Vol. 129, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIoLOGY, Jan. 1977, p. 156-165 Copyright C 1977 American Society for Microbiology
Amino Acid Chemoreceptors of Bacillus subtilis GEORGE W. ORDAL,* DAVID P. VILLANI, AND KATHARINE J. GIBSON School of Basic Medical Sciences and Department of Biochemistry, University of Illinois, Urbana, Illinois 61801 Received for publication 7 July 1976
Specificities of chemoreceptors for the 20 common amino acids, toward which Bacillus subtilis shows chemotaxis, were assessed by competition ("jamming") experiments using a modification of the traditional capillary assay, called the "sensitivity capillary assay." Many amino acids were sensed by at least two chemoreceptors. All the highest affinity chemoreceptors for the amino acids were distinct, except glutamate and aspartate, which may share one chemoreceptor, and tyrosine, for which the data could not be collected due to low solubility. The data suggest the hypothesis that each amino acid-chemoreceptor complex binds to a different signaler (from which signals travel to the flagella to modify behavior appropriately), and that many of the signalers can also bind other attractant-chemoreceptor complexes as antagonists (no signals to flagella).
Bacillus subtilis shows taxis to all 20 amino Ordal and Goldman (4). Assays were carried out for acids (5, 7). How many receptors are used to 15 min at 37°C on bacteria at an optical density at mediate this chemotaxis, and what are the 600 nm of 0.01 unit (3.4 x 106 bacteria/ml). In these assays, each capillary contained 3.16-fold the conspecificities of these receptors? According to de centration of attractant found in its corresponding Jong et al. (1), B. subtilis has an asparagine pond (see Mesibov et al. [2] for discussion of method receptor, which senses asparagine and gluta- and Fig. 1 for diagram of similar experimental mine, an isoleucine receptor, which detects setup). A wide concentration range is broken down isoleucine, valine, leucine, phenylalanine, into V/i0-fold intervals, and the strength of taxis serine, threonine, cysteine, and methionine, within each interval is assessed to construct a "sensiand an alanine receptor, which detects alanine tivity curve." The number of bacteria that enter the and proline. However, their method involved capillary in such an assay is proportional to the putting 1 mM attractant in a capillary and difference in number of receptors titrated with atattempting to inhibit chemotaxis to that at- tractant at the two end points (i.e., the capillary tractant by putting a different substance at 10 concentration and the pond concentration) (2). These numbers of bacteria are graphed as a function mM in both capillary and suspension. Al- of the geometric mean of the concentrations of atthough in a simple system of noninteracting tractant in and pond. Standard deviation chemoreceptors, in which no amino acid is in this assaycapillary was found to be 15% (excluding Poisson detected by two or more chemoreceptors, such sampling error) and is illustrated by the error bars a method is effective, the data in this article in Fig. 2. Day-to-day variations were larger, alindicate that amino acid chemoreception in B. though not quantitated. However, all conclusions subtilis is complex. Indeed, it appears that were drawn from comparison of control and experisome amino acids are sensed by several chem- mental curves from the same day's experiments. Jamming and self-jamming capillary assays. A oreceptors for each, that nearly all the "highest affinity" chemoreceptors for the 20 amino "jamming capillary assay" is one in which a second acid is present at a specified concentration in acids are distinct, and that there is a multi- amino capillaries and ponds in a sensitivity capillary tude of instances in which an amino acid can all assay of the first amino acid. A "self-jamming capilprevent taxis to a different amino acid by lary assay" is the same, except the first amino acid binding at its own receptor (i.e., not competi- itself is used instead of a second one. In the jamming tive inhibition). capillary assay, we refer to this second amino acid as the "ja.mmer" and to the first one, which is serving as the attractant, as the "victim" (see Fig. 1 for diagram of the experimental setup used in the jamming capillary assay). To prevent oxidation of low concentrations of cysteine, 1 mM dithiothreitol was included in all experiments involving a sensitivity capillary assay of cysteine. Taxis to other amino
MATERIALS AND METHODS Bacteria. B. subtilis OI8 has been described (4). Media. Tryptone broth, minimal medium, and chemotaxis buffer were used as described (4). Sensitivity capillary assays. Bacteria were grown and prepared for capillary assays as mentioned in 1
AC
AMINO ACID CHEMORECEPTORS OF B. SUBTILIS
VOL. 129, 1977
Gloss plote
l1ry
3.16XmM victim suspension + X mM
+
157
of the sensitivity curve to 10-fold higher concentrations approximates the value of the dissociation constant, Kd (see Appendix). Thus, Fig. 3 shows self-jamming sensitivity curves
YmM jommer
victim + Y mM jommer)
FIG. 1. Diagram of setup for sample in a jamming capillary assay. See text for definition of terms.
acids, such as threonine, aspartate, and glutamate, not affected by dithiothreitol.
was
-J -J
z
RESULTS
Determination of dissociation constants. The object of determining the dissociation constants for the receptors was twofold: (i) to aid in identifying the receptor in experiments to isolate and study it and (ii) to help us design a rationalized method (see below) for determining specificity of receptors. According to Mesibov et al. (2), one means of determining the dissociation constant of a chemoreceptor for its ligand (attractant) is a sensitivity capillary assay (see Materials and Methods for description). In the experiment shown in Fig. 2, the concentration of threonine in the capillary was always 3.16-fold greater than that in the pond with the bacteria: hence, one was able to measure "strength of chemotaxis" in each of these small intervals. The center of symmetry of the peak is the concentration at which chemotaxis is most potent and hence the value of the dissociation constant (see reference 2 for discussion of this point). Thus, there are two chemoreceptors for threonine, having dissociation constants of about 10 ,uM and about 10 mM.
LI0)
50
-4 cK-5 10-6 10-7 10-' 10-2 10-3 THREONINE GEOMETRIC MEAN MOLARITY
FIG. 2. Sensitivity curve for threonine taxis. See Materials and Methods for procedures.
NO ADDITION
+10
M
2'14 31 M
This method was applied to other amino co acids. However, since many amino acids appeared to have two or three chemoreceptors, locating the centers of symmetry of peaks was often difficult. Therefore, we designed the following procedure for determining the value of the dissociation constant of the receptor having the lowest dissociation constant for its amino acid ligand (highest affinity receptor). This "self-jamming sensitivity" method is a 10-5 10-4 10-3 o-2 lO4 variation of the sensitivity capillary assay (see VALINE GEOMETRIC MEAN MOLARITY Materials and Methods). However, in addition FIG. 3. Self-jamming capillary assays for valine. to the normal contents of capillary and sus- See Materials and Methods for procedures. Symbols: pension, one adds a constant concentration of (0) no addition, (O) 10 pM valine as jammer, (-) 32 amino acid to both. Several concentrations are .M valine as jammer, and (A) 100 pM valine as tried, and the one that shifts the "rising part" jammer.
158
J. BACTERIOL.
ORDAL, VILLANI, AND GIBSON
for valine and indicates the dissociation constant to be about 30 uM. This method was found to be reproducible and to give self-consistent results in competition experiments as indicated below. Table 1 indicates the dissociation constants for each amino acid as obtained by this means. Specificities of chemoreceptors. One purpose of identifying the approximate Kd is the following: if two amino acids are detected by the same chemoreceptor, then the Kd concentration of the first should shift the sensitivity curve of the second 10-fold, and the Kd concentration of the second should shift the sensitivity curve of the first 10-fold. If the two amino acids, however, are seen by different receptors, then the presence of the Kd concentration of one will not interfere with the sensitivity curve for the other in this "jamming sensitivity assay." By seeking "equivalence concentrations" (in this case, approximately the Kd concentration) and then demanding that the jammer (see definition in Materials and Methods) shift the victim's (see definition in Materials and Methods) curve about 10-fold, we minimize the possible problem that the jammer binds to the victim's chemoreceptor, but at significantly higher (or lower) concentrations. By focusing on the shift of the low concentration end of the victim's sensitivity TABLE 1. Approximate values of Kd a Kd (M) Amino acid 3.2 x 10-7 Ala 10-4 Arg 5.6 x 10-5 Asn 10-2 Asp 10-5 Cysb 3.2 x 10-2 Glu 3.2 x 10-5 Gln 3.2 x 10-4 Gly 3.2 x 10-3 His 10-4 Ilu 3.2 x 10-5 Leu 10-3 Lys 1-4 Met 3.2 x 10-4 Phe 1o-6 Pro 3.2 x 10-5 Ser 5.6 x 10-6 Thr 3.2 x 1O-3 Trp 3.2 x 10-4 TyrC 3.2 x 10-5 Val a From self-jamming sensitivity assays. See Materials and Methods, Results, and Appendix. b Dithiothreitol, 1 mM, present. c Shift of sensitivity curve somewhat less than 10fold (problem of insolubility of this amino acid).
curve, we are able to examine the properties of the highest affinity chemoreceptor. Since many amino acids seem to have several chemoreceptors, data using high concentrations of the victim are likely to be hard to interpret since a jammer might bind to some of the chemoreceptors but not others. Jamming sensitivity assays were carried out on most of the 380 possible combinations (20 victims "jammed" by 19 jammers each) (Table 2). As shown in Fig. 4 and Table 2, many amino acids mutually jammed each other's taxis. However, in only two instances-glutamate and aspartate, glutamine and asparagine-was the further expectation fulfilled, that all other amino acids would affect each member of the mutually jamming pair in the same way when each member of the pair was the victim in jamming sensitivity assays. Figure 4 shows a more typical example: phenylalanine, lysine, and serine jammed valine taxis but not isoleucine taxis, even though valine and isoleucine exhibited mutual
jamming.
In general, two criteria were applied to conclude that two amino acids are not sensed by the same chemoreceptor: (i) lack of reciprocal jamming, or (ii) given reciprocal jamming, the jamming by other amino acids of taxis to one but not the other. Table 3 lists all the pairs of amino acids that can be considered under criterion ii, both those for which reciprocal jamming has been observed and those for which it has not been tested. Glutamine and asparagine taxes are jammed by no other amino acids except glutamate and aspartate. However, only glutamine (not asparagine) jams lysine and glycine taxes and, since it presumably does so by binding at the glutamine chemoreceptor, glutamine is not on the same chemoreceptor as asparagine. Finally, the numerous instances of nonreciprocal jamming -when one amino acid inhibits taxis to the second but not vice versa are recorded in Table 4 and illustrated in Fig. 5.
DISCUSSION The main conclusion from this study is that, aside from glutamate and aspartate, which probably share a site, and for tyrosine, for which the data could not be collected due to its low solubility, each of the amino acids is recognized by a different site but that there is extensive interaction among the sites. One might loosely separate the amino acids into five groups, which reflect the degree of mutual
159
AMINO ACID CHEMORECEPTORS OF B. SUBTILIS
VOL. 129, 1977
TABLE 2. Amino acid jamming experimentsa Victim° Jammer
---
-
----
--
_
Asp Glu Asn GLIn His Arg Ala Cys' Gly Ilu Leu Lys Met Phe Pro Ser Thr Trp Val Asp Glu Asn Gln His Arg Ala Cys Gly
flu Leu Lys Met Phe Pro Ser Thr Trp Val Tyr
Yes No No No No No No No No No No No No No
No No No No No
Yes Yes Yes No No Yes No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No
Yes Yes Yes Yes Yes Yes Yes Yes No No No No Yes Yes No No No Yes No No No No Yes No No Yes No Yes Yes No Yes No No Yes No Yes No No No No No No No No
Yes Yes Yes Yes Yes Yes Yes Yes No No Yes No No No No No No No Yes Yes Yes
Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes No Yes Yes Yes Yes No Yes Yes
Yes
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Yes No No Yes No Yes Yes Yes
Yes Yes
Yes Yes Yes
No Yes Yes No Yes No No No No No Yes No Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes Yes No Yes Yes Yes Yes No Yes Yes No Yes Yes No Yes Yes Yes Yes No No No Yes
Yes Yes Yes Yes Yes Yes Yes
Yes Yes No No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No Yes Yes Yes Yes Yes Yes Yes Yes No No No
Yes Yes Yes
aConcentrations used are given in Table 1. "Yes" means jamming occurred. "No" means no significant effect of jammer on taxis to the victim amino acid. All experiments in the top 4 rows of the left-hand 4 columns and all "no's" in the bottom 15 rows of the right-hand 14 columns represent experiments done on two or three separate occasions. Other experiments were done once (usually) or twice. c Dithiothreitol, 1 mM, present in experiments in this column. b
jamming: one containing glutamate and aspartate, one containing glutamine and asparagine, one containing histidine, one containing arginine, and the last containing alanine, proline, glycine, leucine, phenylalanine, isoleucine, threonine, valine, methionine, cysteine, tryptophan, lysine, and serine. The "dominant" group, based upon ability to jam others' taxes but not be itself jammed, is the glutamate-aspartate group, and, to a lesser extent, the glutamine-asparagine group. Histidine is fairly independent. Arginine is least dominant since its taxis is jammed by many other amino acids, but it is itself fairly impotent as a jammer. It should be emphasized that in all instances of jamming, the victim's sensitivity curve has shifted about 10-fold, implying that the Ki for the amino acid as jammer equals the Kd for the same amino acid as attractant (values in Table 1). In view of the 52 instances of nonreciprocal jamming and of the 35 instances of mutual jamming, despite distinctness of receptors, all jamming results from an amino acid binding to its own receptor. The mechanism by which one amino acid interferes with taxis to a second, apparently without binding at the sec-
ond site, is unknown. Recently, Strange and Koshland (6) found that ribose could interfere with galactose taxis in Salmonella typhimurium by binding at its own (ribose) chemoreceptor. They postulated that the ribose-chemoreceptor complex binds to a hypothetical protein and decreases the likelihood that a galactose-chemoreceptor complex will bind to it. Since binding by the galactose-chemoreceptor complex is necessary for chemotactic signals to reach the flagella, the chemotaxis is blocked. This protein may correspond to the trg protein of Ordal and Adler (3), since it is needed for taxis to both galactose and ribose. In a similar vein, there might exist such proteins (termed "signalers" [31) for amino acids in B. subtilis such that binding of one attractant-chemoreceptor complex to the signaler inhibits binding of another attractantchemoreceptor complex. Each attractantchemoreceptor complex would have its own site on a signaler. (Glutamate-chemoreceptor complex and aspartate-chemoreceptor complex might share one.) Inhibition could occur when an attractant-chemoreceptor complex binds to its own signaler site or, as a competitive inhibitor (i.e., as an antagonist: no sig-
160
J. BACTERIOL.
ORDAL, VILLANI, AND GIBSON ;
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JOURNAL OF BACTERIoLOGY, Jan. 1977, p. 156-165 Copyright C 1977 American Society for Microbiology
Amino Acid Chemoreceptors of Bacillus subtilis GEORGE W. ORDAL,* DAVID P. VILLANI, AND KATHARINE J. GIBSON School of Basic Medical Sciences and Department of Biochemistry, University of Illinois, Urbana, Illinois 61801 Received for publication 7 July 1976
Specificities of chemoreceptors for the 20 common amino acids, toward which Bacillus subtilis shows chemotaxis, were assessed by competition ("jamming") experiments using a modification of the traditional capillary assay, called the "sensitivity capillary assay." Many amino acids were sensed by at least two chemoreceptors. All the highest affinity chemoreceptors for the amino acids were distinct, except glutamate and aspartate, which may share one chemoreceptor, and tyrosine, for which the data could not be collected due to low solubility. The data suggest the hypothesis that each amino acid-chemoreceptor complex binds to a different signaler (from which signals travel to the flagella to modify behavior appropriately), and that many of the signalers can also bind other attractant-chemoreceptor complexes as antagonists (no signals to flagella).
Bacillus subtilis shows taxis to all 20 amino Ordal and Goldman (4). Assays were carried out for acids (5, 7). How many receptors are used to 15 min at 37°C on bacteria at an optical density at mediate this chemotaxis, and what are the 600 nm of 0.01 unit (3.4 x 106 bacteria/ml). In these assays, each capillary contained 3.16-fold the conspecificities of these receptors? According to de centration of attractant found in its corresponding Jong et al. (1), B. subtilis has an asparagine pond (see Mesibov et al. [2] for discussion of method receptor, which senses asparagine and gluta- and Fig. 1 for diagram of similar experimental mine, an isoleucine receptor, which detects setup). A wide concentration range is broken down isoleucine, valine, leucine, phenylalanine, into V/i0-fold intervals, and the strength of taxis serine, threonine, cysteine, and methionine, within each interval is assessed to construct a "sensiand an alanine receptor, which detects alanine tivity curve." The number of bacteria that enter the and proline. However, their method involved capillary in such an assay is proportional to the putting 1 mM attractant in a capillary and difference in number of receptors titrated with atattempting to inhibit chemotaxis to that at- tractant at the two end points (i.e., the capillary tractant by putting a different substance at 10 concentration and the pond concentration) (2). These numbers of bacteria are graphed as a function mM in both capillary and suspension. Al- of the geometric mean of the concentrations of atthough in a simple system of noninteracting tractant in and pond. Standard deviation chemoreceptors, in which no amino acid is in this assaycapillary was found to be 15% (excluding Poisson detected by two or more chemoreceptors, such sampling error) and is illustrated by the error bars a method is effective, the data in this article in Fig. 2. Day-to-day variations were larger, alindicate that amino acid chemoreception in B. though not quantitated. However, all conclusions subtilis is complex. Indeed, it appears that were drawn from comparison of control and experisome amino acids are sensed by several chem- mental curves from the same day's experiments. Jamming and self-jamming capillary assays. A oreceptors for each, that nearly all the "highest affinity" chemoreceptors for the 20 amino "jamming capillary assay" is one in which a second acid is present at a specified concentration in acids are distinct, and that there is a multi- amino capillaries and ponds in a sensitivity capillary tude of instances in which an amino acid can all assay of the first amino acid. A "self-jamming capilprevent taxis to a different amino acid by lary assay" is the same, except the first amino acid binding at its own receptor (i.e., not competi- itself is used instead of a second one. In the jamming tive inhibition). capillary assay, we refer to this second amino acid as the "ja.mmer" and to the first one, which is serving as the attractant, as the "victim" (see Fig. 1 for diagram of the experimental setup used in the jamming capillary assay). To prevent oxidation of low concentrations of cysteine, 1 mM dithiothreitol was included in all experiments involving a sensitivity capillary assay of cysteine. Taxis to other amino
MATERIALS AND METHODS Bacteria. B. subtilis OI8 has been described (4). Media. Tryptone broth, minimal medium, and chemotaxis buffer were used as described (4). Sensitivity capillary assays. Bacteria were grown and prepared for capillary assays as mentioned in 1
AC
AMINO ACID CHEMORECEPTORS OF B. SUBTILIS
VOL. 129, 1977
Gloss plote
l1ry
3.16XmM victim suspension + X mM
+
157
of the sensitivity curve to 10-fold higher concentrations approximates the value of the dissociation constant, Kd (see Appendix). Thus, Fig. 3 shows self-jamming sensitivity curves
YmM jommer
victim + Y mM jommer)
FIG. 1. Diagram of setup for sample in a jamming capillary assay. See text for definition of terms.
acids, such as threonine, aspartate, and glutamate, not affected by dithiothreitol.
was
-J -J
z
RESULTS
Determination of dissociation constants. The object of determining the dissociation constants for the receptors was twofold: (i) to aid in identifying the receptor in experiments to isolate and study it and (ii) to help us design a rationalized method (see below) for determining specificity of receptors. According to Mesibov et al. (2), one means of determining the dissociation constant of a chemoreceptor for its ligand (attractant) is a sensitivity capillary assay (see Materials and Methods for description). In the experiment shown in Fig. 2, the concentration of threonine in the capillary was always 3.16-fold greater than that in the pond with the bacteria: hence, one was able to measure "strength of chemotaxis" in each of these small intervals. The center of symmetry of the peak is the concentration at which chemotaxis is most potent and hence the value of the dissociation constant (see reference 2 for discussion of this point). Thus, there are two chemoreceptors for threonine, having dissociation constants of about 10 ,uM and about 10 mM.
LI0)
50
-4 cK-5 10-6 10-7 10-' 10-2 10-3 THREONINE GEOMETRIC MEAN MOLARITY
FIG. 2. Sensitivity curve for threonine taxis. See Materials and Methods for procedures.
NO ADDITION
+10
M
2'14 31 M
This method was applied to other amino co acids. However, since many amino acids appeared to have two or three chemoreceptors, locating the centers of symmetry of peaks was often difficult. Therefore, we designed the following procedure for determining the value of the dissociation constant of the receptor having the lowest dissociation constant for its amino acid ligand (highest affinity receptor). This "self-jamming sensitivity" method is a 10-5 10-4 10-3 o-2 lO4 variation of the sensitivity capillary assay (see VALINE GEOMETRIC MEAN MOLARITY Materials and Methods). However, in addition FIG. 3. Self-jamming capillary assays for valine. to the normal contents of capillary and sus- See Materials and Methods for procedures. Symbols: pension, one adds a constant concentration of (0) no addition, (O) 10 pM valine as jammer, (-) 32 amino acid to both. Several concentrations are .M valine as jammer, and (A) 100 pM valine as tried, and the one that shifts the "rising part" jammer.
158
J. BACTERIOL.
ORDAL, VILLANI, AND GIBSON
for valine and indicates the dissociation constant to be about 30 uM. This method was found to be reproducible and to give self-consistent results in competition experiments as indicated below. Table 1 indicates the dissociation constants for each amino acid as obtained by this means. Specificities of chemoreceptors. One purpose of identifying the approximate Kd is the following: if two amino acids are detected by the same chemoreceptor, then the Kd concentration of the first should shift the sensitivity curve of the second 10-fold, and the Kd concentration of the second should shift the sensitivity curve of the first 10-fold. If the two amino acids, however, are seen by different receptors, then the presence of the Kd concentration of one will not interfere with the sensitivity curve for the other in this "jamming sensitivity assay." By seeking "equivalence concentrations" (in this case, approximately the Kd concentration) and then demanding that the jammer (see definition in Materials and Methods) shift the victim's (see definition in Materials and Methods) curve about 10-fold, we minimize the possible problem that the jammer binds to the victim's chemoreceptor, but at significantly higher (or lower) concentrations. By focusing on the shift of the low concentration end of the victim's sensitivity TABLE 1. Approximate values of Kd a Kd (M) Amino acid 3.2 x 10-7 Ala 10-4 Arg 5.6 x 10-5 Asn 10-2 Asp 10-5 Cysb 3.2 x 10-2 Glu 3.2 x 10-5 Gln 3.2 x 10-4 Gly 3.2 x 10-3 His 10-4 Ilu 3.2 x 10-5 Leu 10-3 Lys 1-4 Met 3.2 x 10-4 Phe 1o-6 Pro 3.2 x 10-5 Ser 5.6 x 10-6 Thr 3.2 x 1O-3 Trp 3.2 x 10-4 TyrC 3.2 x 10-5 Val a From self-jamming sensitivity assays. See Materials and Methods, Results, and Appendix. b Dithiothreitol, 1 mM, present. c Shift of sensitivity curve somewhat less than 10fold (problem of insolubility of this amino acid).
curve, we are able to examine the properties of the highest affinity chemoreceptor. Since many amino acids seem to have several chemoreceptors, data using high concentrations of the victim are likely to be hard to interpret since a jammer might bind to some of the chemoreceptors but not others. Jamming sensitivity assays were carried out on most of the 380 possible combinations (20 victims "jammed" by 19 jammers each) (Table 2). As shown in Fig. 4 and Table 2, many amino acids mutually jammed each other's taxis. However, in only two instances-glutamate and aspartate, glutamine and asparagine-was the further expectation fulfilled, that all other amino acids would affect each member of the mutually jamming pair in the same way when each member of the pair was the victim in jamming sensitivity assays. Figure 4 shows a more typical example: phenylalanine, lysine, and serine jammed valine taxis but not isoleucine taxis, even though valine and isoleucine exhibited mutual
jamming.
In general, two criteria were applied to conclude that two amino acids are not sensed by the same chemoreceptor: (i) lack of reciprocal jamming, or (ii) given reciprocal jamming, the jamming by other amino acids of taxis to one but not the other. Table 3 lists all the pairs of amino acids that can be considered under criterion ii, both those for which reciprocal jamming has been observed and those for which it has not been tested. Glutamine and asparagine taxes are jammed by no other amino acids except glutamate and aspartate. However, only glutamine (not asparagine) jams lysine and glycine taxes and, since it presumably does so by binding at the glutamine chemoreceptor, glutamine is not on the same chemoreceptor as asparagine. Finally, the numerous instances of nonreciprocal jamming -when one amino acid inhibits taxis to the second but not vice versa are recorded in Table 4 and illustrated in Fig. 5.
DISCUSSION The main conclusion from this study is that, aside from glutamate and aspartate, which probably share a site, and for tyrosine, for which the data could not be collected due to its low solubility, each of the amino acids is recognized by a different site but that there is extensive interaction among the sites. One might loosely separate the amino acids into five groups, which reflect the degree of mutual
159
AMINO ACID CHEMORECEPTORS OF B. SUBTILIS
VOL. 129, 1977
TABLE 2. Amino acid jamming experimentsa Victim° Jammer
---
-
----
--
_
Asp Glu Asn GLIn His Arg Ala Cys' Gly Ilu Leu Lys Met Phe Pro Ser Thr Trp Val Asp Glu Asn Gln His Arg Ala Cys Gly
flu Leu Lys Met Phe Pro Ser Thr Trp Val Tyr
Yes No No No No No No No No No No No No No
No No No No No
Yes Yes Yes No No Yes No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No
Yes Yes Yes Yes Yes Yes Yes Yes No No No No Yes Yes No No No Yes No No No No Yes No No Yes No Yes Yes No Yes No No Yes No Yes No No No No No No No No
Yes Yes Yes Yes Yes Yes Yes Yes No No Yes No No No No No No No Yes Yes Yes
Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes No Yes Yes Yes Yes No Yes Yes
Yes
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Yes No No Yes No Yes Yes Yes
Yes Yes
Yes Yes Yes
No Yes Yes No Yes No No No No No Yes No Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes Yes No Yes Yes Yes Yes No Yes Yes No Yes Yes No Yes Yes Yes Yes No No No Yes
Yes Yes Yes Yes Yes Yes Yes
Yes Yes No No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No Yes Yes Yes Yes Yes Yes Yes Yes No No No
Yes Yes Yes
aConcentrations used are given in Table 1. "Yes" means jamming occurred. "No" means no significant effect of jammer on taxis to the victim amino acid. All experiments in the top 4 rows of the left-hand 4 columns and all "no's" in the bottom 15 rows of the right-hand 14 columns represent experiments done on two or three separate occasions. Other experiments were done once (usually) or twice. c Dithiothreitol, 1 mM, present in experiments in this column. b
jamming: one containing glutamate and aspartate, one containing glutamine and asparagine, one containing histidine, one containing arginine, and the last containing alanine, proline, glycine, leucine, phenylalanine, isoleucine, threonine, valine, methionine, cysteine, tryptophan, lysine, and serine. The "dominant" group, based upon ability to jam others' taxes but not be itself jammed, is the glutamate-aspartate group, and, to a lesser extent, the glutamine-asparagine group. Histidine is fairly independent. Arginine is least dominant since its taxis is jammed by many other amino acids, but it is itself fairly impotent as a jammer. It should be emphasized that in all instances of jamming, the victim's sensitivity curve has shifted about 10-fold, implying that the Ki for the amino acid as jammer equals the Kd for the same amino acid as attractant (values in Table 1). In view of the 52 instances of nonreciprocal jamming and of the 35 instances of mutual jamming, despite distinctness of receptors, all jamming results from an amino acid binding to its own receptor. The mechanism by which one amino acid interferes with taxis to a second, apparently without binding at the sec-
ond site, is unknown. Recently, Strange and Koshland (6) found that ribose could interfere with galactose taxis in Salmonella typhimurium by binding at its own (ribose) chemoreceptor. They postulated that the ribose-chemoreceptor complex binds to a hypothetical protein and decreases the likelihood that a galactose-chemoreceptor complex will bind to it. Since binding by the galactose-chemoreceptor complex is necessary for chemotactic signals to reach the flagella, the chemotaxis is blocked. This protein may correspond to the trg protein of Ordal and Adler (3), since it is needed for taxis to both galactose and ribose. In a similar vein, there might exist such proteins (termed "signalers" [31) for amino acids in B. subtilis such that binding of one attractant-chemoreceptor complex to the signaler inhibits binding of another attractantchemoreceptor complex. Each attractantchemoreceptor complex would have its own site on a signaler. (Glutamate-chemoreceptor complex and aspartate-chemoreceptor complex might share one.) Inhibition could occur when an attractant-chemoreceptor complex binds to its own signaler site or, as a competitive inhibitor (i.e., as an antagonist: no sig-
160
J. BACTERIOL.
ORDAL, VILLANI, AND GIBSON ;
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