and tumble when the motors briefly reverse to clockwise rotation (normal random motility). The cheU mutation causes a shift in response regulator level relative to the critical threshold value, resulting in extended clockwise operation so that the flagella are fully converted to the right-handed helical form. These cells therefore swim smoothly by clockwise rotation of a right-handed bundle and tumble when the motor briefly reverses to counterclockwise rotation (inverse random motility). Thus, tumbling is associated with brief reversals and not with a particular sense of rotation. A wild-type cell, with its steady-state response regulator level placing it initially in normal random motility, will swim smoothly on addition of attractant, whereas a cheU mutant with inverse random motility will tumble given the same stimulus. The phenomenon illustrates the profound behavioral consequences that can result from a single mutation in a key gene.

Chemotaxis↗