A Temperature-Sensitive trpS Mutation Interferes with trp RNA ...

JOURNAL OF BACTERIOLOGY, Nov. 1996, p. 6518–6524 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 178, No. 22

A Temperature-Sensitive trpS Mutation Interferes with trp RNA-Binding Attenuation Protein (TRAP) Regulation of trp Gene Expression in Bacillus subtilis ALFRED IAN LEE, JOSEPH P. SARSERO,

AND

CHARLES YANOFSKY*

Department of Biological Sciences, Stanford University, Stanford, California 94305-5020 Received 17 June 1996/Accepted 9 September 1996

each consisting of 75 amino acid residues. There is one molecule of L-tryptophan bound per subunit (2, 21). When activated by bound L-tryptophan, the protein binds to RNA via recognition of GAG and UAG trinucleotide repeats in the trp and trpG transcripts (2, 3). L-Tryptophan is not necessary for oligomerization of the TRAP monomer. TRAP binding to (G/ U)AG trinucleotide repeats has been confirmed by footprinting experiments and deletion analyses (4). Eleven (G/U)AG repeats separated by 2 to 3 nucleotides are present in the trp leader transcript; many of these repeats are in the transcript segment that forms the antiterminator structure. In the presence of L-tryptophan, binding of TRAP to these sequences disrupts or prevents formation of the antiterminator, thus allowing terminator formation and resulting in cessation of transcription (2, 5, 21, 26). This form of transcription attenuation does not require the synthesis of a leader peptide, or ribosome stalling, as is seen in some other examples of attenuation control in bacteria (34). It has also been shown that when TRAP is bound to trp operon mRNA, transcripts that escape termination are also subject to translational regulation via the formation of an RNA secondary structure that sequesters the trpE ribosome binding site (16, 19). Access to an initiating ribosome is presumably blocked, and translation initiation is hindered. Nine (G/U)AG trinucleotide repeats overlap the trpG ribosome binding site (4, 33). TRAP binding to this segment of folate operon mRNA interferes with translation initiation (4, 33). Some years ago, Steinberg and Anagnostopoulos described a temperature-sensitive, 5-fluorotryptophan-resistant mutant of B. subtilis harboring a defect in trpS, the gene encoding tryp-

Expression of seven genes is required for biosynthesis of the amino acid L-tryptophan in Bacillus subtilis (13). Six of these genes are arranged in a cotranscribed gene cluster designated the trpEDCFBA operon, which is located in the aromatic supraoperon. The seventh trp gene, trpG, is within the unlinked folate operon. trpG encodes an amphibolic enzyme required for both p-aminobenzoate and o-aminobenzoate (anthranilate) synthesis (27). Expression of all seven tryptophan biosynthetic genes is regulated in response to the intracellular level of L-tryptophan by the product of the mtrB gene, trp RNA-binding attenuation protein (TRAP) (4, 10, 21). TRAP regulates transcription of the trp operon by a novel form of transcription attenuation; it regulates TrpG polypeptide synthesis translationally (16, 21, 33). The leader segment of the trp operon transcript is capable of forming alternative secondary structures (26). One of these structures is a rho-independent transcription terminator which, when formed, directs RNA polymerase to terminate transcription at a site preceding the trp structural genes. The alternate RNA hairpin, the antiterminator, contains several of the bases required for terminator formation; thus, the existence of the antiterminator precludes formation of the terminator. When the terminator does not form, transcription proceeds into the trp operon structural genes (5). The three-dimensional structure of TRAP has been determined (2). This protein is composed of 11 identical subunits,

* Corresponding author. Phone: (415) 725-1834. Fax: (415) 7258221. Electronic mail address: [email protected]. 6518

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In Bacillus subtilis, the tryptophan-activated trp RNA-binding attenuation protein (TRAP) regulates expression of the seven tryptophan biosynthetic genes by binding to specific repeat sequences in the transcripts of the trp operon and of the folate operon, the operon containing trpG. Steinberg observed that strains containing a temperature-sensitive mutant form of tryptophanyl-tRNA synthetase, encoded by the trpS1 allele, produced elevated levels of the tryptophan pathway enzymes, when grown at high temperatures in the presence of excess L-tryptophan (W. Steinberg, J. Bacteriol. 117:1023–1034, 1974). We have confirmed this observation and have shown that expression of two reporter gene fusions, trpE*-*lacZ and trpG*-*lacZ, is also increased under these conditions. Deletion of the terminator or antiterminator RNA secondary structure involved in TRAP regulation of trp operon expression eliminated the trpS1 effect, suggesting that temperature-sensitive expression was mediated by the TRAP protein. Analysis of expression of mtrB, the gene encoding the TRAP subunit, both by examination of a lacZ translational fusion and by measuring the intracellular levels of TRAP by immunoblotting, indicated that the trpS1-induced increase in trp gene expression was not due to inhibition of mtrB expression or to alteration of the amount of TRAP present per cell. Increasing the cellular level of TRAP by overexpressing mtrB partially counteracted the trpS1 effect, demonstrating that active TRAP was limiting in the trpS1 mutant. We also showed that elevated trp operon expression was not due to increased transcription initiation at the upstream aroF promoter, a promoter that also contributes to trp operon expression. We postulate that the increase in trp gene expression observed in the trpS1 mutant is due to the reduced availability of functional TRAP. This could result from inhibition of TRAP function by uncharged tRNATrp molecules or by increased synthesis of some other transcript capable of binding and sequestering the TRAP regulatory protein.

trpS MUTATION AFFECTS TRAP REGULATION

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TABLE 1. Strains of B. subtilis used in this study Genotypea

Source or reference

1A1 1A353 1A605 WB3164 CYBS223 CYBS350 CYBS360 CYBS361 CYBS365 CYBS366 CYBS370 CYBS371 CYBS372 CYBS375 CYBS376 CYBS380 CYBS381 CYBS404 CYBS411 CYBS413 CYBS415 CYBS416 CYBS417 CYBS418 CYBS422 PGBS11 PGBS31

trpC2 trpS1 trpC2 argF83::Tn917 Emr (SPbc2) mtrB3115 mtrBVTc trpS1 amyE::[Ptrp (trpE9-9lacZ)] Cmr trpS1 amyE::[Ptrp (trpE9-9lacZ)] Cmr amyE::[Ptrp (D29–95) (trpE9-9lacZ)] Cmr amyE::[Ptrp (D29–95) (trpE9-9lacZ)] Cmr amyE::[Pspac (mtrA mtrB) lacI] Cmr trpS1 amyE::[Pspac (mtrA mtrB) lacI] Cmr mtrB3115 amyE::[Pspac (mtrA mtrB) lacI] Cmr amyE::[Pmtr (mtrA mtrB9-9lacZ)] Cmr trpS1 amyE::[Pmtr (mtrA mtrB9-9lacZ)] Cmr amyE::[Paro (aroF9-9lacZ)] Cmr trpS1 amyE::[Paro (aroF9-9lacZ)] Cmr trpS1 argF83::Tn917 Emr mtrB264 amyE::[Ptrp (trpE9-9lacZ)] Cmr mtrB264 trpS1 amyE::[Ptrp (trpE9-9lacZ)] Cmr mtrBVTc amyE::[Ptrp (D29–95) (trpE9-9lacZ)] Cmr amyE::[Ptrp (D65–147) (trpE9-9lacZ)] Cmr trpS1 amyE::[Ptrp (D65–147) (trpE9-9lacZ)] Cmr mtrBVTc amyE::[Ptrp (D65–147) (trpE9-9lacZ)] Cmr argC4 trpS1 amyE::[Ppab (pab-trpG9-9lacZ)] Cmr argF83::Tn917 Emr argC4 amyE::[Ppab (pab-trpG9-9lacZ)] Cmr argC4 DmtrB amyE::[Ppab (pab-trpG9-9lacZ)] Cmr

BGSCb 30 31 14 19 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study 33 33

a Cmr, chloramphenicol resistance; Emr, erythromycin resistance; Ptrp, the trp operon promoter; Ppab, the folate operon promoter; Pmtr, the mtr operon promoter; Paro, the aroFBH operon promoter; Pspac, the heterologous spac promoter. The segment of the trp leader region that was deleted is shown in parentheses. Numbers are relative to the transcription start site. b BGSC, Bacillus Genetic Stock Center, Ohio State University, Columbus.

tophanyl-tRNA synthetase (30). In studies of this trpS mutant (allele trpS1) grown with L-tryptophan at a high temperature, it was shown that the trpS1 alteration resulted in production of elevated levels of the trp operon-specified enzymes, anthranilate synthase and tryptophan synthetase (29). Strains containing the temperature-sensitive trpS1 allele could not grow in minimal medium at high temperatures, presumably because there was insufficient tryptophanyl-tRNA synthetase activity to charge tRNATrp at an acceptable rate. However, growth was normal at the elevated temperature in the presence of excess L-tryptophan, probably because the amino acid stabilized the thermolabile tRNA synthetase. Relatively little is known about the role of tRNAs and aminoacyl-tRNA synthetases in regulation of amino acid biosynthetic operons of B. subtilis. However, some of the aminoacyltRNA synthetase genes (including trpS) do appear to be regulated by a different form of transcription attenuation in which interactions between leader RNA and uncharged tRNA modulate transcription termination in the leader region of the respective operon (11, 12). In this study, we examine the effects of the trpS1 alteration on TRAP-mediated regulation of trp gene expression in B. subtilis. MATERIALS AND METHODS Bacterial strains and transformations. The relevant genotypes of the B. subtilis strains used in this study are given in Table 1. Transformation was carried out by natural competence (1). Strain CYBS350 (trpS1) was constructed by transforming 1A353 (trpS1) with chromosomal DNA prepared from 1A1 (trpS1) and selecting for growth on minimal medium at 428C. CYBS404 was constructed by transformation of 1A353 with chromosomal DNA of 1A605 (in which the transposon Tn917 and trpS1 exhibit approximately 25% cotransformation), followed by selection for erythromycin resistance and screening for temperature sensitivity. The trpS1 allele was then introduced into other strains by transformation with chromosomal DNA prepared from CYBS404.

Gene fusions were integrated into the chromosomal amyE locus by homologous recombination. The DNA used in transformations was either linearized plasmid DNA containing the gene fusion or chromosomal DNA prepared from strains in which the gene fusion had previously been integrated into the amyE locus. Following selection for chloramphenicol resistance, disruption of amyE was confirmed by testing amylase production by iodine staining as described by Sekiguchi et al. (24). Southern blot analysis of representative strains indicated that only one copy of each gene fusion was present in the chromosome (data not shown). Media and reagents. The minimal media used were those described by Vogel and Bonner (32) and Spizizen (28), supplemented with 0.5% glucose and appropriate growth factors. Chloramphenicol and erythromycin were used at final concentrations of 5 and 2 mg/ml, respectively. 5-Bromo-4-chloro-3-indolyl-b-Dgalactopyranoside (X-Gal) was used at a concentration of 25 mg/ml in solid media, and isopropyl-b-D-thiogalactopyranoside (IPTG) was used at a concentration of 1 mM. Construction of an mtrB*-*lacZ translational fusion. Standard recombinant DNA procedures were used essentially as described by Sambrook et al. (22). Plasmid pSI45 contains the entire mtrAB operon (10). Sequence analysis of this vector revealed that the 235 region of the mtrAB promoter contained an undesired mutation (18). A 440-bp DNA fragment encompassing the mtrAB promoter region was amplified by PCR, with chromosomal DNA from a wild-type strain as a template, and completely sequenced. This was used to replace the corresponding region of pSI45, to produce the vector pAL1. An mtrB9-9lacZ translational fusion was constructed by subcloning the 1.1-kb EcoRI-HaeIII fragment of pAL1 containing the mtr promoter, mtrA, and the first 23 codons of mtrB into pKG7 (9) digested with EcoRI and SmaI. The 1.9-kb EcoRI-ClaI fragment of this plasmid (pAL6) containing the gene fusion was cloned into the EcoRI and ClaI sites of ptrpBG1 (25), resulting in a B. subtilis integration vector. The construct was then introduced into the chromosomal amyE locus. Enzyme assays. Cultures were grown overnight in Vogel-Bonner or Spizizen minimal salts containing 0.5% glucose at 328C. These were then subcultured into the same medium in the presence or absence of 50 mg of L-tryptophan per ml and grown to the mid-exponential phase at various temperatures. Anthranilate synthase activity was assayed as described previously (8), and b-galactosidase activity was assayed in permeabilized cells as described by Miller (20). One unit of anthranilate synthase specific activity represents the enzyme activity required to synthesize 0.01 nmol of anthranilate in 20 min at 378C per 0.1 ml of a washed, resuspended cell suspension (OD600 5 0.5). Each assay was performed in duplicate on at least four separate occasions.


Strain

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LEE ET AL.

J. BACTERIOL. TABLE 2. Effects of the trpS1 mutation on trp gene expressiona

Strain

CYBS360 CYBS360 CYBS361 CYBS361 CYBS411 CYBS411 CYBS413 CYBS413

Genetic background 1

1

mtrB trpS mtrB1 trpS1 mtrB1 trpS1 mtrB1 trpS1 mtrB264 trpS1 mtrB264 trpS1 mtrB264 trpS1 mtrB264 trpS1

b

L-Tryptophan

2 1 2 1 2 1 2 1

Anthranilate synthase specific activityc of strain grown at:

b-Galactosidase activityd of strain grown at:

328C

428C

328C

428C

35 12 41 8.0 2,330 2,900 2,410 2,520

28 9.0 NGe 390 1,380 1,430 1,620 1,920

31 0.20 8.9 0.20 1,820 1,830 1,850 1,820

1.3 0.10 NG 4.5 120 180 160 160

Western blot (immunoblot) analysis. Cultures were grown under the same conditions described for enzyme assays and adjusted to an A600 reading of 0.5. Cells were concentrated 50-fold and disrupted by sonication. Samples containing 50 mg of total cell protein were electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels (17) and then electrophoretically transferred to a nitrocellulose membrane (PH79; Schleicher & Schuell, Inc.). Immunoblotting was performed as described previously (6) with polyclonal antisera directed against the MtrB protein, provided by Paul Gollnick (State University of New York at Buffalo). Bound antibody was visualized by use of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Pierce) and enhanced chemiluminescence Western blotting detection reagents (Amersham).

RESULTS Elevated expression of the trp operon in a trpS1 strain. Steinberg demonstrated that strains containing the temperature-sensitive trpS1 allele produced elevated levels of tryptophan pathway enzymes when cultured at high temperatures in the presence of L-tryptophan (29). To confirm this observation, we examined trp operon expression by measuring anthranilate synthase activity in isogenic trpS1 and trpS1 strains (Table 2). At the low, permissive temperature, 328C, anthranilate synthase activities in both trpS1 and trpS1 cultures were comparable, and the presence of excess L-tryptophan resulted in a four- to fivefold reduction of expression. At 428C, the anthranilate synthase specific activities of the trpS1 cultures were similar to those observed at the lower temperature. However, in the trpS1 strain grown at 428C in the presence of L-tryptophan, anthranilate synthase activity was appreciably elevated, some 40-fold higher than that seen in the trpS1 strain under the same conditions. This level is approximately one-fourth that observed in an mtrB strain. These initial experimental results support the basic findings of Steinberg (29). One possible complication in the interpretation of the results in Table 2 is that expression of the trp operon is driven by two promoters, the promoter for the upstream aroFBH operon (13) and the promoter that immediately precedes the trp operon (13). It has been shown that transcription initiated at the aroF promoter does continue into the trp operon and contributes trp operon transcripts (26). To examine the level of expression due solely to transcription from the trp operon promoter, we constructed a trpE9-9lacZ translational fusion driven by the intact trp promoter-leader regulatory region and integrated this construct into the amyE locus. We measured expression of this fusion, and overall, the same trends were observed in b-galactosidase production, although the extent of inhibition of expression of the trp operon caused by the addition of L-tryptophan, a 40-fold reduction, was more marked

(Table 2). The greater L-tryptophan-mediated inhibition of expression observed when measuring b-galactosidase activity in comparison with that observed when measuring anthranilate synthase activity could be due to the absence of the transcripts of the trp operon initiated at the aroF promoter. It should also be noted that we observed a 10- to 20-fold decrease in b-galactosidase activity under all conditions examined at 428C (Table 2). This was also observed in all mtrB mutants. Diminished b-galactosidase activity at elevated temperatures has been observed with a number of lacZ fusions in B. subtilis and does not appear to be unique to this trpE fusion (23). Escherichia coli b-galactosidase appears to be labile at elevated temperatures in B. subtilis. However, most importantly, the level of b-galactosidase produced by the trpS1 strain grown at the elevated temperature in the presence of L-tryptophan was also 45-fold higher than that for the trpS1 strain grown under the same conditions. This level was greater than that observed in the trpS1 strain grown in the absence of L-tryptophan, as was also found for anthranilate synthase levels (Table 2). However, the b-galactosidase levels of the trpS1 mutant are some 30-fold lower than those obtained with mtrB strains, whereas the anthranilate synthase levels were only 4-fold lower. To determine whether the trpS1 effect was additive to TRAP regulation, we also measured expression in trpS1 strains which lacked the TRAP regulatory protein due to a mutation in the mtrB gene. Anthranilate synthase levels were comparable in the mtrB trpS1 and mtrB trpS1 strains. The trpS1-induced elevation in trp gene expression was not exhibited in mtrB strains. These results indicate that the trpS1 allele strongly influences trp operon expression, whether transcription is initiated solely at the trp promoter or at both the trp promoter and the aroFBH promoter. This increase could be due to effects on the RNA structures involved in transcription attenuation, changes in the level of mtrB gene expression, or reduction of the intracellular TRAP concentration. In the following sections, we examine these possibilities in an effort to explain how the trpS1 mutation increases expression of the tryptophan pathway genes. Deletion of the antiterminator or terminator structures of the trp leader transcript eliminates the trpS1-induced increase in trp operon expression. The observation that trp gene expression in the trpS1 strain was elevated at high temperatures in the presence of excess L-tryptophan raised the question whether TRAP, the trp attenuation system, or both might be affected. To examine these possibilities, two additional trpE9-9lacZ


a Strains were grown overnight in Spizizen minimal medium at 328C, subcultured in the same medium with or without 50 mg of L-tryptophan per ml at 32 or 428C, as indicated, and grown for three to four generations. b 1, present; 2, absent. c Encoded by chromosomal trpE and trpG genes. The units of anthranilate synthase specific activity are given in Materials and Methods. d Encoded by trpE9-9lacZ translational fusion in amyE locus. Values are in Miller units. e NG, no growth. mtrB1 strains containing the trpS1 allele are unable to grow at 428C in the absence of L-tryptophan.


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TABLE 3. Effects of the trpS1 mutation on expression of trpE9-9lacZ translational fusions lacking the terminator or antiterminator structuresa Fusionb

Strain

CYBS416 CYBS416 CYBS417 CYBS417 CYBS418 CYBS418 CYBS365 CYBS365 CYBS366 CYBS366 CYBS415 CYBS415

(D65–147) trpE9-9lacZ (D65–147) trpE9-9lacZ (D65–147) trpE9-9lacZ (D65–147) trpE9-9lacZ (D65–147) trpE9-9lacZ (D65–147) trpE9-9lacZ (D29–95) trpE9-9lacZ (D29–95) trpE9-9lacZ (D29–95) trpE9-9lacZ (D29–95) trpE9-9lacZ (D29–95) trpE9-9lacZ (D29–95) trpE9-9lacZ

1

b-Galactosidase activityd of strain grown at:

c

L-Tryptophan

1

2 1 2 1 2 1 2 1 2 1 2 1

mtrB trpS mtrB1 trpS1 mtrB1 trpS1 mtrB1 trpS1 mtrBVTc trpS1 mtrBVTc trpS1 mtrB1 trpS1 mtrB1 trpS1 mtrB1 trpS1 mtrB1 trpS1 mtrBVTc trpS1 mtrBVTc trpS1

328C

428C

400 320 440 360 620 660 0.12 ,0.1 0.11 ,0.1 ,0.1 ,0.1

41 38 NGe 73 99 88 ,0.1 ,0.1 NG ,0.1 ,0.1 ,0.1

a Strains were grown overnight in Vogel-Bonner minimal medium at 328C, subcultured in the same medium with or without 50 mg of L-tryptophan per ml at 32 or 428C, as indicated, and grown for three to four generations. b The region of the trp leader transcript which has been deleted is shown in parentheses. c 1, present; 2, absent. d Values are in Miller units. e NG, no growth.

translational fusions were examined. In these constructs, the region of DNA encoding either the antiterminator structure (nucleotides 29 to 95 relative to the start point of transcription) or both the antiterminator and terminator structures (nucleotides 65 to 147) had been deleted (26). Each was separately integrated into the amyE locus of strains CYBS350 (trpS1), 1A353 (trpS1), and CYBS223 (mtrB). If the trpS1-induced increase in trp gene expression was the result of changes in TRAP and/or the trp attenuation system, removal of the RNA secondary structures necessary for TRAP-mediated regulation would eliminate the effect. However, if the trpS1-induced increase in trp gene expression was independent of TRAP and attenuation, removal of these structures might not affect the increase in expression associated with growth at a high temperature. In strains containing the deletion (of nucleotides 65 to 147 [D65–147]) that removes both the terminator and antiterminator, expression was high and the presence of L-tryptophan had no effect on trp operon expression (Table 3). The b-galactosidase levels of CYBS416 (mtrB1 trpS1) and CYBS417 (mtrB1 trpS1) grown at the high temperature in the presence of Ltryptophan differed by only 2-fold in comparison with the 45fold difference observed with the unaltered trpE9-9lacZ fusion. Essentially the same elevated levels were observed in strains CYBS417 (mtrB1 trpS1) and CYBS418 (mtrBVTc trpS1) (Table 3). In the antiterminator deletion construct (D29–95), most of the antiterminator and the TRAP binding sites on the trp leader are absent, leaving the terminator intact. The terminator hairpin would therefore be expected to form under all conditions. Low b-galactosidase levels were present in all the strains with this deletion grown under various conditions of temperature or L-tryptophan availability. No significant differences were seen between the trpS1 and trpS1 strains. These results suggest that the elevated level of trp operon expression observed in the trpS1 strain at high temperatures is attributable to effects mediated by TRAP and/or the trp attenuation system. Expression of trpG is elevated in a trpS1 strain. Anthranilate synthase is composed of two polypeptides, specified by the unlinked trpE and trpG genes. Both polypeptides are required for the conversion of chorismate plus glutamine to anthranilate. Anthranilate synthase activity was shown to be elevated in the trpS1 strain grown at the higher temperature, implying that

expression of trpG also was elevated. To verify this interpretation, trpG expression was measured by use of a trpG9-9lacZ translational fusion (33). At the lower temperature, regulation of trpG expression in the presence of L-tryptophan was evident in the mtrB1 strains, and the presence of the trpS1 allele had no significant effect on gene expression (Table 4). At 428C, the b-galactosidase activity produced by the translational fusion construct was too low to allow comparisons to be made (data not shown). However, growth of the strains at 408C did reveal an eightfold-higher level of TrpG–b-galactosidase activity for the trpS1 strain grown in excess L-tryptophan than for the trpS1 strain. This level was some 20-fold lower than that obtained in an mtrB deletion strain (Table 4). These results extend the findings of Steinberg regarding the trpS1-induced elevation of trp operon expression to the unlinked trpG gene. Overexpression of TRAP partially reverses the trpS1-induced increase in trp operon expression. One explanation that would account for the high expression of both the trp operon and trpG gene in the trpS1 strain at increased temperatures is that the level of functional TRAP is decreased. If this is the primary cause of elevation of trp operon expression in the trpS1

TABLE 4. Effects of the trpS1 mutation on expression of the trpG genea

Strain

PGBS11 PGBS11 CYBS422 CYBS422 PGBS31 PGBS31 a

Genetic background 1

1

mtrB trpS mtrB1 trpS1 mtrB1 trpS1 mtrB1 trpS1 DmtrB trpS1 DmtrB trpS1

b

L-Tryptophan

2 1 2 1 2 1

b-Galactosidase activityc of strain grown at: 328C

408C

17.0 4.3 13 5.0 210 160

2.4 0.28 NGd 2.4 64 49

Encoded by Ppab (pab-trpG9-9lacZ) translational fusion in amyE locus. 1, present; 2, absent. Strains were grown overnight in Vogel-Bonner minimal medium at 328C, subcultured in the same medium with or without 50 mg of L-tryptophan per ml at 32 or 408C, as indicated, and grown for three to four generations. Values are in Miller units. d NG, no growth. b c


Ptrp Ptrp Ptrp Ptrp Ptrp Ptrp Ptrp Ptrp Ptrp Ptrp Ptrp Ptrp

Genetic background

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TABLE 5. Effects of TRAP overproduction on trp operon expressiona

Strain [fusion]

Anthranilate synthase specific activityb

Genetic background

2IPTG

CYBS372 [Pspac (mtrA mtrB)] CYBS371 [Pspac (mtrA mtrB)] CYBS370 [Pspac (mtrA mtrB)]

TABLE 6. Effects of the trpS1 mutation on expression of the mtrB and aroF genesa

1

mtrB3115 trpS mtrB1 trpS1 mtrB1 trpS1

770 680 9.7

Strain

Fusion

Genetic background

1IPTG

7.8 120 6.9

a Strains were grown overnight in Spizizen minimal medium containing 50 mg of L-tryptophan per ml at 328C, subcultured in the same medium with or without 1 mM IPTG, and grown at 428C for four generations. b The units of activity are given in Materials and Methods.

mtrB9-9lacZ mtrB9-9lacZ aroF9-9lacZ aroF9-9lacZ

trpS trpS1 trpS1 trpS1

328C

428C

24 23 54 43

1.6 2.3 11 2.2

a Strains were grown overnight in Vogel-Bonner minimal medium at 328C, subcultured in the same medium containing 50 mg of L-tryptophan per ml at 32 or 428C, as indicated, and grown for three to four generations. b Values are in Miller units.

pared from strains CYBS350 (trpS1) and 1A353 (trpS1) following growth under various conditions of temperature and L-tryptophan supplementation. Samples containing equal amounts of protein were electrophoresed on SDS-polyacrylamide gels and subjected to Western blot analysis, with antisera directed against the TRAP subunit (Fig. 1). Visual inspection suggested that in a wild-type strain, the levels of TRAP were comparable under the various conditions of growth temperature and L-tryptophan availability. The presence of the trpS1 allele had no effect on the amount of the regulatory protein in the cell. (Multiple bands are due to the presence of partially dissociated TRAP complex. As observed previously [4], it is difficult to completely denature the TRAP complex in SDS sample buffer.) Thus, the trpS1-induced effect on trp operon expression does not appear to be the result of changes in the level of TRAP in the cell. The TRAP level was not influenced by the presence or absence of excess L-tryptophan in the culture medium. Expression of the aroFBH operon is not affected by the trpS1 mutation. As mentioned above, the aroFBH operon is located immediately upstream of the trp operon and forms part of the larger aromatic supraoperon; transcription from the aroF promoter can continue into the trp operon (13, 26). If the trpS1 mutation resulted in increased transcription of the aroFBH operon, this would lead to a direct increase in trp operon

FIG. 1. Cellular levels of TRAP measured by immunoblotting. Cell extracts containing equal amounts of protein prepared from trpS1 and trpS1 strains were electrophoresed on an SDS–15% polyacrylamide gel, and TRAP was visualized by immunoblotting with antiserum directed against the MtrB protein. Cultures were grown in minimal medium in the absence (2) of L-tryptophan or in the presence (1) of 50 mg of L-tryptophan per ml at the indicated temperatures. The positions of molecular weight standards (kilodaltons) are shown on the left.


strain, then overexpression of TRAP should reverse the effect. To overproduce TRAP in the trpS1 strain, we utilized a construct consisting of the mtrAB structural genes driven by the Pspac promoter. The plasmid also contains the lacI gene (10). Expression from the Pspac promoter is controlled by the lac repressor and is inducible by the addition of IPTG (36). The lacI Pspac-mtrAB construct was integrated into the amyE locus of strains CYBS350 (trpS1), 1A353 (trpS1), and WB3164 (mtrB) to give strains CYBS370, CYBS371, and CYBS372, respectively (Table 5). Measurements of the anthranilate synthase levels of mtrB3115 mutant cultures (CYBS372) grown in the presence of L-tryptophan at 428C with or without the inducer, IPTG, showed that addition of inducer reduced the anthranilate synthase level 100-fold. This result establishes that the PspacmtrAB construct was functional and that it responded to inducer (Table 5). Expression in the mtrB1 trpS1 strain containing the Pspac-mtrAB construct (CYBS370) was very low, with or without inducer, indicating that this strain contained sufficient levels of TRAP to inhibit trp operon expression. In the absence of IPTG, the anthranilate synthase activity of the mtrB1 trpS1 strain (CYBS371) was high and comparable to that of the mtrB mutant that was examined (Table 5), but addition of IPTG to the growing culture resulted in a sevenfold decrease in anthranilate synthase activity. This result demonstrates that overexpression of mtrB partially reverses the trpS1induced increase in trp operon expression that is typical of trpS1 strains growing at high temperatures. Levels of TRAP are not affected by the trpS1 mutation. One possible explanation for the finding that increasing the intracellular level of TRAP partially overcomes the trpS1-induced elevation of trp operon expression is that mtrB is poorly expressed or that TRAP is destroyed in the trpS1 strain. To examine mtrB expression in trpS1 and trpS1 strains, an mtrB99lacZ translational fusion was constructed (as described in Materials and Methods) and integrated into the amyE locus of CYBS350 and 1A353. b-Galactosidase assays were performed on strains CYBS375 and CYBS376 grown at 32 and 428C in the presence of L-tryptophan (Table 6). At both temperatures, the b-galactosidase activities of the trpS1 and trpS1 strains were comparable. Thus, the trpS1-induced increase in trp operon expression does not appear to be due to inhibition of mtrB expression. Although the data suggest that the mtrB expression levels are identical in both the trpS1 and trpS1 strains growing at high temperatures in the presence of L-tryptophan, these findings do not rule out the possibility that the actual concentration of TRAP itself is lower in the trpS1 strain. Thus, the trpS1 effect could be the result of an increased rate of degradation of TRAP. To address this possibility, total cell extracts were pre-

CYBS375 CYBS376 CYBS380 CYBS381

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expression. Increased expression of the aroFBH operon could result in greater expression of the trpE9-9lacZ fusion as a consequence of sequestration of some of the available TRAP by the supraoperon transcript; sequestration would reduce the concentration of free TRAP that could regulate the trpE9-9lacZ fusion. We examined expression from the aroF promoter by using an aroF9-9lacZ translational fusion integrated at amyE (15). Our results showed that the presence of the trpS1 allele did not lead to an increase in expression of the aroF9-9lacZ fusion (Table 6). When grown at 428C in the presence of L-tryptophan, the trpS1 strain demonstrated expression that was in fact reduced in comparison with that of the trpS1 strain. DISCUSSION

trp operon. trpG9-9lacZ expression also was about 20-fold higher in an mtrB than in an mtrB1 background. While use of the two assays reveals a quantitative discrepancy that will ultimately have to be explained, the general conclusion that the trpS1 mutation has a marked effect on trp gene expression is confirmed. The increase in trp gene expression observed in trpS1 strains could be due to a trpS1-mediated reduction in the cellular TRAP level. Overexpression of TRAP did partially reverse the trpS1-induced increase in trp operon expression, supporting this conjecture. However, examination of expression of an mtrB9-9lacZ fusion construct and of the cellular levels of TRAP by immunoblotting in trpS1 and trpS1 strains revealed that there was no apparent difference in TRAP levels or levels of expression between the two strains (Table 6 and Fig. 1). Therefore, the trpS1 effect must not be due to inhibition of mtrB expression or alteration of the intracellular level of TRAP. The 5-fluorotryptophan resistance phenotype exhibited by strains with the trpS1 allele is most likely due to a change in tryptophanyl-tRNA synthetase, which alters its affinity for this tryptophan analog. Resistance is not due to dilution of the analog by increased L-tryptophan production in the cell, since trpS1 strains are capable of growth in the presence of 5-fluorotryptophan at 328C, conditions under which trp gene expression is fully regulated by TRAP (Tables 2 and 4). The trpS1 change is thought to affect the enzyme’s ability to bind Ltryptophan and perform the subsequent charging of tRNATrp at elevated temperatures. Even when subjected to the stabilizing effects of L-tryptophan at high temperatures, the thermolabile tryptophanyl-tRNA synthetase probably is at least partially defective in catalytic activity. This would alter the cellular levels of charged versus uncharged tRNATrp, either of which may be sensed in an event that influences trp gene expression. Many bacterial operons are regulated by attenuation in response to the availability of a specific charged tRNA. In grampositive species, aminoacyl-tRNA synthetase genes and a number of amino acid biosynthetic operons are regulated by uncharged tRNA-induced readthrough beyond a transcription terminator located in the mRNA leader segment, upstream of the coding sequence (11, 12). In enteric bacteria, a deficiency of a charged tRNA can result in ribosome stalling during synthesis of a leader peptide; transcript site-specific ribosome stalling prevents transcription termination (34). This attenuation mechanism is used to regulate expression of the trp operon of E. coli. The intracellular level of L-tryptophan is independently sensed by the trp repressor in this organism (35). The findings described in this paper suggest that both L-tryptophan availability and tRNATrp charging are sensed during regulation of trp gene expression in B. subtilis. In this organism, the regulatory protein TRAP may mediate a response to either signal. There are several hypotheses that could explain how an increase in the concentration of uncharged tRNATrp could lead to elevated trp gene expression. Uncharged tRNATrp could interact with TRAP and inhibit its ability to bind Ltryptophan or its target transcripts. Since TRAP is an RNAbinding protein, it is conceivable that it could bind tRNATrp. Alternatively, a defect in tRNATrp charging could lead to induction of expression of some other operon that encodes a transcript with GAG repeats that is capable of binding TRAP. It is also possible that defective tRNATrp charging has some indirect effect on TRAP activation or function. Whatever the explanation, it is conceivable that as in E. coli, the trp gene regulatory mechanisms of B. subtilis are designed to detect and respond to changes in the intracellular levels of either tryptophan or tryptophanyl-tRNATrp. The DNA sequence upstream of the trpS coding region (7)


Aminoacyl-tRNA synthetases catalyze the charging of their cognate amino acid onto uncharged tRNA, thus playing an essential role in protein synthesis. In addition to serving as a component of the cell’s translation machinery, the tRNA substrates of these enzymes can be utilized in a regulatory capacity (11, 12, 34). In this study, we have shown that a mutation in the trpS gene of B. subtilis, which encodes a partially defective temperature-sensitive tryptophanyl-tRNA synthetase, results in the elevated expression of the genes encoding the enzymes of the L-tryptophan biosynthetic pathway. These genes are known to be regulated by the tryptophan-activated RNA-binding protein, TRAP (2–4, 10, 21, 33). In the presence of excess L-tryptophan, and following growth at 428C, the anthranilate synthase activity of a mutant containing the temperature-sensitive trpS1 allele was some 40-fold higher than that of a trpS1 strain grown under the same conditions (Table 2), confirming the early findings of Steinberg (29). Since anthranilate synthase is a complex of TrpE and TrpG polypeptides, and since TRAP regulates expression of the genes encoding these polypeptides, we also examined the effect of the trpS1 mutation on expression of trpG. Using a trpG9-9lacZ reporter gene fusion, we showed that expression of trpG also was increased by the presence of the trpS1 allele, at elevated temperatures (Tables 2 and 4). In our analyses, we also measured trpE expression by use of a trpE9-9lacZ reporter construct under the exclusive control of the trp promoter. We observed the same relative increase in trp gene expression (40-fold) in the presence of the trpS1 allele that was observed when anthranilate synthase activity was measured. The findings that at high temperatures the trpS1 mutation results in elevated expression of the trp operon, the trpE9-9lacZ fusion, and the trpG9-9lacZ fusion (Tables 2 and 4) and that deletions of or within the terminator-antiterminator region of the trp operon abolish this effect (Table 3) indicate that the trpS1-induced increase in trp gene expression is mediated by TRAP. In support of this conclusion, mtrB trpS1 strains, when grown at high temperatures, did not exhibit higher levels of trp gene expression than that observed with the mtrB strain alone (Table 2); thus, the trpS1 effect is not additive to TRAP regulation. With these strains, we observed an appreciable difference when anthranilate synthase and b-galactosidase assays were used; trpE9-9lacZ activity was some 30-fold lower in the mtrB1 trpS1 strain than in the mtrB trpS1 strain, whereas anthranilate synthase activity was only fourfold lower (Table 2). The discrepancy may be due to the fact that anthranilate synthase is a complex composed of two different polypeptides, with the TrpE polypeptide being translated from transcripts initiated at the trp promoter and the aroF promoter. Another possibility is that the trp operon transcript originating from the aroF promoter is less sensitive to the regulatory effects of TRAP or that there are differences in the stability of the two transcripts of the

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is similar to the leader region of a number of gram-positive aminoacyl-tRNA synthetase genes known to be regulated by the transcription antitermination mechanism first described by Grundy and Henkin (11, 12). Expression of these genes is regulated in response to the availability of their cognate amino acid by using uncharged tRNA as a positive regulator. If the trpS1 mutation does result in accumulation of uncharged tRNATrp, then expression of the mutant tryptophanyl-tRNA synthetase should also be elevated at high temperatures. Whether this is responsible for or contributes to the temperature-induced increase in trp gene expression in the trpS1 mutant remains to be determined. ACKNOWLEDGMENTS

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The first two authors contributed equally to the work presented in this article. We are grateful to P. Gollnick for kindly providing bacterial strains and antisera, J. Klein for bacterial strains, and M.-C. Yee for advice on immunoblotting procedures. P. Babitzke and E. Merino provided valuable advice during the initial stages of the project. We thank J. Kuhn and M.-C. Yee for critical reading of the manuscript. This study was supported by National Institutes of Health grant GM09738. J.S. is supported by a Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship (DRG-1315).

13.

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