Jul 6, 1981 - allowed assignment of all mutants to the trpE, trpD, trpB, or trpA gene, confirming earlier resultswith the same mutants (Johnston et al., Mol. Gen ...
JOURNAL OF BACTERIOLOGY, Mar. 1982, 0021-9193/82/031135-03$02.00/0
p. 1135-1137
Vol. 149, No.
3
Regulation of TrVptophan Genes in Rhizobium leguminosarum ERIK HOLMGRENt AND IRVING P. CRAWFORD* Department of Microbiology, University of Iowa, Iowa City, Iowa 52242
Received 6 July 1981/Accepted 9 November 1981
Twelve tryptophan auxotrophs of Rhizobium leguminosarum were characterized biochemically. They were grown in complex and minimal media with several carbon sources, in both limiting and excess tryptophan. Missing enzyme activities allowed assignment of all mutants to the trpE, trpD, trpB, or trpA gene, confirming earlier results with the same mutants (Johnston et al., Mol. Gen. Genet. 165:323-330, 1978). In regulatory experiments, only the first enzyme of the pathway, anthranilate synthase, responded (about 15-fold) to tryptophan excess or limitation.
Genetic markers in Rhizobium leguminosarum have been mapped on a single circular linkage group (2). Chromosomal genes can be transferred in heterospecific crosses from R. leguminosarum to R. meliloti, but when crosses are made in the opposite direction, only rare recombinants are formed. These recombinants usually contain an R-prime plasmid where the plasmid has acquired part of the R. meliloti chromosome (7). Johnston et al. (6) isolated and characterized three such R primes which as a group appear to carry all of the structural genes of the tryptophan pathway. Although one or another of these R primes can complement any Pseudomonas aeruginosa trp auxotroph, their activities are not expressed in Escherichia coli. Rhizobial trp genes have been mapped by conjugation (2, 8, 9) and are found in three widely separate locations. Each R-prime plasmid appears to carry the genes from one such location. From complementation experiments in P. aeruginosa, the respective R primes carry genes for the first enzyme (trpE), the second and fourth enzymes (trpD and trpC), or the third and fifth enzymes (trpF, and trpB and trpA) (6). Our study was undertaken to confirm the rhizobial gene-enzyme assignments deduced from genetic tests across a wide evolutionary distance and to examine the regulation of the trp genes in a representative fast-growing Rhizobium species. R. leguminosarum mutants (Table 1) obtained from J. E. Beringer and A. W. B. Johnston were grown in complete (TY) or minimal (Y) medium (1). For extract preparation, cells were grown for 48 h at 30°C (absorbance at 540 nm = 0.25 to 0.50), centrifuged, washed twice in 0.1 M potassium phosphate (pH 7.8), and disrupted by sonication after suspension in the same buffer. A t Present address: KabiGen AB, 11287 Stockholm, Sweden.
30,000 x g supernatant was used for enzyme assays. Because of instability, the anthranilate synthase (AS) activities, ASG (glutamine dependent) and ASN (ammonia dependent), were both assayed on the same day after overnight dialysis against buffer containing 30% glycerol. The other enzymes were stable. All activities were assayed by standard methods (3, 12). For complementation tests, donor and recipient cells were washed off 3-day TY slants in 2 ml of TY, mixed, and incubated at 30°C overnight before plating on selective media. A modification of this procedure was to wash cells off 24-h TY plates with 5 ml of sterile water. One milliliter of donor and 1 ml of recipient were mixed, centrifuged, suspended in 0.2 ml of water, and spread on a TY plate. After 24 h at 30°C, the cells were washed off the plate with 5 ml of sterile water, diluted, and plated on selective media. Crude extracts of the R. leguminosarum tryptophan auxotrophs isolated and partially characterized by Beringer et al. (2) and Johnston et al. (6, 7) were prepared, and the activities of the tryptophan pathway enzymes were measured. Enzyme activity levels were similar whether cells were grown in TY medium or in Y medium with succinate or glucose as the carbon source. Three R-prime-containing strains, ML24, ML73, and ML88, were used in gene complementation analysis. As shown by Johnston et al. (6) and confirmed in this laboratory, ML24 carries R-prime plasmid pAJ24JI bearing the trpF, trpA, and trpB genes, ML73 carries pAJ73JI bearing trpD and trpC, and ML88 carries pAJ88JI bearing trpE. These R-prime plasmids were transferred to each auxotroph by conjugation as described above. Both Trp+ and kana-
mycin-resistant transconjugants were selected. When the chromosomal trp marker was comple-
1135
J. BACTERIOL.
NOTES
1136
TABLE 1. Bacterial strains used in this study Genotype
Strain"
phe-J ser-l str-36 met-14 trpEI7 str-87 p1e-I trpD4O str-196 ura-14 trpD12 str-37 ura-14 trpD14 str-69 phe-I trpB41 str-203 ura-14 trpBI6 str-86 phe-l trpBJ3 str41 phe-I trpAl5 str-82 phe-I trpA42 str-188 phe-I trp-18 str-94 phe-I trp-19 rif-86 rif trp49 spc ura-14 trpB16 str-86 spc-18/pAJ24J1
893 1699 1560 1323 1446 1575 1062 905 1050 1544
1156" 1159" 1741h ML24 ML73 ML88
iura-14 trpD14/pAJ73JI met-14 trpEI7 str/pAJ88JI
a All strains are auxotrophic derivatives of R. leguminosarum. The plasmids and their derivation are described in the text. ' These mutations may be located in either trpA or trpB (see text).
mented by the R plasmid, the frequency of transfer of the Trp+ and kanamycin markers was about the same, ca. 5 x 10-5 per donor cell. No discrepancies were found between genetic and biochemical characterizations of the mutants. Of the 12 mutants studied, 9 were defective in only one enzyme activity and could therefore be classified unambiguously (Table 2). No mutants defective in trpC, trpF, or trpG were found. Strain 1699 had no ASG or ASN activity and is therefore a trpE mutant. Strains 1560, 1323, and 1446 lacked anthranilate phosphoribosyltransferase activity, so by definition they are defective in the trpD gene. Strains 905, 1062, and 1575 are trpB mutants, and strains 1050 and 1544 are trpA mutants, so assigned by the absence of one of the two tryptophan synthase half-reactions. Strains 1156, 1159, and
1741 showed very little activity in either tryptophan synthase half-reaction. These mutations may be located in either trpA or trpB. However, no convincing complementation was seen when crude extracts of strains 1156 and 1159 were mixed with extracts of tryptophan synthase mutants having good tryptophan synthase A or tryptophan synthase B activity (data not shown). Most of the strains used in this study were studied in parallel cultures grown with excess and limiting amounts of tryptophan. Enzyme assays done with cells grown in Y medium with 0.2% glucose and 20 or 3 pg of tryptophan per ml are shown in Table 3. Enzyme specific activities are presented along with the ratio of the activity means from mutants grown in limiting versus excess tryptophan. In calculating those means, assays of the defective enzymes were omitted. Although variation in the activity levels between different strains was sometimes considerable, it is obvious that only the first enzyme, AS, clearly responded to the level of tryptophan in the growth medium. All other enzyme activities appeared to be unregulated. This same result was also obtained with dialyzed extracts; however, the activity of AS was drastically reduced when the extracts were dialyzed or frozen and thawed. Activities of the other enzymes were unaffected when extracts were treated in the same way.
This type of regulation, affecting only the first in the pathway, was reported in the initial studies of Acinetobacter calcoaceticus trp mutants (13), but that interpretation had to be revised later when the enzymes were stabilized with glycerol (4, 10, 11). All seven of the Acinetobacter trp gene products showed measurable derepression when enzyme activity was determined in stabilized extracts of trp auxotrophs (3). Chromobacterium violaceeum does not regulate the synthesis of the tryptophan synthetic
enzyme
TABLE 2. Patterns of complementation by R primes and enzyme content of tryptophan auxotrophs Enzyme presence" Complementation' Strain
893 1699 trpE 1560 trpD 905 trpB 1050 trpA 1156 trpA or trpB
pAJ88 + -
-
pAJ73
+ -
AS
PRT
PRAI
InGPS
+ -
±
+
±
+
+
+ + +
-
+
+ +
+ +
+
+ + + +
+
+
+
pAJ24
-
+ +
TS-A
TS-B
+ +
+
± +
+
-
+
+
a From complementation of P. aeruginosa mutants (6; also confirmed in this laboratory), it is known that pAJ88 carries trpE, pAJ73 carries trpD and trpC, and pAJ24 carries trpF, trpB, and trpA. Identical patterns of complementation and enzyme presence were shown by strains 1560, 1323, and 1446; 905, 1062, and 1575; 1050 and 1544; and 1156, 1159, and 1741. hAS, Anthranilate synthase; PRT, anthranilate phosphoribosyltransferase; PRAI, phosphoribosylanthranilate isomerase; InGPS, indole-3-glycerol phosphate synthase; and TS-A and TS-B. tryptophan synthase (halfreactions).
NOTES
VOL. 149, 1982
1137
TABLE 3. Enzyme levels in R. leguminosarum strains grown in limiting and excess tryptophan Enzyme sp actb Strain
Growth conditiona
ASG, trpEG
ASN, trpE
PRT, trpD
PRAI,
InGPS,
TS-A,
TS-B,
trpF
trpC
trpA
trpB
893 1699 1560 905 1050 Meanc
Tryptophan limit Tryptophan limit Tryptophan limit Tryptophan limit Tryptophan limit Tryptophan limit
