Escherichia Coli Mutants Defective in Chemotaxis Toward Specific Chemicals* by Gerald L

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ESCHERICHIA COLI MUTANTS DEFECTIVE IN CHEMOTAXIS TOWARD SPECIFIC CHEMICALS* BY GERALD L. HAZELBAUER, ROBERT E. 1\IESIBOV, AND JULIUS ADLER

DEPARTMENTS OF BIOCHEMISTRY AND GENETICS, UNIVERSITY OF WISCONSIN, MADISON Communicated by R. H. Burris, October 6, 1969 Abstract.-Mutants of Escherichia coli K12 have been found which fail to carry out chemotaxis toward certain chemicals only. One mutant exhibits greatly reduced chemotaxis toward L-serine but has no detectable defect either in uptake or in oxidative metabolism of that compound. Another mutant is not attracted to D-galactose and certain related sugars. There is a correlation between the galactose chemotaxis defect and a defect in galactose uptake, perhaps indicating a common component for chemotaxis and uptake systems. The results are discussed in terms of a model for chemotaxis in which attractants are detected by specific "chemoreceptors."

Escherichia coli are chemotactic, that is, they are attracted to certain chemicals.1, 2 A model for bacterial chemotaxis has been proposed2 in which classes of sterically related compounds are recognized by specific "chemoreceptors." In- formation from all chemoreceptors is channeled through a common pathway to the flagella where a response is initiated. The model predicts two classes of nonchemotactic mutants. One class is defective in the common pathway, reducing chemotaxis to all chemicals ("generally nonchemotactic mutants"). The other has defects in specific chemoreceptors, reducing chemotaxis to certain chemicals ("specifically nonchemotactic mutants"). Generally nonchemotactic mutants of Escherichia coli K12 have been isolated, characterized,3 and studied genetically4' 5 in our laboratory. In this report we describe two specifically nonchemotactic mutants, one defective in taxis to a group of amino acids and the other to a group of sugars. Materials and Methods.-Bacteria: The serine taxis mutant, AW518, was isolated from AW405 (E. coli K12, wild type for chemotaxis, gal-i, gal-2, thr, leu, his-4, thi, lac, xyl, ara, str-r, ton A-r, tsx-r) by means of a selection procedure described previously.3 In this procedure, cells are placed at the center of a tryptone semisolid plate. Rings of chemotactic bacteria swarm out from the center in response to amino acid gradients created by bacterial growth.1 Cells remaining at the center after swarming has occurred are used to inoculate a fresh plate. After several such transfers, the center is enriched in non- flagellated, abnormally flagellated, paralyzed, and nonchemotactic mutants. W3109 (gal-9), a galactose operon extreme polar mutant of E. coli K12 obtained from Dr. E. Lederberg, was found to be a galactose taxis mutant. AW520, a gal+ revertant of W3109 (also lami-r and ton A-r), was used in the experiments described in this paper. To select for revertants to normal galactose taxis, about 109 cells of AW520 (grown, harvested, and washed as in chemotaxis experiments) in 0.1 ml of chemotaxis medium were spread in a streak on a semisolid galactose plate. On such a plate, bacteria chemotactic toward galactose form a dense ring' easily visible after 12 hr. After 3 days at 350C in a wet incubator, chemotaxis rings were faintly visible along the border of the AW520 streak. A single colony isolate, AW521, from one of the rings showed normal chemotaxis and had the same bacteriophage sensitivity as AW520 (ttw-s, tfr-s, ton A-r, 1300 Downloaded by guest on September 27, 2021 Voi.. 64, 4BIOCHEMISTRY:196') HAZELBA JER ET AL. 1301 and lamt-r). There are only small quantitative differences between chemotaxis by the revertant AW521 and the wild-type K12 strain, W3110, routinely used in this laboratory. W4611 (gal-3), a galactose operon extreme polar mutant of E. coli K12, was obtained from Dr. E. Lederberg. Media: Tryptone broth contains 1% Difco Bacto-Tryptone and 0.5% NaCl. The inorganic portion of the minimal media contains6 11.2 gm K2HPO4, 4.8 gm KH2PO4, 2.0 gm (NH4)2SO4, 0.25 gm MgSO4 X 7 H20, and 0.5 mg Fe2(SO4)3 per liter of distilled water (finally adjusted to pH 7.0). Separately autoclaved or filtered sugar or glycerol (5 gm/ liter), serine (2.5 gm/liter), or 20 amino acids (0.25 gm/liter of each of the 20 common L-amino acids) serve as the carbon and energy source. The media are supplemented with 0.25 gm/liter of L-threonine, L-leucine, and L-histidine for growth of AW405 and AW518. Growth on L-serine requires an additional supplement of 0.25 gm/liter i-methionine (as well as threonine already present) (L. Pizer, personal communication). Eosin-methylene blue plates are prepared with Difco eosin-methylene blue agar and contain 1% glucose. Streptomycin, when present, is at 100 ,ug/ml. Tryptone semisolid plates contain 0.35% agar in tryptone broth. Galactose semisolid plates contain 10-2 M potassium phosphate buffer, pH 7.0, 10-3 M (NH4)2SO4, 10-3 M MgSO4, and 1.6 X 10-4 M D-galactose in 0.35% agar. Chemotaxis medium consists of 10-4 M EDTA and 10-2 M potassium phosphate buffer, pH 7.0. All solutions used in chemotaxis experiments are prepared with glass- distilled water. Chemicals: Sugars (D configuration, except where noted) used as attractants were obtained from Pfanstiehl Laboratories Inc., Waukegan, Ill., or Calbiochem, Los Angeles, Calif. They were tested for purity by chromatography and when necessary were chro- matographically purified (see ref. 2 footnotes for details). The amino acids (all L configuration) used as attractants were obtained from Calbiochem. uL-14C-D-galactose (26.6 mCi/mmole) was obtained from Mallinckrodt Chemical Works, Orlando, Fla.; 6-H3-D-fucose (200 mCi/mmole) from Calbiochem; and uL-14C-L-serine (115.5 mCi/ mmole) from New England Nuclear Corp., Boston, Mass. Solutions containing L-cysteine are prepared from L-cysteine-HCl neutralized with NaOH just prior to the assay. Aspartic and glutamic acid solutions are neutralized with KOH. Chemotaxis assay: Cells adapted to growth in minimal medium (350C with rotatory shaking at 200 rpm) are inoculated into fresh minimal medium at an initial O.D.59o nm of 0.05 (3.5 X 107 cells/ml). When the culture reaches an O.D.590 nm between 0.2 and 0.4, approximately 7 X 108 cells are harvested by centrifugation at 8000 X g at 40C for 10 min. The cells are then washed twice with 5 ml of chemotaxis medium by centrifugation and resuspended in chemotaxis medium at a concentration of approximately 7 X 107 cells/ml. In the assay, about 0.25 ml of cell suspension is maintained at 30°C (on a Fisher slide warmer) in a small chamber formed by a glass slide, a glass coverslip, and a U-shaped 1.5-mm glass tube separating the two. A micropipette ("Microcap" 1-Al capacity, 3-cm long, 0.2 mm inside diameter; Drummond Scientific Co., Broomall, Pa.) is sealed at one end in a flame, passed quickly several times through the flame, and plunged, open end down, into a small beaker containing attractant dissolved in chemotaxis medium. As the micropipette cools, liquid is drawn ca. 1 cm into the pipette. The micropipette is inserted open end first into the suspension on the slide at zero time, and is removed after 45 or 60 min of incubation. After rinsing off the micropipette with a thin stream of water, the sealed end is broken off and the contents are transferred to tryptone broth, diluted, and plated on eosin-methylene blue glucose plates. Assay points are done at least in duplicate. Error in the assay is in the order of 10 to 20%. Background accumulation (accumulation in a capillary containing chemotaxis medium without attractant) is strain dependent but is generally less than 7000 bacteria. A more complete discussion of the assay can be found elsewhere.7 Oxygen consumption: Oxygen consumption at 30°C was measured directly using a Gilson respirometer. Cells are grown, harvested, and washed as in chemotaxis experiments, and resuspended in chemotaxis medium at a concentration of 3.5 X 108 cells/ml. Downloaded by guest on September 27, 2021 1302 BIOCHEMISTRY: HAZELBAUER ET AL. PROC. N. A. S.

Two milliliters of this suspension are added to the main compartment of ca. 15-ml flasks. The sidearm and center well contain, respectively, 0.5 ml of 5 X concentrated substrate in chemotaxis medium, and 0.2 ml of 10% KOH on fluted filter lpaper. Equilibration time is 10 min. Uptake: Cells are grown, harvested, and washed as in chemotaxis experiments. Up- take of labeled fucose or galactose is determined by Millipore filtration (type HA filters, 0.45 ,u pore size) of 0.3-ml aliquots of an incubation mixture containing bacteria (ca. 108 cells/ml) and labeled sugar in chemotaxis medium at 30'C. Cells on the filter are immediately washed with 2 ml of 300C chemotaxis medium containing unlabeled sugar at a concentration 5X greater than in the incubation mixture. Washing with from 1 to 10 ml under these conditions yields the same number of counts. Nonspecific adsorption is controlled by subtracting counts retained on the filter after washing cells treated with 30%O formaldehyde, then incubated under the same conditions as un- treated cells. Filters are dried under a heat lamp and counted in 10 ml of cold scintillation fluid (3 gm 2,5-diphenyloxazole (PPO) and 100 mg 1,4-bis-2-(4-methyl-5 phenyl- oxazolyl)-benzene (dimethyl POPOP) in 1 liter toluene) using a Packard Tri-Carb liquid scintillation counter. Uptake of labeled serine is followed using the same procedure except that the aliquot and wash volumes are 0.4 and 1.5 ml, respectively, the wash medium contains 10-3 M serine, and the cell concentration in the incubation mixture is approximately 3.5 X 108 cells/ml. Also, chloramphenicol (200 i.g/ml) was present in the incubation mixture. Results.-A mutant for serine taxis: The defect of the serine taxis mutant appears as an anomaly in swarming behavior on a tryptone semisolid plate (Fig. 1). The first, or serine-consuming ring,' formed by the parent strain AW405 is absent from the mutant swarm (the residual swarming by the mutant results from aspartate and oxygen taxis'). Responses to serine, glycine, alanine, and cysteine in the chemotaxis assay are altered in the mutant with both threshold and peak concentrations shifted to higher values (Fig. 2). (The threshold concentration is the attractant concentration at which bacterial accumulation in the chemotaxis assay first exceeds background accumulation. It is determined by extrapolating a double log plot of number of bacteria accumulated versus attractant concentration to the background accumulation "base line." Peak concentration is the attractant concentration which yields the largest bacterial accumulation. These parameters are discussed in greater detail elsewhere.7) The defect in serine taxis is the most

FIG. 1.-Chemotactic response of the serine taxis mutant and its parent on a tryptone semisolid plate. Approximately 108 cells of each strain were spotted on the plate and incubated 5 hr at 350C in a wet incubator. The outermost ring formed by the parent is absent from the mutant swarm (mutant on right). Downloaded by guest on September 27, 2021 VOL. 64, 1969 BIOCHEMISTRY: HAZELBAUER ET AL. 1303

FIG. 2.-Chemotaxis assays cormi @ b ;2 =-04< paring responses of the serine taxis 7CSTE 3 t A-SP4ATEG3U2M3 mutant (A-A) and of its wild- pa type parent (0 -). All cells 3W- 4\ 3W were grown in glycerol minimal 40 _ medium, and prepared and assayed U 2WI §2 as described in Materials and 2i Methods. Incubation was for 45 O 0 - 5 3 i min at 30'C. All amino acids were -6-5-4 -3 2 1-6i- -4 -2 0 3 ,STOVEE ofthetheD configuraion.L configuration andsugars of ih5075 G3 150- RIB-M -

LO)GI MOLARTY OF ATTRACTA

severe, the threshold being more than 103 times greater in the mutant than in the parent. Taxis toward aspartate and glutamate by mutant and parent is identical. Taxis toward galactose, glucose, and ribose is characterized by larger responses by the mutant than by the parent. This phenomenon is presently un- explained. The growth rates of the two strains on minimal medium with 20 amino acids, glycerol, or serine as carbon and energy source are identical (generation times 1.3, 1.7, and 2.7 hours, respectively). Oxygen consumption rates in the presence of serine, alanine, and aspartate are the same for both strains whether they are grown on minimal medium with 20 amino acids or glycerol (Table 1). Thus, the defect in taxis toward serine does not appear to be correlated with a defect in the oxidative metabolism of this compound. The rapid utilization of serine makes the results of uptake experiments some- what ambiguous. However, the initial rates of incorporation of 14C-serine counts into cells of the two strains are identical even at very low external serine concentration (Table 2), suggesting that the mutant is not defective in uptake of this compound. TABLE 1. Oxygen consumption by the serine taxis mutant. - ~Oxygen Consumption (Mmoles 02/hr/109 Cells) --Grown on Glycerol-. Grown on 20 Amino Acids Substrate Parent Mutant Parent Mutant Serine 2.3 2.3 1.5 1.5 Alanine 0. 74 0.72 0.34 0.36 Aspartate 0.30 0.24 0.19 0.27 Rates were determined from the straight line portions of plots of 02 consumed versus time over at least 90 min. Substrates were present at 5 X 10-3 M. Details of the procedure appear in Ma- tertals and Methods. Downloaded by guest on September 27, 2021 13040IOCHEMISTRY: HAZELBA UER ET A L. PRtOc. N. A. S.

TABLE 2. Uptake of labeled serine by the serine taxis mutant. I--- --Serine Uptake (pmoles/min/107 Cells) . - Serine concentration Parent Mutant 2.3 X 10-8AI 0.14 0.14 4.6 X 10-8Al 0.24 0.26 1.9 X 10-7M 0.64 0.56 3.1 X 10-7M 0.98 1.04 4.6 X 10-7M 1.50 1.88 Uptake rates are based on a sample taken 30 sec after adding labeled serine. Cells were grown on glycerol minimal medium. Details of the procedure appear in Materials and Methods. A mutant for galactose taxis: The mutant defective in galactose taxis, AW520, is unable to form on a galactose semisolid plate (Fig. 3) the chemotaxis ring characteristically formed by wild-type E. colil or the galactose taxis revertant, AW521. In the chemotaxis assay, the galactose mutant differs from its chemotactic revertant and wild-type E. coli in the lack of any detectable chemotactic response toward galactose, fucose, L-arabinose, xylose, and L-sorbose. Tactic response toward glucose, 2-deoxyglucose and a-methylglucoside is altered by shifts in the threshold and peak concentrations to higher values. Taxis toward fructose, ribose, serine, and aspartate is normal. Examples of these responses appear in Figure 4. Growth rates of the mutant and revertant are indistinguishable in minimal medium with 0.5 per cent glycerol, glucose, or galactose as sole carbon and energy source (generation times 1.8, 1.4, and 1.6 hours, respectively). Oxygen consumption rates in the presence of galactose at concentrations from 10-4 M to 10-1 M suggest that the mutant is defective in galactose uptake (Table 3). As the concentration of galactose is lowered, the ratio (revertant rate)/ (mutant rate) increases, implying an uptake defect in contrast to a metabolism defect. At all concentrations of glucose tested, the ratio is approximately 1.5. It is not yet clear whether this reflects an influence of the galactose uptake defect or a separate defect in glucose metabolism or uptake. The two strains consume 02 at identical rates in the presence of ribose or serine (Table 3).

FIG. 3.-Chemotactic response of the galactose taxis mutant and its revertant on a galactose semisolid plate. Approximately 109 cells of each strain were spotted on the plate and incubated 12 hr at 350C in a wet incubator. The chemotaxis ring of the revertant is not present for the mutant (mutant on right). Downloaded by guest on September 27, 2021 Voi..VIOCHEMISTRY:64, 1969 HAZELBA UER Et' AL. 1305

FIG. 4.-Chemotaxis assays corn- 100 20I10 paring responses of the* galactose no taxis mutant (A-A) and of its revertant (e4.Cells for fructose 06- -5-4 -3 -2 0 43 -2 01V4 3 -2 and ribose assays were grown on f L-SORSOSE GLUCOSE atMTUELULUCOSN)E fructose or ribose minimal medium. M IW50 The other cells were grown in loo- galactose minimal medium, and all cells were prepared and assayed as described in Materials and Meth- E 1- ods. Incubation was for 60 min 0 -4 -3 -2 - 1 0 at 30'C. All sugars were of the D 100 FRU E 6 K configuration except L-arabinose 200 400 and L-sorbose. Serine was the L- \ isomer. 50 00- 200

0 -4 -3 -2 -I -2 0- -4-3 -2-1 -5 LOG10 0-6-5-4-3MOLAPflY OF ATTRACTANT

Galactose uptake cannot be measured separately from galactose metabolism since the strains are galactose+. However, at low concentrations of galactose (10-6 M or 10-7 M) where uptake might be limiting, the nonchemotactic mutant has an initial rate of uptake less than one-fourteenth of the revertant rate (Table 4). The galactose- parent, W3109, of the galactose taxis mutant accumulates labeled galactose from a 10-6 M solution to less than 5 per cent the level of W4611 (gal-3), another galactose operon extreme polar mutant, which exhibits normal galactose taxis. These results indicate that the mutant does not take up galactose as well as bacteria chemotactic toward galactose. Fucose is not a carbon or energy source for E. coli2 and thus accumulation can be measured independently of metabolism. The mutant, nonchemotactic toward fucose, accumulates labeled fucose from a 5 X 10-4 M solution to one-third TABLE 3. Oxygen consumption by the galactose taxis mutant. Oxygen Consumption (pmoles O2/hr/109 Cells) Revertant/ Substrate Mutant Revertant mutant 0-1 M galactose 1.6 2. 1 1.3 0-2 M galactose 0.90 1.6 1.8 10-3 M galactose 0.59 1.3 2.2 10-4 M galactose 0.34 1.0 2.9 10-1 M glucose 1.8 2.6 1.4 10-2 M glucose 1.2 1.7 1.4 10-3 M glucose 1.0 1.6 1.6 10-4Mglucose 1.1 1.7 1.6 0-2 M ribose 0.86 0.77 0.9 10-3 M ribose 0.80 0.72 0.9 0-4 M ribose 0.64 0.60 0.9 10-2 M serine 1.5 1.5 1.0 10-3Mserine 1.1 1.1 1.0 l0-4 M serine 0.89 0.90 1.0 Rates were determined from the straight line portions of plots of 02 consumed versus time over at least 90 min. Cells were grown on galactose minimal medium. Details of the procedure appear in Materials and Methods. Downloaded by guest on September 27, 2021 130)6 0BOCHEMISTRY: HAZELBAUER ET AL. PROC. N. A. S.

TABLE 4. Uptake of labeled galactose and fucose by the galactose taxis mutant. Galactose Galactose Initial Taxis +/ Substrate Strain metabolism taxis rate* Plateaut Taxis- 10- M galactose AW520 + - 7.8 ... 14.5 10M galactose AW521 + + 113 ... -7 M galactose AW520 + - 1.15 ... 14.5 10-7 M galactose AW521 + + 16.7 10- M galactose W3109 - - ... 2.0 22.2 106 M galactose W4611 - + ... 44.5 5 X 10-4 M fucose AW520 + - ... 5.6 3.3 5 X 10-4Mfucose AW521 + + ... 18.4 * Picomoles/min/107 cells. t Picomoles/107 cells. Rates of galactose uptake were determined from initial slopes of plots of uptake versus time. Usually at least two points could be taken during the time of linear rise. Galactose accumulation by gal - strains reached a plateau after 10 min. Fucose accumulation reached a plateau after 15 min. Gal+ strains were grown on galactose minimal medium. Gal - strains were grown on glycerol minimal medium plus 10-3 M fucose. Details of the procedure appear in Materials and Methods. the level of the chemotactic revertant, again indicating a difference in the ability to concentrate an attractant. Discussion.-The chemotactic responses of the mutants described in this paper may be explained in terms of the chemoreceptor theory, which states that attractants are detected by specific receptors.2 Thus, the serine taxis mutant is defective in a receptor (the "serine receptor") that detects serine and, less well, cysteine, alanine, and glycine; and the galactose taxis mutant is defective in a receptor (the "galactose receptor") that detects galactose and glucose, and, to a lesser extent, some related compounds. The existence of these mutants is some of the most compelling evidence for the chemoreceptor theory. Serine and galactose do not inhibit motility or chemotaxis in the respective taxis mutants.8' 9 Thus, the taxis defects cannot be explained by such inhibition. If only one chemoreceptor exists for the detection of a compound, then a single mutation can totally eliminate the response to that compound. The behavior of the galactose taxis mutant suggests that this is the case for galactose. If more than one chemoreceptor is involved in a response, a mutation in one receptor would be expected to alter, but not abolish, chemotaxis toward that compound. This seems to be true for the glucose, 2-deoxyglucose, and a-methylglucoside responses of the galactose mutant and the alanine, glycine, cysteine, and serine responses of the serine mutant. In fact, there is evidence2' 8 9 for a "glucose receptor" which detects glucose but not galactose, and an "aspartate receptor" which detects aspartate, glutamate less well, and cysteine, alanine, glycine, and serine weakly. Tactic responses in the mutants are drastically altered or eliminated without concomitant alterations in attractant metabolism, demonstrating that utilization of an attractant is not sufficient for the existence of a chemotactic response to that compound. The evidence that metabolism of attractants is not required for chemotaxis has been discussed in detail.2 The correlation between chemotaxis and transport defects is unclear. It has been shown that mutations in several components of the transport systems for galactose and glucose do not affect chemotaxis,2 and as far as can be determined, Downloaded by guest on September 27, 2021 VOL. 64, 1969 BIOCHEMISTRY: HAZELBAUER ET AL. 1307

the serine taxis mutant has no defect in serine uptake. The galactose taxis mutant does have defects in both chemotaxis toward and uptake of galactose and fucose, but the two defects are not of equal severity. The chemotactic response is abolished but the attractant is still concentrated in the cells. Coordinate cor- rection of the defects in chemotaxis and uptake by reversion or conjugation with a wild-type male (G. L. Hazelbauer, unpublished results) indicates that these defects are the result of a single mutation. This mutation is probably not closely linked to the galactose operon since elimination of the galactose metabolism defect in the original galactose- strain (W3109) by transduction with Xdg does not correct the chemotaxis defect. It is therefore probable that there is a component which is involved in both chemotaxis and uptake systems. Normal function of this component might be a stringent requirement for chemotaxis, while the attractant still could be taken up by either a defective or an alternative uptake system. Other components of the' uptake system are not required for chemotaxis.2 The serine taxis mutant would then either be defective in a component not involved in transport or have a transport defect masked by rapid metabolism of seine. Study of the relationship between chemotaxis and transport of attractants is in progress. Selection procedures have recently been developed to obtain additional mutants defective in chemotaxis toward various chemicals. We have isolated several more serine taxis mutants and a mutant apparently defective in the aspartate receptor. This mutant shows no chemotaxis toward aspartate and glutamate; reduced taxis toward alanine, cysteine, glycine, and serine; and normal taxis toward sugars.8 Further study of such specifically nonchemotactic mutants should help to reveal the mechanisms of chemoreception and behavior in bacteria. We thank M. M. Dahl for isolating the serine taxis mutants. * This research was supported by a grant from the U.S. Public Health Service. 1 Adler, J., Science, 153, 708 (1966). 2Ibid., 166, 1588 (1969). 8 Armstrong, J. B., J. Adler, and M. M. Dahl, J. Bacteriol., 93, 390 (1967). 4Armstrong, J. B., and J. Adler, J. Bacteriol., 97, 156 (1969). 6 Armstrong, J. B., and J. Adler, Genetics, 61, 61 (1969). 6 Kaiser, A. D., and D. S. Hogness, J. Mol. Biol., 2, 392 (1960). 7Adler, J., in preparation. 8 Mesibov, R. E., and J. Adler, in preparation. I Adler, J., Hazelbauer, G. L., and M. M. Dahl, in preparation. Downloaded by guest on September 27, 2021