JOURNAL OF BACTERIOLOGY, May 1977, p. 787-792 Copyright 0 1977 American Society for Microbiology
Vol. 130, No. 2 Printed in U.S.A.
Flagellar Formation in Escherichia coli Electron Transport Mutants J. BAR TANA,1 BARBARA J. HOWLETT,
AND
D. E. KOSHLAND, JR.*
Department of Biochemistry, University of California, Berkeley, California 94720
Received for publication 9 February 1977
Mutants of Escherichia coli lacking ubiquinone or heme have been tested for motility and found to be essentially immotile. The loss of motility is identified with the loss of flagellum synthesis. Motility of Escherichia coli was shown by Sherris et al. (17) and by Adler and Templeton (2) to be sustained either by oxidative phosphorylation or by substrate level phosphorylation. Larsen et al. have pointed out that the immediate energy source for motility is probably the high-energy intermediate of oxidative phosphorylation, not adenosine 5'-triphosphate (10). Thus, E. coli adenosine triphosphatase mutants were shown to be motile aerobically, but they became immotile anaerobically (10). Electron transport mutants ofE. coli that contain a functioning Mg-Ca adenosine triphosphatase are expected, therefore, to be motile under growth conditions that maintain substrate level phosphorylation. The present study describes the motility pattern observed aerobically with two types of electron transport mutants, due to ubi mutations, which are deficient in ubiquinone synthesis (7), and hemA mutants, which are defective in functional cytochrome synthesis (8, 16).
cient mutants were selected by analyzing the quinone content of single colonies grown on nutrient agar plates. Growth and incubations were carried out aerobically to mid-logarithmic phase ((Dmo 0.4 to 0.8). Ubiquinone and cytochrome mutants are unable to grow on succinate, and, therefore, succinate was used as a test for possible reversion of mutants. Quinone determination. Bacteria were grown to mid-log phase in nutrient broth, with additions as described, harvested, washed twice in 0.9% KCl, then washed in water, and lyophilized to dryness. A weighed sample of the bacterial powder (20 to 50 mg) was extracted three times successively in 5 ml of acetone at 65°C. The combined acetone extract was evaporated to dryness under N2, suspended in a small volume of petroleum ether (20 to 60°C)-ether (1:1), and was subjected to thin-layer chromatography in benzene. Commercial ubiquinone-10 (Rf, 0.21), ubiquinone-6 (R,, 0.28) and menaquinone-10 (R,, 0.58) served as markers. The ubiquinone spot (Rf, 0.13 to 0.30) and the menaquinone spot (Rf, 0.55 to 0.65) (visible as yellow bands) were scraped off the plates and were eluted three times successively in 2.5 ml of diethylether. The ether eluate was evaporated to dryness under N2. Ubiquinone was deterMATERIALS AND METHODS mined by the (275ox-275NaBH4 reduced) difference Chemicals and media. Ubiquinone-10, ubiqui- spectrum in ethanol (AE275 in ethanol, 12.4) (5). Menone-6, menaquinone-10 and 8-amino levulinic acid naquinone was determined by the (245NaBH4 re(8ALA) were obtained from Sigma Chemical Co. duced-245ox) difference spectrum in ethanol (A245 in Silic-AR CC-4 100- to 200-mesh silicic acid was ob- ethanol, 25.8) (6). tained from Mallinckrodt. Adsorbil-5-Prekotes thinMotility assay. Motility was measured by a modilayer plates were purchased from Applied Science fication of the tumble frequency assay described by Laboratories Inc. and Koshland (18). Bacteria were grown in Spudich H minimal medium was prepared according to 22 mM pyruvate in nutrient broth medium with Kaiser and Hogness (9). VB minimal medium was additions as described to mid-logarithmic phase, diprepared as described by Vogel and Bonner (21). VB- luted in VB-EDTA minimal medium to ca. 3 x 107 EDTA medium was VB minimal medium with 10-5 cells/ml, and equilibrated by shaking at 30°C for M ethylenediaminetetraacetate (EDTA) added. Nu- 15 min. Portions (0.9 ml) of these bacteria were trient broth medium consisted of 0.8% nutrient mixed rapidly with 0.1 ml of 50 mM -serine, a broth plus 100 mM potassium phosphate buffer, pH chemotactic attractant, causing the smooth swim7.0. ming of all motile bacteria (18). This suspension (8 Bacterial strains. The strains used are described ,ul) was delivered under a cover slip, which was in Table 1. Motile revertants of immotile mutants supported by two other cover slips, to yield a chamwere selected by swarming on semisolid tryptone ber of 30-/sm depth, and 1 min later a 0.8-s-exposure plates (1). Spontaneous revertants of quinone-defi- photograph (Kodak recording film 2475) was taken I Present address: by dark-field illumination with a stroboscopic, highDepartment of Biochemistry, Hebrew pressure xenon arc lamp (flashing rate, 5 Hz) as a University, Jerusalem, Israel. 787 -
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TABLE 1. Strains ofE. coli used Strain no.
Genetic description
Source
AB2154 AN66 AN661 AN662 AN59 AN592 AN151 AN1512 AN98 AN99 AN187 AN1871 AN1872 SAS x 76
ubi+ ubiD Quinone revertant of AN66 Motility revertant of AN66 ubiB Motility revertant of AN59 ubiG Motility revertant of AN151 men+ ubi+ menA menA ubiD Menaquinone revertant of AN187 Quinone revertant hemA
Cox et al. (4) Cox et al. (4) This study This study; motility revertant of AN66 Cox et al. (4) This study Stroobant et al. (19) This study Newton et al. (12) Newton et al. (12) Cox et al. (3) This study; spontaneous revertant of AN187 This study; spontaneous revertant of AN187 Sasarman et al. (16)
light source. The number of motile tracks, consisting of four successive bacterial images, and the number of immotile bacterial spots were counted. The percentage of motile bacteria was defined as: number of motile tracks x 100/number of motile tracks plus number of nonmotile spots. Differences in the measured percentage of motility of the same culture were usually in the range of ±5%. Characteristic photomicrographs are shown in Fig. 1. The qualitative terms "motile" and "immotile" are used on occasions when more than 60% or less than 5% ofthe bacteria are motile, respectively. Respiration measurements. Bacteria were grown to mid-log phase in nutrient broth with additions as described. The culture was harvested, washed once in 100 mM phosphate, pH 7.0, and suspended in H medium-22 mM glucose with additions as described. Respiration was followed in an oxygen electrode chamber (Yellow Springs Instruments Co., no. 5331), which was calibrated with the reduced nico tenamide adenine dinucleotide-phenazine methyl sulfonate catalase couple described by Robinson and Cooper (14). The bacterial density was estimated by measuring the optical density at 650 nm. Electron microscopy studies. Bacteria were grown in nutrient broth-22 mM pyruvate to mid-logarithmic phase. A drop of the culture was placed in a carbon-coated grid, drained, washed with distilled water, and then stained with 1% uranyl acetate for 40 s. The grid was loaded and examined in a Siemans electron microscope. The flagella emanating from 50 individual, nonoverlapping bacteria were counted.
tile, irrespective of the specific mutation concerned. The isogenic parents as well as spontaneous Ubi+ revertants are motile when grown under the same conditions as the mutants. Furthermore, revertants selected for motility from the immotile Ubi- mutants by swarming on semisolid agar plates were shown to revert concomitantly with the return of ubiquinone formation. It appears, therefore, that the phenotypic expression of motility and that of ubiquinone synthesis are strongly related. On the other hand, menaquinone is not related to motility (Table 3). A double mutant bearing ubiD menA is immotile; its Men+ revertant is immotile, whereas its Ubi+ revertant is motile. Motility of hemA mutants. Immotility observed with E. coli ubi mutants is also observed with mutants of E. coli bearing hemA, which are defective in the synthesis of 8ALA (16). E. coli SAS x 76 is immotile when grown aerobically in the absence of 8ALA. Under the same growth conditions and in the presence of 10-5 M of 8ALA, the mutant becomes motile (Table 4). The ubiquinone content is not affected by the hemA locus. A mutant of E. coli (1004A) bearing hemA, isolated by Haddock and Schairer (8), forms only the apocytochrome when grown in the absence of 8ALA but synthesizes the protoporphyrin in the presence of 8ALA under nongrowth RESULTS conditions. It was tempting, therefore, to follow Motility of ubiquinone and menaquinone the time course of motility attainment, as well mutants. The motility pattern of mutants in as respiration, upon the addition of 8ALA in the pathway of ubiquinone synthesis in E. coli the presence and absence of chloramphenicol. is shown in Table 2. The pathway starts with As shown in Fig. 2A, respiration is restored at chorismic acid, the common precursor of ubi- about 2 to 4 h of incubation with 8ALA, whereas quinone, and aromatic amino acids and mu- motility is restored at about 10 to 20 h. Respiratants in all the intermediate steps have been tion is still restored in the presence of chloramisolated and characterized by Gibson and asso- phenicol (Fig. 2B), but protein synthesis seems ciates (3, 4, 7, 12, 19). The Ubi- mutants (kindly to be obligatory for restoring motility. The late supplied by F. Gibson) all appear to be immo- decline in respiration, which was observed in
VOL. 130, 1977
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FIG. 1. Quantitative assay of motitity. Bacterial motility is measured by counting the tracks after exposure intervals with five stroboscopic flashes per second. Details are described in the text. Illustrative examples show bacteria swimming (shown by tracks) versus immotile or tumbling (bright splotches) bacteria. Illustrative examples show 3%, 21%, 53%, and 84% motility.
BAR TANA, HOWLETT, AND KOSHLAND
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TABLE 2. Motility of E. coli ubiquinone mutant8a Motility
Strain
Ubiquinone Menaquinone (nmolVmg [dry (nmollmg [dry wt]) wt])
0.26 0.71 Motile AB2154 0.17 0.05 Immotile AN66 0.19 0.70 Motile AN661 0.06 0.87 Motile AN662 0.10 0.01 Immotile AN59 0.03 0.47 Motile AN592 0.06 0.05 Immotile AN151 NDb 0.48 Motile AN1512 a Bacteria were grown in nutrient broth to midlog phase. Motility and quinone contents were determined as described in the text. b ND, Not detectable.
TABLz 3. Motility of E. coli menaquinone mutantsa Motility
Strain
Ubiquinone
Menaquinone
wt])
wt])
(nmolVmg [dry (nmoVmg [dry
J. BACTERIOL.
nificant reduction in flagellum number of ubi or hemA mutants was observed in correlation with their motility pattern. The percentage of motility is shown to be related to the percentage of bacteria that possess three or more flagella per bacterium. This agrees with the findings of Quadling and Stocker, who limited flagellum synthesis by environmental conditions (13).
DISCUSSION The lack of motility observed for mutants deficient in ubiquinone or heme synthesis is evidently ascribed to their failure to form adequate numbers of flagella. The observed correlation should then be analyzed in terms of flagellum synthesis and/or assembly and should not be deduced from the prevailing concept concerning the immediate energy source for bacterial motility. The obligatory role played by electron transport in flagellum synthesis (assembly) in E.
0.02 0.63 Motile AN98 NDb 0.94 Motile AN99 ND ND Immotile AN187 ND 0.41 Immotile AN1871 ND 0.89 AN1872 Motile a Bacteria were grown in nutrient broth to midlog phase. Motility and quinone contents were determined as described in the text. b ND, Not detectable.
TABLz 4. Characteristics of E. coli hemA mutanta Additions to broth b
Motility Immotile
Respiration ( 0
Ubiquinone
(nmol/mg (dry wt]) 0.74
0.76 100 Motile 10-5 M 8ALA a E. coli SAS x 76 was grown in nutrient broth-22 mM glucose to mid-log phase with addition as described. Motility, ubiquinone content, and respiration were determined as described in the text. -, No addition.
the presence of chloramphenicol, is probably due to turnover of the cytochrome apoprotein under these conditions. Neither respiration nor motility was reconstituted during the first 12 h of incubation in the absence of 8ALA, and reversion often occurred after this time. Flagellar patterns of ubi and hemA E. coli mutants. The time lag between respiration reconstitution and motility reconstitution observed with the mutant bearing hemA, as well as the differential effect of chloramphenicol upon the reconstitution of motility and respiration, has indicated a possible role of oxidative electron transport in the synthesis or assembly of bacterial flagella. As shown in Table 5, sig-
Time (hours) FIG. 2. Reconstitution of respiration and motility in E. coli hemA mutant. E. coli SAS x 76 was grown to mid-log phase in nutrient broth plus 22 mM glucose. The culture was haruested, washed once in 100 mM phosphate buffer, pH 7.0, and suspended at time zero in H medium-0.5% Casamino Acids-22 mMglucose-10-5 M MALA in the presence (B) and absence (A) of 30 pg of chloramphenicol per ml. Incubation was carried out at 30°C with continuous gyratory ) and motility (-----) were shaking. Respiration ( determined on portions at particular times. The respiration rate of E. coli SAS x 76 grown in the presence of 10-5 M 8ALA was taken to be 100%.
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TABLE 5. Distribution of flagella of E. coli strains useda Bacterial strain
1
Bacteria 2
(%) with flagellum no. indicated 3
4
5
6
7
Mean flagellum no.
Motility (%)
3 1 0 0 1.5 1.5 15 47 24 11 AN59 60 2.5 3.3 26 5 21 8 16 11 11 AN592 0 0 0 0 0 1.5 84 12 1.5 AN66 1.5 50 2.6 9 11 2 17 13 13 28 9 AN662 1 1 0 0.8 3 3 1.5 18 10 65 SAS x 76 75 28 6 2 3.5 16 18 2 12 16 SAS x 76 + 8ALA a Electron microscopy examination and motility assays were as described in the text. For each bacterial strain, about 50 individual bacteria were examined. The number of flagella emanating from each individual was recorded and counted.
coli pertains to aerobically as well as anaero- grant no. AM-09765 from the National Institute of Arthribically grown bacteria. Anaerobic growth leads tis, Metabolism and Digestive Diseases. to motile bacteria (as reported by Adler and LITERATURE CITED Templeton [2] and confirmed by us), but nonJ. 1966. Chemotaxis in bacteria. Science 153:708leaky mutants in anaerobic electron transport 1. Adler, 716. are devoid of flagellum synthesis when grown 2. Adler, J., and B. Templeton. 1967. The effect of environanaerobically (R. Hertz and J. Bar Tana, mental conditions on the motility of Escherichia coli. J. Gen. Microbiol. 46:175-184. manuscript in preparation). G. B., N. A. Newton, J. D. Butlin, and F. Gibson. The observed correlation between oxidative 3. Cox, 1971. The energy-linked transhydrogenase reaction electron transport and anaerobic flagellum synin respiratory mutants of Escherichia coli K12. Biothesis (assembly) may be analyzed in the light chem. J. 125:489-493. of the following considerations. The shift in 4. Cox, G. B., I. G. Young, L. M. McCann, and F. Gibson. 1969. Biosynthesis of ubiquinone in Escherichia coli metabolic pathways in the absence of oxidative K-12: location of genes affecting the metabolism of 3electron transport (23) may eliminate a crucial octaprenyl-4-hydroxybenzoic acid and 2-octaprenylintermediate in flagellum synthesis, or the rephenol. J. Bacteriol. 99:450-458. sulting reducing environment may inhibit 5. Crane, F. L., and R. Barr. 1971. Determination of ubiquinones, p. 137-165. In D. B. McCormick and L. some rate-limiting step in flagellum synthesis. D. Wright (ed.), Methods in enzymology, vol. 18, part The effect may be mediated through cyclic nuC. Academic Press Inc., New York. cleotide levels involved in catabolite repression 6. Dunphy, P. J., and A. F. Brodie. 1971. The structure and function of quinones in respiratory metabolism, of flagellum synthesis (24). However, it is also p. 407-461. In D. B. McCormick and L. D. Wright possible that a specific step in flagellum synthe(ed.), Methods in enzymology, vol. 18, part C. Acasis or assembly is energized by a high-energy indemic Press Inc., New York. termediate of oxidative phosphorylation (3, 15, 7. Gibson, F. 1973. Chemical and genetic studies on the biosynthesis of ubiquinone by Escherichia coli. Bio20). An alternative explanation would mainchem. Soc. Trans. 1:317-326. tain that a specific chain-lengthened ubiqui- 8. Haddock, B. A., and H. U. Schairer. 1973. Electronnone (22) and a specific cytochrome (11), apart transport chains of Escherichia coli. Reconstitution of from their possible relevant role in oxidative respiration in a 5-aminolaevulinic acid-requiring mutant. Eur. J. Biochem. 35:34-45. electron transport, are involved in flagellum A. D., and D. S. Hogness. 1960. The transformasynthesis by serving as cofactors in a defined 9. Kaiser, tion of Escherichia coli with deoxyribonucleic acid oxidation-reduction step. The specific role of isolated from bacteriophage Adg. J. Mol. Biol. 2:392menaquinone in anaerobic pyrimidine biosyn415. thesis in E. coli may serve as a model system 10. Larsen, S. H., J. Adler, J. J. Gargus, and R. W. Hogg. 1974. Chemomechanical coupling without ATP: the for such a requirement (12). source of energy for motility and chemotaxis in bactecorrelation The ubiquinone-heme-motility ria. Proc. Natl. Acad. Sci. U.S.A. 71:1239-1243. reported here is of considerable interest, since 11. Meyer, D. J., and C. W. Jones. 1973. Oxidative phosphorylation in bacteria which contain different cytoit involves a key relationship between flachrome oxidases. Eur. J. Biochem. 36:144-151. gellum synthesis and intermediates in meta- 12. Newton, N. A., G. B. Cox, and F. Gibson. 1971. The bolic pathways. function of menaquinone (vitamin K2) in EscherACKNOWLEDGMENTS We are particularly grateful to F. Gibson, I. Young, P. D. Bragg, and B. A. Haddock for their generous gifts of mutants. This work was supported by a Public Health Service
ichia coli K-12. Biochim. Biophys. Acta 244:155-166. 13. Quadling, C., and B. Stocker. 1962. An environmentally induced transition from the flagellated to nonflagellated state in Salmonella typhimurium: the fate of parental flagella at cell division. J. Gen. Microbiol. 28:257-270.
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14. Robinson, J., and J. M. Cooper. 1970. Method of determining oxygen concentrations in biological media, suitable for calibration of the oxygen electrode. Anal. Biochem. 33:390-399. 15. Rosenberg, H., G. B. Cox, J. D. Butlin, and S. J. Gutowski. 1975. Metabolite transport in mutants of Escherichia coli K12 defective in electron transport and coupled phosphorylation. Biochem. J. 146:417423. 16. Sasirman, A., M. Surdeanu, and T. Horodniceanu. 1968. Locus determining the synthesis of a 8-aminolevulinic acid in Escherichia coli K-12. J. Bacteriol. 96:1882-1884.
17. Sherris, J. C., N. W. Preston, and J. G. Shoesmith. 1957. The influence of oxygen and arginine on the motility of a strain ofPseudomonas sp. J. Gen. Microbiol. 16:86-96. 18. Spudich, J. L., and D. E. Koshland, Jr. 1975. Quantitation of the sensory response in bacterial chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 72:710-713. 19. Stroobant, P., I. G. Young, and F. Gibson. 1972. Mu-
J. BACTERIOL. tants of Escherichia coli K-12 blocked in the final reaction of ubiquinone biosynthesis: characterization
and genetic analysis. J. Bacteriol. 109:134-139. 20. Vallin, I., and H. Low. 1968. The effect of piericidin A on energy-linked processes in submitochondrial particles. Eur. J. Biochem. 5:402-408. 21. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase ofEscherichia coli: partial purification and some properties. J. Biol. Chem. 218:97-106. 22. Wan, Y.-P., R. H. Williams, and K. Folkers. 1975. Low molecular weight analogs of coenzyme Q as hydrogen acceptors and donors in systems of the respiratory chain. Biochem. Biophys. Res. Commun. 63:11-15. 23. Wimpenny, J. W. T., and J. A. Cole. 1967. The regulation of metabolism in facultative bacteria. IIIL. The effect of nitrate. Biochim. Biophys. Acta 148:233-242. 24. Yokota, T., and J. S. Gots. 1970. Requirement of adenosine 3',5'-cyclic phosphate for flagella formation in Escherichia coli and Salmonella typhimurium. J. Bacteriol. 103:513-515.
Vol. 130, No. 2 Printed in U.S.A.
Flagellar Formation in Escherichia coli Electron Transport Mutants J. BAR TANA,1 BARBARA J. HOWLETT,
AND
D. E. KOSHLAND, JR.*
Department of Biochemistry, University of California, Berkeley, California 94720
Received for publication 9 February 1977
Mutants of Escherichia coli lacking ubiquinone or heme have been tested for motility and found to be essentially immotile. The loss of motility is identified with the loss of flagellum synthesis. Motility of Escherichia coli was shown by Sherris et al. (17) and by Adler and Templeton (2) to be sustained either by oxidative phosphorylation or by substrate level phosphorylation. Larsen et al. have pointed out that the immediate energy source for motility is probably the high-energy intermediate of oxidative phosphorylation, not adenosine 5'-triphosphate (10). Thus, E. coli adenosine triphosphatase mutants were shown to be motile aerobically, but they became immotile anaerobically (10). Electron transport mutants ofE. coli that contain a functioning Mg-Ca adenosine triphosphatase are expected, therefore, to be motile under growth conditions that maintain substrate level phosphorylation. The present study describes the motility pattern observed aerobically with two types of electron transport mutants, due to ubi mutations, which are deficient in ubiquinone synthesis (7), and hemA mutants, which are defective in functional cytochrome synthesis (8, 16).
cient mutants were selected by analyzing the quinone content of single colonies grown on nutrient agar plates. Growth and incubations were carried out aerobically to mid-logarithmic phase ((Dmo 0.4 to 0.8). Ubiquinone and cytochrome mutants are unable to grow on succinate, and, therefore, succinate was used as a test for possible reversion of mutants. Quinone determination. Bacteria were grown to mid-log phase in nutrient broth, with additions as described, harvested, washed twice in 0.9% KCl, then washed in water, and lyophilized to dryness. A weighed sample of the bacterial powder (20 to 50 mg) was extracted three times successively in 5 ml of acetone at 65°C. The combined acetone extract was evaporated to dryness under N2, suspended in a small volume of petroleum ether (20 to 60°C)-ether (1:1), and was subjected to thin-layer chromatography in benzene. Commercial ubiquinone-10 (Rf, 0.21), ubiquinone-6 (R,, 0.28) and menaquinone-10 (R,, 0.58) served as markers. The ubiquinone spot (Rf, 0.13 to 0.30) and the menaquinone spot (Rf, 0.55 to 0.65) (visible as yellow bands) were scraped off the plates and were eluted three times successively in 2.5 ml of diethylether. The ether eluate was evaporated to dryness under N2. Ubiquinone was deterMATERIALS AND METHODS mined by the (275ox-275NaBH4 reduced) difference Chemicals and media. Ubiquinone-10, ubiqui- spectrum in ethanol (AE275 in ethanol, 12.4) (5). Menone-6, menaquinone-10 and 8-amino levulinic acid naquinone was determined by the (245NaBH4 re(8ALA) were obtained from Sigma Chemical Co. duced-245ox) difference spectrum in ethanol (A245 in Silic-AR CC-4 100- to 200-mesh silicic acid was ob- ethanol, 25.8) (6). tained from Mallinckrodt. Adsorbil-5-Prekotes thinMotility assay. Motility was measured by a modilayer plates were purchased from Applied Science fication of the tumble frequency assay described by Laboratories Inc. and Koshland (18). Bacteria were grown in Spudich H minimal medium was prepared according to 22 mM pyruvate in nutrient broth medium with Kaiser and Hogness (9). VB minimal medium was additions as described to mid-logarithmic phase, diprepared as described by Vogel and Bonner (21). VB- luted in VB-EDTA minimal medium to ca. 3 x 107 EDTA medium was VB minimal medium with 10-5 cells/ml, and equilibrated by shaking at 30°C for M ethylenediaminetetraacetate (EDTA) added. Nu- 15 min. Portions (0.9 ml) of these bacteria were trient broth medium consisted of 0.8% nutrient mixed rapidly with 0.1 ml of 50 mM -serine, a broth plus 100 mM potassium phosphate buffer, pH chemotactic attractant, causing the smooth swim7.0. ming of all motile bacteria (18). This suspension (8 Bacterial strains. The strains used are described ,ul) was delivered under a cover slip, which was in Table 1. Motile revertants of immotile mutants supported by two other cover slips, to yield a chamwere selected by swarming on semisolid tryptone ber of 30-/sm depth, and 1 min later a 0.8-s-exposure plates (1). Spontaneous revertants of quinone-defi- photograph (Kodak recording film 2475) was taken I Present address: by dark-field illumination with a stroboscopic, highDepartment of Biochemistry, Hebrew pressure xenon arc lamp (flashing rate, 5 Hz) as a University, Jerusalem, Israel. 787 -
788
BAR TANA, HOWLETT, AND KOSHLAND
J. BACTERIOL.
TABLE 1. Strains ofE. coli used Strain no.
Genetic description
Source
AB2154 AN66 AN661 AN662 AN59 AN592 AN151 AN1512 AN98 AN99 AN187 AN1871 AN1872 SAS x 76
ubi+ ubiD Quinone revertant of AN66 Motility revertant of AN66 ubiB Motility revertant of AN59 ubiG Motility revertant of AN151 men+ ubi+ menA menA ubiD Menaquinone revertant of AN187 Quinone revertant hemA
Cox et al. (4) Cox et al. (4) This study This study; motility revertant of AN66 Cox et al. (4) This study Stroobant et al. (19) This study Newton et al. (12) Newton et al. (12) Cox et al. (3) This study; spontaneous revertant of AN187 This study; spontaneous revertant of AN187 Sasarman et al. (16)
light source. The number of motile tracks, consisting of four successive bacterial images, and the number of immotile bacterial spots were counted. The percentage of motile bacteria was defined as: number of motile tracks x 100/number of motile tracks plus number of nonmotile spots. Differences in the measured percentage of motility of the same culture were usually in the range of ±5%. Characteristic photomicrographs are shown in Fig. 1. The qualitative terms "motile" and "immotile" are used on occasions when more than 60% or less than 5% ofthe bacteria are motile, respectively. Respiration measurements. Bacteria were grown to mid-log phase in nutrient broth with additions as described. The culture was harvested, washed once in 100 mM phosphate, pH 7.0, and suspended in H medium-22 mM glucose with additions as described. Respiration was followed in an oxygen electrode chamber (Yellow Springs Instruments Co., no. 5331), which was calibrated with the reduced nico tenamide adenine dinucleotide-phenazine methyl sulfonate catalase couple described by Robinson and Cooper (14). The bacterial density was estimated by measuring the optical density at 650 nm. Electron microscopy studies. Bacteria were grown in nutrient broth-22 mM pyruvate to mid-logarithmic phase. A drop of the culture was placed in a carbon-coated grid, drained, washed with distilled water, and then stained with 1% uranyl acetate for 40 s. The grid was loaded and examined in a Siemans electron microscope. The flagella emanating from 50 individual, nonoverlapping bacteria were counted.
tile, irrespective of the specific mutation concerned. The isogenic parents as well as spontaneous Ubi+ revertants are motile when grown under the same conditions as the mutants. Furthermore, revertants selected for motility from the immotile Ubi- mutants by swarming on semisolid agar plates were shown to revert concomitantly with the return of ubiquinone formation. It appears, therefore, that the phenotypic expression of motility and that of ubiquinone synthesis are strongly related. On the other hand, menaquinone is not related to motility (Table 3). A double mutant bearing ubiD menA is immotile; its Men+ revertant is immotile, whereas its Ubi+ revertant is motile. Motility of hemA mutants. Immotility observed with E. coli ubi mutants is also observed with mutants of E. coli bearing hemA, which are defective in the synthesis of 8ALA (16). E. coli SAS x 76 is immotile when grown aerobically in the absence of 8ALA. Under the same growth conditions and in the presence of 10-5 M of 8ALA, the mutant becomes motile (Table 4). The ubiquinone content is not affected by the hemA locus. A mutant of E. coli (1004A) bearing hemA, isolated by Haddock and Schairer (8), forms only the apocytochrome when grown in the absence of 8ALA but synthesizes the protoporphyrin in the presence of 8ALA under nongrowth RESULTS conditions. It was tempting, therefore, to follow Motility of ubiquinone and menaquinone the time course of motility attainment, as well mutants. The motility pattern of mutants in as respiration, upon the addition of 8ALA in the pathway of ubiquinone synthesis in E. coli the presence and absence of chloramphenicol. is shown in Table 2. The pathway starts with As shown in Fig. 2A, respiration is restored at chorismic acid, the common precursor of ubi- about 2 to 4 h of incubation with 8ALA, whereas quinone, and aromatic amino acids and mu- motility is restored at about 10 to 20 h. Respiratants in all the intermediate steps have been tion is still restored in the presence of chloramisolated and characterized by Gibson and asso- phenicol (Fig. 2B), but protein synthesis seems ciates (3, 4, 7, 12, 19). The Ubi- mutants (kindly to be obligatory for restoring motility. The late supplied by F. Gibson) all appear to be immo- decline in respiration, which was observed in
VOL. 130, 1977
5SIo
FLAGELLAR FORMATION IN E. COLI
789
I 484U
FIG. 1. Quantitative assay of motitity. Bacterial motility is measured by counting the tracks after exposure intervals with five stroboscopic flashes per second. Details are described in the text. Illustrative examples show bacteria swimming (shown by tracks) versus immotile or tumbling (bright splotches) bacteria. Illustrative examples show 3%, 21%, 53%, and 84% motility.
BAR TANA, HOWLETT, AND KOSHLAND
790
TABLE 2. Motility of E. coli ubiquinone mutant8a Motility
Strain
Ubiquinone Menaquinone (nmolVmg [dry (nmollmg [dry wt]) wt])
0.26 0.71 Motile AB2154 0.17 0.05 Immotile AN66 0.19 0.70 Motile AN661 0.06 0.87 Motile AN662 0.10 0.01 Immotile AN59 0.03 0.47 Motile AN592 0.06 0.05 Immotile AN151 NDb 0.48 Motile AN1512 a Bacteria were grown in nutrient broth to midlog phase. Motility and quinone contents were determined as described in the text. b ND, Not detectable.
TABLz 3. Motility of E. coli menaquinone mutantsa Motility
Strain
Ubiquinone
Menaquinone
wt])
wt])
(nmolVmg [dry (nmoVmg [dry
J. BACTERIOL.
nificant reduction in flagellum number of ubi or hemA mutants was observed in correlation with their motility pattern. The percentage of motility is shown to be related to the percentage of bacteria that possess three or more flagella per bacterium. This agrees with the findings of Quadling and Stocker, who limited flagellum synthesis by environmental conditions (13).
DISCUSSION The lack of motility observed for mutants deficient in ubiquinone or heme synthesis is evidently ascribed to their failure to form adequate numbers of flagella. The observed correlation should then be analyzed in terms of flagellum synthesis and/or assembly and should not be deduced from the prevailing concept concerning the immediate energy source for bacterial motility. The obligatory role played by electron transport in flagellum synthesis (assembly) in E.
0.02 0.63 Motile AN98 NDb 0.94 Motile AN99 ND ND Immotile AN187 ND 0.41 Immotile AN1871 ND 0.89 AN1872 Motile a Bacteria were grown in nutrient broth to midlog phase. Motility and quinone contents were determined as described in the text. b ND, Not detectable.
TABLz 4. Characteristics of E. coli hemA mutanta Additions to broth b
Motility Immotile
Respiration ( 0
Ubiquinone
(nmol/mg (dry wt]) 0.74
0.76 100 Motile 10-5 M 8ALA a E. coli SAS x 76 was grown in nutrient broth-22 mM glucose to mid-log phase with addition as described. Motility, ubiquinone content, and respiration were determined as described in the text. -, No addition.
the presence of chloramphenicol, is probably due to turnover of the cytochrome apoprotein under these conditions. Neither respiration nor motility was reconstituted during the first 12 h of incubation in the absence of 8ALA, and reversion often occurred after this time. Flagellar patterns of ubi and hemA E. coli mutants. The time lag between respiration reconstitution and motility reconstitution observed with the mutant bearing hemA, as well as the differential effect of chloramphenicol upon the reconstitution of motility and respiration, has indicated a possible role of oxidative electron transport in the synthesis or assembly of bacterial flagella. As shown in Table 5, sig-
Time (hours) FIG. 2. Reconstitution of respiration and motility in E. coli hemA mutant. E. coli SAS x 76 was grown to mid-log phase in nutrient broth plus 22 mM glucose. The culture was haruested, washed once in 100 mM phosphate buffer, pH 7.0, and suspended at time zero in H medium-0.5% Casamino Acids-22 mMglucose-10-5 M MALA in the presence (B) and absence (A) of 30 pg of chloramphenicol per ml. Incubation was carried out at 30°C with continuous gyratory ) and motility (-----) were shaking. Respiration ( determined on portions at particular times. The respiration rate of E. coli SAS x 76 grown in the presence of 10-5 M 8ALA was taken to be 100%.
FLAGELLAR FORMATION IN E. COLI
VOL. 130, 1977
791
TABLE 5. Distribution of flagella of E. coli strains useda Bacterial strain
1
Bacteria 2
(%) with flagellum no. indicated 3
4
5
6
7
Mean flagellum no.
Motility (%)
3 1 0 0 1.5 1.5 15 47 24 11 AN59 60 2.5 3.3 26 5 21 8 16 11 11 AN592 0 0 0 0 0 1.5 84 12 1.5 AN66 1.5 50 2.6 9 11 2 17 13 13 28 9 AN662 1 1 0 0.8 3 3 1.5 18 10 65 SAS x 76 75 28 6 2 3.5 16 18 2 12 16 SAS x 76 + 8ALA a Electron microscopy examination and motility assays were as described in the text. For each bacterial strain, about 50 individual bacteria were examined. The number of flagella emanating from each individual was recorded and counted.
coli pertains to aerobically as well as anaero- grant no. AM-09765 from the National Institute of Arthribically grown bacteria. Anaerobic growth leads tis, Metabolism and Digestive Diseases. to motile bacteria (as reported by Adler and LITERATURE CITED Templeton [2] and confirmed by us), but nonJ. 1966. Chemotaxis in bacteria. Science 153:708leaky mutants in anaerobic electron transport 1. Adler, 716. are devoid of flagellum synthesis when grown 2. Adler, J., and B. Templeton. 1967. The effect of environanaerobically (R. Hertz and J. Bar Tana, mental conditions on the motility of Escherichia coli. J. Gen. Microbiol. 46:175-184. manuscript in preparation). G. B., N. A. Newton, J. D. Butlin, and F. Gibson. The observed correlation between oxidative 3. Cox, 1971. The energy-linked transhydrogenase reaction electron transport and anaerobic flagellum synin respiratory mutants of Escherichia coli K12. Biothesis (assembly) may be analyzed in the light chem. J. 125:489-493. of the following considerations. The shift in 4. Cox, G. B., I. G. Young, L. M. McCann, and F. Gibson. 1969. Biosynthesis of ubiquinone in Escherichia coli metabolic pathways in the absence of oxidative K-12: location of genes affecting the metabolism of 3electron transport (23) may eliminate a crucial octaprenyl-4-hydroxybenzoic acid and 2-octaprenylintermediate in flagellum synthesis, or the rephenol. J. Bacteriol. 99:450-458. sulting reducing environment may inhibit 5. Crane, F. L., and R. Barr. 1971. Determination of ubiquinones, p. 137-165. In D. B. McCormick and L. some rate-limiting step in flagellum synthesis. D. Wright (ed.), Methods in enzymology, vol. 18, part The effect may be mediated through cyclic nuC. Academic Press Inc., New York. cleotide levels involved in catabolite repression 6. Dunphy, P. J., and A. F. Brodie. 1971. The structure and function of quinones in respiratory metabolism, of flagellum synthesis (24). However, it is also p. 407-461. In D. B. McCormick and L. D. Wright possible that a specific step in flagellum synthe(ed.), Methods in enzymology, vol. 18, part C. Acasis or assembly is energized by a high-energy indemic Press Inc., New York. termediate of oxidative phosphorylation (3, 15, 7. Gibson, F. 1973. Chemical and genetic studies on the biosynthesis of ubiquinone by Escherichia coli. Bio20). An alternative explanation would mainchem. Soc. Trans. 1:317-326. tain that a specific chain-lengthened ubiqui- 8. Haddock, B. A., and H. U. Schairer. 1973. Electronnone (22) and a specific cytochrome (11), apart transport chains of Escherichia coli. Reconstitution of from their possible relevant role in oxidative respiration in a 5-aminolaevulinic acid-requiring mutant. Eur. J. Biochem. 35:34-45. electron transport, are involved in flagellum A. D., and D. S. Hogness. 1960. The transformasynthesis by serving as cofactors in a defined 9. Kaiser, tion of Escherichia coli with deoxyribonucleic acid oxidation-reduction step. The specific role of isolated from bacteriophage Adg. J. Mol. Biol. 2:392menaquinone in anaerobic pyrimidine biosyn415. thesis in E. coli may serve as a model system 10. Larsen, S. H., J. Adler, J. J. Gargus, and R. W. Hogg. 1974. Chemomechanical coupling without ATP: the for such a requirement (12). source of energy for motility and chemotaxis in bactecorrelation The ubiquinone-heme-motility ria. Proc. Natl. Acad. Sci. U.S.A. 71:1239-1243. reported here is of considerable interest, since 11. Meyer, D. J., and C. W. Jones. 1973. Oxidative phosphorylation in bacteria which contain different cytoit involves a key relationship between flachrome oxidases. Eur. J. Biochem. 36:144-151. gellum synthesis and intermediates in meta- 12. Newton, N. A., G. B. Cox, and F. Gibson. 1971. The bolic pathways. function of menaquinone (vitamin K2) in EscherACKNOWLEDGMENTS We are particularly grateful to F. Gibson, I. Young, P. D. Bragg, and B. A. Haddock for their generous gifts of mutants. This work was supported by a Public Health Service
ichia coli K-12. Biochim. Biophys. Acta 244:155-166. 13. Quadling, C., and B. Stocker. 1962. An environmentally induced transition from the flagellated to nonflagellated state in Salmonella typhimurium: the fate of parental flagella at cell division. J. Gen. Microbiol. 28:257-270.
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17. Sherris, J. C., N. W. Preston, and J. G. Shoesmith. 1957. The influence of oxygen and arginine on the motility of a strain ofPseudomonas sp. J. Gen. Microbiol. 16:86-96. 18. Spudich, J. L., and D. E. Koshland, Jr. 1975. Quantitation of the sensory response in bacterial chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 72:710-713. 19. Stroobant, P., I. G. Young, and F. Gibson. 1972. Mu-
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