Journal of Bacteriology, February 2004, p . 818-828, Vol . 186, No . 3

Regulation of the Tryptophan Biosynthetic Genes in Bacillus halodurans: Common Elements but Different Strategies than Those Used by Bacillus subtilis

Reka Szigeti, Mirela Milescu, and Paul Gollnick^*

Department of Biological Sciences, State University of New York, Buffalo, New York 14260

Received 27 August 2003/ Accepted 23 October 2003

 
In Bacillus subtilis, an RNA binding protein called TRAP regulates both transcription and translation of the tryptophan biosynthetic genes . Bacillus halodurans is an alkaliphilic Bacillus species that grows at high pHs . Previous studies of this bacterium have focused on mechanisms of adaptation for growth in alkaline environments . We have characterized the regulation of the tryptophan biosynthetic genes in B . halodurans and compared it to that in B . subtilis . B . halodurans encodes a TRAP protein with 71% sequence identity to the B . subtilis protein . Expression of anthranilate synthetase, the first enzyme in the pathway to tryptophan, is regulated significantly less in B . halodurans than in B . subtilis . Examination of the control of the B . halodurans trpEDCFBA operon both in vivo and in vitro shows that only transcription is regulated, whereas in B . subtilis both transcription of the operon and translation of trpE are controlled . The attenuation mechanism that controls transcription in B . halodurans is similar to that in B . subtilis, but there are some differences in the predicted RNA secondary structures in the B . halodurans trp leader region, including the presence of a potential anti-antiterminator structure . Translation of trpG, which is within the folate operon in both bacilli, is regulated similarly in the two species .

 
Bacillus halodurans is an alkaliphilic bacterium that grows optimally above pH 9.5 . It is the second Bacillus species whose entire genomic sequence has been completely defined (39). B . halodurans is similar to Bacillus subtilis in terms of genome size, G+C content, and physiological properties, except for its alkaliphilic phenotype (39) . Since the majority of published studies on B . halodurans have focused on mechanisms of adaptation to alkaline environments, little is known about the regulatory mechanisms this bacterium uses to control gene expression in response to changes in its environment .

Regulation of the genes involved in tryptophan biosynthesis (trp) has been studied extensively in B . subtilis (for a review, see reference 5) and to a lesser extent in Bacillus pumilus (24, 27) and Bacillus stearothermophilus (13) . The regulatory elements and strategies used are similar for all of these bacilli, although they differ from the well-characterized mechanism that regulates the trp genes in Escherichia coli (reviewed in reference 45) . The B . subtilis trpEDCFBA operon contains six of the seven genes required for the biosynthesis of L-tryptophan from chorismic acid (the common aromatic amino acid precursor) and is contained within an aromatic supraoperon together with aroFBH, hisC, tyrA, and aroE (23) . The remaining trp gene, pabA (formerly known as trpG), is located in the folic acid operon (37) and encodes an amidotransferase subunit that participates in both tryptophan and folate biosynthesis (25) . Expression of both the trp operon and pabA is regulated in response to changes in the levels of intracellular tryptophan by an 11-subunit RNA-binding protein called TRAP (trp RNA-binding attenuation protein) (3-5, 20) .

TRAP regulates both transcription and translation of the B . subtilis trp genes . Transcription of the trp operon is regulated by an attenuation mechanism that controls elongation through a 203-nucleotide leader region upstream of trpE. In the presence of excess tryptophan, TRAP is activated to bind to a site in the trp leader transcript (7, 8, 11, 30) . This binding induces formation of a transcription terminator, and the structural genes are not expressed . When tryptophan is limiting, TRAP does not bind RNA; an alternative antiterminator RNA structure forms in the trp leader region, allowing transcription to continue into the structural genes . TRAP also regulates the translation of trpE and pabA, as well as that of yhaG, which encodes a putative tryptophan transporter (34) . In the case of trpE, TRAP binding to the leader region of trp operon mRNAs that have extended beyond the terminator results in formation of an RNA secondary structure that sequesters the trpE Shine-Dalgarno (SD) sequence, thereby inhibiting translation initiation (17, 29) . TRAP binding to pabA (18, 44) and yhaG (33, 34) MRNAs inhibits translation initiation by directly blocking ribosome access to the SD sequence . It also appears that TRAP regulates translation of ycbK, a gene of unknown function, by a similar mechanism, since there is a putative TRAP binding site that overlaps its SD sequence (33) . In all cases TRAP recognizes RNAs that contain multiple (between 9 and 11) GAG, UAG, and occasionally AAG trinucleotide repeats (3, 6) . These triplets are separated from each other by nonconserved nucleotides termed spacers . Two-nucleotide spacers are optimal, although larger spacers occur in the pabA, yhaG, and ycbK binding sites (9, 42) . Pyrimidines are generally favored over purines in the spacers (12, 42) .

The genome sequence of B . halodurans shows that it contains a gene predicted to encode a TRAP protein whose 76-amino-acid sequence is approximately 71% identical to the sequence of B . subtilis TRAP (21, 39) . In this report we characterize the regulation of the tryptophan biosynthetic genes in B . halodurans and demonstrate that, as seen in B . subtilis, both the trpEDCFBA operon and pabA (trpG) are regulated by TRAP in response to changes in tryptophan levels . We also show that although the regulatory elements of these genes are similar in B . halodurans and B . subtilis, the levels of regulation in response to the availability of tryptophan are significantly different . Additionally, we demonstrate that the strategies used to control these genes in the two species differ .

 
Bacterial strains and transformations. The B . subtilis and B . halodurans strains used in this study are described in Table 1 . E . coli K802 and LE392 were used as hosts for plasmid constructions . B . subtilis BG2087 (argC4) and BG4233 (argC4 mtrB) were used as hosts for transformation and integration of gene fusions . BG4233 contains a deletion of codons 7 to 62 of mtrB, which encodes TRAP (44) .

 
E . coli was transformed by the calcium heat shock procedure (14), and transformants were selected on Luria-Bertani agar plates containing 100 µg of ampicillin/ml . B . subtilis was transformed by natural competence (1) as described previously (26) . Transformants were selected on plates containing Vogel and Bonner (VB) minimal salts (41), 0.2% acid-hydrolyzed casein (ACH), 0.2% glucose, 10 µg of L-arginine/ml, 50 µg of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal)/ml, and 5 µg of chloramphenicol/ml .

Plasmids and gene fusions. Figure 1 outlines the transcriptional and translational fusions between B . halodurans trpE and lacZ, or between pabA and lacZ, driven by the trp promoter or the pab promoter (26, 37), respectively . Using PCR, we introduced EcoRI and BamHI (transcriptional fusion) or EcoRI and XbaI (translational fusion) restriction endonuclease sites at the ends of an 810-bp DNA fragment (from -680 to + 130 relative to the trpE start codon) containing the trp promoter, the regulatory region (trpL), and the first 45 codons of trpE . A similar strategy was used to create the pabA-lacZ fusions by generating a 1,710-bp DNA fragment (from -1620 to + 90 relative to the pabA start codon), with EcoRI/BamHI (transcriptional fusion) or EcoRI/HindIII (translational fusion) restriction sites, containing the pab promoter, the pabB structural gene, and the first 30 codons of pabA. For the translational fusions, the restriction sites were designed to make in-frame fusions between the 45th codon of trpE or the 30th codon of pabA and lacZ . The PCR products were digested with the appropriate restriction endonucleases and ligated into similarly cut plasmids; pDH32 (31) was used to create transcriptional fusions with lacZ, and ptrpBG1 (36) was used to create translational fusions . The resulting plasmids were linearized with PstI and transformed into B . subtilis BG2087 or BG4233 . The gene fusions were integrated into the amyE locus by homologous recombination (36), and transformants were selected as chloramphenicol-resistant blue colonies on X-Gal plates . The sequence of the integrated region in each strain was confirmed by PCR amplification using genomic DNA and primers complementary to the 5' end of the insert and the 19th to 24th codons of lacZ, followed by sequencing of the amplified DNA fragments (USB Sequenase) .

 
Enzyme assays. For anthranilate synthetase (ASase) assays, B . subtilis and B . halodurans cells were grown in minimal medium containing VB minimal salts, 0.2% glucose, and 0.2% ACH in the presence or absence of 50 µg of L-tryptophan/ml . The pH of the medium for B . halodurans was adjusted to 9.5 to 10.0 with 70 mM Na₂CO₃ . Samples were assayed for ASase activity as previously described (15) . ß-Galactosidase was assayed as described previously (26) from B . subtilis cells grown in minimal medium in the presence or absence of 50 µg of L-tryptophan/ml .

Selection for B . halodurans mtrB mutants on 5FTrp. B . halodurans mutants resistant to 5-fluorotryptophan (5FTrp) were selected on minimal agar plates containing VB minimal salts, 0.2% ACH, 0.2% glucose, 100 µg of shikimic acid, 70 mM Na₂CO₃, and 600 µg of 5FTrp . The mutations were identified by sequencing the mtrB locus as a PCR-amplified product by using primers complementary to the sequences at -76 to -54 and at +262 to +283 relative to the mtrB start codon, with genomic DNA from the mutant strains as the template (USB Sequenase) .

Preparation of total cellular RNA and primer extension mapping. Cells were grown as described above for ASase assays . Total cellular RNA was isolated by using the Qiagen RNeasy kit . Thirty micrograms of total cellular RNA and 1 µM primer (complementary to the B . halodurans trp operon regulatory region from position 166 to 136 upstream of the start codon of trpE) were mixed, heated to 80°C for 5 min, and then chilled on ice . Reverse transcription was carried out for 50 min at 42°C in 500 nM (each) dCTP, dGTP, and dTTP, 25 nM dATP, 10 µCi of [-³²P]dATP, and 20 µM dithiothreitol in First Strand buffer, with 200 U of Superscript II reverse transcriptase (Invitrogen) .

Expression and purification of B . halodurans TRAP. The B . halodurans mtrB gene was amplified by PCR using genomic DNA and primers complementary to regions 10 bp upstream of the start codon and 103 bp downstream of the stop codon of mtrB, respectively . The primers introduced restriction sites for NdeI and XhoI upstream and downstream of the gene, allowing the 333-bp PCR product to be ligated into similarly cut pET17b (Novagen) . The resulting plasmid, pETbhmtrB, was transformed into E . coli BL21(DE3) . The cells were grown, and TRAP expression was induced by the addition of isopropyl-ß-D-thiogalactopyranoside (IPTG) to 1 mM as described previously for expression of B . subtilis TRAP (2) . TRAP was purified by immunoaffinity chromatography using rabbit anti-B . stearothermophilus TRAP antibodies coupled to CNBr-activated Sepharose 4B as described previously (30) .

In vitro transcription attenuation assay. For the in vitro transcription attenuation assay, the EcoRI/BamHI and EcoRI/HindIII 820-bp restriction fragments described above, containing the B . halodurans and B . subtilis trp promoter-leader regions, respectively, were used as templates . Reactions were carried out for 30 min at 37°C in 100 mM potassium-glutamate, 40 mM Tris-Cl (pH 8), 4 mM MgCl₂, 1 mM dithiothreitol, 4 mM spermidine, 2.5% glycerol, 500 µM (each) ATP, CTP, and GTP, 25 µM UTP, 5 µCi of [-³²P]UTP, and B . subtilis vegetative (_A) RNA polymerase purified as described previously (32); 0.5 µM TRAP and 1 mM L-tryptophan were added as indicated . Samples were electrophoresed on 6% polyacrylamide-8 M urea gels . Gels were exposed to a phosphorstorage screen and developed on a Molecular Dynamics PhosphorImager . Radiolabeled RNA bands were quantified by using ImageQuant (version 5.1) software . The percentage of the terminated transcript was calculated as the ratio between the terminated product and the total RNA detected in the lane .

RNA binding assays. RNA binding to TRAP was measured by a nitrocellulose filter binding assay as described previously (11), and the data were analyzed using a nonlinear regression algorithm (GraphPad Prism, version 3.0; GraphPad Software Incorporated, San Diego, Calif.) .

 
Organization of the B . halodurans aromatic supraoperon. The order of genes in the aro-trp region of the B . halodurans chromosome is the same as that in B . subtilis: aroFBHtrpEDCFBA-hisC-tyrA-aroE (23) . The protein sequences of the B . halodurans TrpE, TrpD, TrpC, TrpF, TrpB, and TrpA polypeptides are 40 to 70% identical to those of their homologues from B . subtilis and B . stearothermophilus; identities with the B . stearothermophilus polypeptides are slightly higher (Table 2) .

 
The initiation codons of all the B . halodurans trp polypeptides are preceded by recognizable SD sequences . The start codon for trpD is GUG, and that for trpF is UUG . In B . halodurans the adjacent coding sequences of all the trp genes overlap by 8 to 23 bp . A similar situation exists in B . subtilis, where the coding sequences overlap by 8 to 29 bp, except for trpC and trpF, which are separated by 6 bp .

The B . halodurans trp promoter and regulatory region. Primer extension using RNA isolated from B . halodurans and a primer complementary to residues 136 to 166 upstream of the trpE start codon identified two sites for initiation of transcription of the trp operon 234 and 238 nucleotides upstream of the trpE start codon (Fig . 2) . The residue at the upstream site of transcription initiation is an A, while the downstream residue is a C . It is unusual for transcription to initiate with a pyrimidine . There was no difference in the sites of transcription initiation or in the levels of trp mRNA produced in response to tryptophan . This observation indicates that transcription initiation from the trp promoter is not regulated in response to tryptophan, which is also true of B . subtilis (26) . Based on these start sites, the proposed trp promoter is indicated in Fig. 2B . As seen with the B . subtilis (10, 35) and B . stearothermophilus (13) trp promoters, there is an excellent match to the -35 region consensus sequence, whereas the -10 region matches less well . The -35 region of the B . halodurans trp promoter is contained within the aroH coding region (Fig . 2B), which also occurs in B . subtilis (11) and in B . stearothermophilus (13) .

 
In B . subtilis both transcription and translation of the trp operon are regulated through formation of alternative RNA secondary structures in the leader region of the trp mRNAs prior to trpE (5) . We used computer analysis (Mfold [46]) to examine whether similar RNA structures could form in the leader regions of B . halodurans trp mRNAs . These analyses predicted RNA structures similar to the transcription terminator (a stem-loop structure followed by 5 U residues interrupted by a G residue) and to the antiterminator upstream of the terminator (Fig . 3) . These two structures overlap by 8 nucleotides, and hence their formation is mutually exclusive . The predicted stabilities of both structures are significantly lower than those of the analogous structures from B . subtilis . Furthermore, the putative terminator contains an internal loop just above the base of the stem . Both the low stability and the interruption of the base-paired stem make this an unusual transcription terminator . The predicted antiterminator is also small and has a predicted stability lower than that of the putative terminator, suggesting that in the absence of any other influence, the putative terminator would be likely to form in the leader transcript . This situation differs from that seen in trp leader regions from several other bacilli, where the predicted antiterminator structure is at least as stable as the terminator structure (13, 26), leading to a high level of transcriptional readthrough in the absence of any other influencing factor .

 
In addition to the putative terminator and antiterminator, there is another potential stem-loop structure in the B . halodurans trp leader region upstream of the antiterminator (Fig . 3A and B) . This structure overlaps the 5' portion of the antiterminator by 9 nucleotides and hence could potentially function as an anti-antiterminator (AAT), since its formation would prevent formation of the antiterminator and thus favor formation of the terminator structure . If this structure exists in the default case (in the absence of TRAP binding), then it would reduce readthrough transcription into the structural genes even in the absence of tryptophan . AAT structures are not seen in the leader regions of trp operons of other bacilli that have been characterized (5), although such a system exists in the B . subtilis pyr operon (28) .

The trp leader transcripts from several bacilli, including B . subtilis, B . pumilus, B . stearothermophilus, and "Bacillus caldotenax," also contain a stem-loop structure at the 5' end of the RNA (5) . Moreover, Du et al . (19) showed that this structure is important for proper attenuation control of the B . subtilis trp operon . In B . halodurans there is a predicted stem-loop structure upstream of the putative AAT (Fig . 3A) . In this case the 5' end of the stem is at residue 12, which is further downstream than in the other bacilli, where this structure starts at residue 2 (5) . The predicted stability of this structure is very low, and hence this putative 5' stem-loop may not be stable .

The TRAP protein regulates transcription and translation of the B . subtilis trp operon by binding to the leader transcript in a tryptophan-dependent manner (17, 26) . TRAP binds to a site containing 11 triplet repeats consisting of GAG and UAG separated from each other by two or three nonconserved residues termed spacers (3, 9) . This binding prevents the antiterminator structure from forming and thus induces formation of the terminator, halting transcription of the operon . The leader region of the B . halodurans trp transcript also contains multiple NAG triplet repeats (Fig . 3) . In this case there are potentially as many as 19 GAG, UAG, and AAG repeats between the start of transcription and the antiterminator, which is more than the 11 to 12 repeats seen in the trp leader regions of other bacilli (5, 13, 24) .

The arrangement of the triplet repeats in the B . halodurans trp leader is also different from that in the TRAP binding sites of other trp operon leader regions . In the cases characterized previously, the repeats start shortly after the 5' stem-loop and continue through the 5' portion of the antiterminator (Fig . 3C) . In B . halodurans, the (G/U/A)AG repeats begin immediately at the start of the trp operon transcript and continue through the 5' stem-loop and the AAT, and there is one UAG in the 5' portion of the antiterminator as well as a GAG in the 3' portion of the antiterminator stem (Fig . 3A) . It is not clear whether all of these triplet repeats play a role in TRAP binding . However, if the function of TRAP binding in regulating transcription attenuation of this operon is similar to that in B . subtilis, then binding to the repeat(s) in the antiterminator would be most critical (See Discussion) .

In addition to controlling the transcription of the trp operon, TRAP also regulates the translation of trpE, the first structural gene of the operon in B . subtilis (17) . This regulation is accomplished by the binding of TRAP to the same 11 (G/U)AGs in the leader region of trp readthrough mRNAs that have extended beyond the attenuator region . TRAP binding to these transcripts alters the structure of the leader region such that the trpE SD sequence is sequestered in a stable RNA hairpin, which inhibits translation initiation (17) . Our computer search did not reveal any potential similar structure for translational control of trpE in B . halodurans .

The B . halodurans pabA (trpG) gene. In both B . subtilis and B . halodurans, the only tryptophan biosynthetic gene that is not in the trp operon is pabA (formerly known as trpG), which is located in the folic acid operon (37) . TRAP regulates the translation of pabA in B . subtilis by binding to a site containing 9 NAG repeats (7 GAG repeats, 1 UAG repeat, and 1 AAG repeat) that in part overlaps the SD sequence (44) (Fig . 4) . TRAP binding to this site directly inhibits translation initiation (18) . There is a putative TRAP binding site upstream of pabA in B . halodurans that also contains 9 NAG repeats (4 GAG, 2 UAG, and 3 AAG repeats) (Fig . 4) . The arrangement of the repeats is slightly different in B . halodurans: only the last GAG is within the SD sequence, and there is no repeat between the SD sequence and the pabA start codon .

 
Additional genes regulated by TRAP. Two other B . subtilis genes, yhaG (encoding a putative tryptophan transport protein) and ycbK (whose function is unknown), have TRAP binding sites overlapping their SD sequences (5) . ycbK is part of a two-gene operon with rtpA, which encodes a protein called anti-TRAP (AT) (40) . Expression of AT is induced in response to uncharged tRNA^Trp (34) . AT influences the regulation of the trp genes by binding to tryptophan-activated TRAP and preventing it from binding RNA, thus increasing the expression of the trp genes (40) . A computer search failed to identify genes in B . halodurans with sequence homology to yhaG, ycbK, or rtpA . Interestingly, to date, the gene encoding AT, rtpA, has been identified only in B . subtilis .

B . halodurans TRAP. Sequence analysis identified a B . halodurans gene predicted to encode a 76-amino-acid polypeptide with 71% identity to B . subtilis TRAP (Fig. 5) . As seen with B . subtilis, B . stearothermophilus, and B . pumilus TRAPs, homology is greatest in the middle portion of the sequence and weakest at the N and C termini (14). B . halodurans TRAP was expressed in E . coli by using a T7 promoter expression system (38) . The recombinant E . coli cells expressed TRAP to 15 to 20% of total soluble protein . Purification of B . halodurans TRAP by immunoaffinity chromatography using antibodies against B . stearothermophilus TRAP yielded approximately 15 mg of TRAP per liter of cell culture, and the protein was more than 95% pure based on staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels with Coomassie blue or silver (data not shown) .

 
Selection for B . halodurans mtrB mutants. 5FTrp is a potent tryptophan analog that inhibits the growth of B . subtilis by activating TRAP to suppress expression of the trp genes while failing to substitute for tryptophan in protein synthesis . B . subtilis mutants selected for growth on 5FTrp contained mutations in the mtrB gene, which encodes TRAP, resulting in constitutive overexpression of the trp genes (21) . We selected B . halodurans mutants that were able to grow on high levels of 5FTrp . One such mutant (PGBH18) contained a point mutation in the stop codon (TAACAA) of the mtrB gene, thus extending the open reading frame by 13 codons . This mutation is similar to the previously characterized mtrB allele 264 of B . subtilis (21) .

Regulation of ASase activity in response to tryptophan availability. ASase is a complex of two polypeptides, encoded by trpE and pabA (trpG), which catalyzes the first step in tryptophan synthesis (23) . Examining the levels of ASase activity in cells grown in the absence and presence of tryptophan provides an indication of the regulation of the production of these polypeptides . We assayed ASase activity in wild-type B . halodurans C-125 grown in the absence and presence of tryptophan, and we found approximately threefold down-regulation in response to tryptophan (Table 3) . As seen previously (27), under the same conditions wild-type B . subtilis showed nearly 80-fold down-regulation of ASase .

 
There was very little regulation of ASase activity in the B . halodurans mtrB mutant strain (PGBH18) in response to tryptophan, and expression was elevated approximately fourfold over that seen in wild-type B . halodurans grown in the absence of tryptophan . These results are similar to those observed for the B . subtilis mtrB mutant BG2086 (Table 3) (27), and they show that regulation of the expression of ASase in wild-type B . halodurans in response to tryptophan is mediated by TRAP .

Transcriptional regulation of the trp operon in B . halodurans in vivo. To further examine the regulatory mechanisms of the B . halodurans trp operon, we compared transcriptional and translational control of the B . halodurans and B . subtilis trp operons in vivo . We integrated B . halodurans trpE'-'lacZ transcriptional or translational fusions into the amyE locus of the B . subtilis chromosome and examined ß-galactosidase expression in the absence and presence of tryptophan . We compared these results to those from similar fusions using the B . subtilis regulatory region . These studies were performed with B . subtilis, because B . halodurans C-125 competence has not been demonstrated (39), and we, like others, were unable to transform this strain despite extensive efforts .

ß-Galactosidase expression from a transcriptional fusion of the B . halodurans trp leader sequence to lacZ showed approximately 2.4-fold regulation in BG2087 (mtrB⁺) in response to tryptophan (Table 4) . In comparison, 80-fold regulation was observed for the B . subtilis trpE'-'lacZ transcriptional fusion in this strain in response to tryptophan . For both transcriptional fusions, expression was not affected by tryptophan in BG4233 (mtrB) (Table 4), indicating that TRAP is required for regulating transcription in both cases . The level of ß-galactosidase expression from each transcriptional fusion in BG4233 was approximately twofold higher than that seen in the wild-type strain BG2087 grown in the absence of added tryptophan . This difference presumably reflects partial activation of TRAP in response to the intracellular levels of tryptophan produced in BG2087, which is trp⁺ .

 
We also examined translational fusions of trpE to lacZ under the control of the trp promoter and regulatory region . In this case, the observed regulation of lacZ reflects both transcriptional and translational control . Expression of the B . halodurans translational fusion was regulated approximately 2.7-fold in response to tryptophan (Table 4) . This is nearly the same amount of regulation that was seen with the transcriptional fusion; thus, it suggests no additional translational control . In the wild-type strain, the B . subtilis translational fusion showed 118-fold regulation in response to tryptophan, which indicates additional translational regulation over that seen with the transcriptional fusion (80-fold), a finding similar to previous observations (29) . No regulation was seen for either translational fusion in the mtrB strain (Table 4) .

Transcription attenuation in the 5' leader region of the B . halodurans trp operon in vitro. To further examine the role of the leader region in controlling the transcription of the B . haldurans trp operon, we tested in vitro transcription of a DNA template containing the trp promoter, the leader region, and the start of trpE by using B . subtilis RNA polymerase . We examined the transcription of this template in the absence and presence of TRAP and tryptophan and compared these results to those obtained by using an analogous template from B . subtilis (Fig. 6) . With the B . halodurans template alone, two prominent transcripts were observed (Fig . 6, lane 6): a major product of 370 nucleotides, corresponding to the full-length runoff transcript initiating at the trp promoter, and a minor product of 150 nucleotides consistent with a transcript ending at the putative trp attenuator . This observation indicates that the B . halodurans trp attenuator does function as a terminator for B . subtilis RNA polymerase . The size of the terminated transcript was determined by comparison with several RNA markers of 130, 140, 155, and 160 nucleotides . The B . halodurans terminated transcript ran just under the 155-nucleotide marker and above the 140-nucleotide RNA (data not shown) . Similar results were seen with the B . subtilis template (Fig . 6, lane 1) . There was approximately 2% termination at the B . subtilis attenuator, whereas with the B . halodurans template there was approximately 30% termination . In either case, adding either TRAP or tryptophan alone had no effect on termination (Fig . 6; compare lane 1 with lanes 2 and 3 and lane 6 with lanes 7 and 8) .

 
Adding both TRAP and tryptophan to the reaction mixture containing the B . halodurans trp leader template increased termination at the attenuator from 30% to approximately 90% (Fig . 6, lanes 9 and 10) irrespective of whether TRAP from B . halodurans or TRAP from B . subtilis was used . This result shows that the terminator in the B . halodurans trp leader is rather strong even though it contains an unusual internal loop . As seen previously (7), transcription termination increased to more than 95% with the B . subtilis trp leader template in the presence of tryptophan-activated TRAP (Fig . 6, lanes 4 and 5) . Again, TRAP from B . halodurans and TRAP from B . subtilis were equally effective at inducing termination in the leader region for this template . Hence, the presence of excess tryptophan-activated TRAP increased termination 50-fold for the B . subtilis template, a finding similar to previous observations (7, 29), but only 3-fold for the B . halodurans template . The observation of a 3-fold increase in transcription termination in the presence of tryptophan-activated TRAP is similar to the degree of transcriptional regulation we observed in vivo (Table 4) .

RNA binding properties of B . halodurans TRAP. Our studies, both in vitro and in vivo, show that transcription of the B . halodurans trp operon is regulated less (3-fold) than that of the B . subtilis trp operon (80-fold) . This difference could potentially be due to differences either in the trp leader regions or in the TRAP proteins from the two species . The amino acid sequences of the TRAPs from the two bacilli are rather similar (71% identical) (Fig . 5), whereas the nucleotide sequences and the predicted RNA structures of the two trp leader regions are less similar (Fig. 3), suggesting that the differences in regulation are more likely due to the cis-acting sequences of the trp leader transcripts . To further probe this question, we examined the RNA binding properties of B . halodurans TRAP compared to those of B . subtilis TRAP . We found no significant differences in the binding affinity of either protein for trp leader RNA from either B . subtilis or B . halodurans; the K_d values were 4.0 ± 1.0 nM in every case . These results further suggest that the differences in the regulation of transcription of the trp operon between the two bacilli are due to variations in the leader regions .

Translational regulation of pabA in vivo. We examined the regulation of B . halodurans pabA in vivo by using pabA'-'lacZ transcriptional and translational fusions integrated into the amyE locus of the B . subtilis chromosome . We compared the regulation of ß-galactosidase production in response to tryptophan to that from similar constructs with B . subtilis pabA (44) . Expression of the transcriptional fusion of either the B . halodurans or the B . subtilis pab promoter region to lacZ was not significantly affected (less than 1.6-fold) by tryptophan in either the wild-type (BG2087) or the mtrB mutant (BG2086) strain (Table 5) . In contrast, expression of the translational fusion of pabA was regulated by tryptophan for both the B . halodurans and B . subtilis pabA translational fusions, but only in the wild-type strains . As seen previously (44), the B . subtilis pabA'-'lacZ fusion was regulated approximately sixfold in response to tryptophan . Likewise, ß-galactosidase activity from the B . halodurans pabA'-'lacZ translational fusion in BG2087 (mtrB⁺) was regulated fourfold in response to tryptophan . These findings indicate that TRAP regulates the translation of pabA in response to tryptophan in B . halodurans .

 
Regulation of the genes involved in tryptophan metabolism has been studied extensively in B . subtilis (5) . An RNA binding protein called TRAP regulates expression of these genes by at least three distinct mechanisms: (i) transcription attenuation control of the trpEDCFBA operon, (ii) translational control of trpE, altering RNA secondary structure, and (iii) translational control of pabA, yhaG, and likely ycbK through direct competition with ribosomes for binding to these mRNAs . In all cases TRAP binds to targets in the mRNAs that contain multiple UAG, GAG, and AAG repeats .

We have investigated the regulation of the tryptophan genes in the alkaliphilic bacterium B . halodurans . This organism contains a TRAP with 71% amino acid sequence identity to the B . subtilis TRAP (Fig . 5), suggesting that it could use similar mechanisms to regulate the trp genes . The arrangements of the trpEDCFBA operon and of pabA are the same in B . halodurans and B . subtilis . However, based on analysis of the genome (39), there do not appear to be B . halodurans homologues of yhaG (encoding a putative tryptophan transport protein), ycbK (a TRAP-regulated gene of unknown function), or rtpA (encoding AT) .

The leader region of the B . halodurans trp operon shows some similarities to, and some distinct differences from, the B . subtilis trp leader region . Both leaders are predicted to be capable of folding into transcription terminators and overlapping antiterminator structures, which are characteristic of transcription attenuation . However, the predicted stabilities of these structures in B . halodurans are significantly lower than those for the analogous structures in B . subtilis (Fig . 3) . The predicted structure of the terminator is also unusual in that the base-paired stem is interrupted by an internal loop . Gusarov and Nudler have shown that an intrinsic terminator that is incapable of forming a fully base-paired stem-loop can still induce transcription termination, at least in vitro (22) . Moreover, our in vivo studies indicate that transcription of the B . halodurans trp operon is regulated and that this regulation is dependent on TRAP (Table 4) . Moreover, our in vitro transcription studies of the B . halodurans trp leader region show a transcript consistent with termination at this attenuator, and addition of tryptophan-activated TRAP increases the fraction of transcripts that terminate at this attenuator (Fig . 6) . Together, these studies indicate that the B . halodurans trp operon is regulated by an attenuation mechanism similar to that which controls the B . subtilis trp operon .

Given the unusual nature of the predicted B . halodurans trp leader RNA structures compared to those from other bacilli, we investigated other possible foldings of this region by using Mfold (44) . We focused on looking for potential terminators, since we have data regarding where the attenuated B . halodurans trp transcript ends (between nucleotides 140 and 155) . The only other potential terminator in this region is a large stem-loop beginning at residue 96 and ending at residue 159, followed by 6 U's . In this case the structure that we have labeled the AAT would now function as the antiterminator, since it overlaps the putative terminator by 11 nucleotides . However, we have discounted this arrangement for two reasons: (i) the predicted terminator is rather large (24 bp and two internal loops) to function as an intrinsic terminator, and (ii) the predicted terminated transcript would be approximately 165 nucleotides, which is clearly larger than what we observe . Hence, we present the structures in Fig . 3 as our best prediction . Clearly, more work needs to be done to demonstrate the existence and relevance of these structures .

The amount of regulation of the B . halodurans trp operon (2- to 3-fold) is far less than that for the B . subtilis operon ( 80-fold) . This difference is seen both in vivo (Tables 3 and 4) and in our in vitro transcription studies (Fig. 6) . The major difference is the basal level of termination in the absence of TRAP . The B . halodurans template shows approximately 30% termination, whereas the B . subtilis template shows only 2% . This difference could be due to the lower stability of the B . halodurans antiterminator and/or the presence of a potential AAT in the B . halodurans trp leader transcript . The AAT is predicted to be the most stable secondary structure in the B . halodurans trp leader RNA, and since it overlaps the antiterminator by 9 nucleotides, its formation would prevent formation of the antiterminator and favor formation of the terminator, leading to an increased level of basal termination .

The B . halodurans trp leader transcript potentially contains as many as 19 (G/U/A)AG repeats starting at +2 and continuing through the antiterminator . This raises the question of where TRAP binds in the B . halodurans trp leader RNA . We have not yet determined this experimentally, but based on comparison with the B . subtilis system, we can predict where TRAP will bind in order to function in attenuation control . Formally, there are two possible mechanisms by which TRAP binding could increase transcription termination in the B . halodurans trp leader region: by disrupting formation of the antiterminator structure or by stabilizing the AAT structure . The AAT structure is stabilized when PyrR binds to the leader transcript of the B . subtilis pyr operon (28); however, given the mechanism by which TRAP binds RNA to regulate attenuation in the B . subtilis system, this mechanism seems unlikely to occur in the B . halodurans trp operon . Hence we propose that TRAP will function in the B . halodurans system by binding to at least one of the triplet repeats in the antiterminator structure . There are two such repeats: a UAG in the 5' portion of the antiterminator stem and a GAG in the 3' portion of the stem (Fig . 3) . However, if TRAP bound to the 3' GAG, it would prevent formation of the terminator . Given that TRAP binding enhances transcription termination both in vivo and in vitro, it does not appear that TRAP binds to this repeat . Therefore, we propose that TRAP binds to the UAG in the 5' portion of the antiterminator and the 10 adjacent repeats prior to it . Based on this assumption, we present a model for TRAP regulation of transcription attenuation in the B . halodurans trp operon (Fig . 7) .

 
In B . subtilis the trpEDCFBA operon is subject to 13-fold translational control of trpE in addition to the 80-fold regulation by transcription attenuation (29) . Furthermore, expression of the downstream genes is affected by translational coupling and transcriptional polarity (43) . We were unable to detect any predicted RNA secondary structures in the B . halodurans trp leader region that would sequester the trpE SD sequence . In agreement with this observation, we found the same level of regulation (two- to threefold) in vivo for both transcriptional and translational gene fusions of the B . halodurans trp promoter and regulatory regions to lacZ (Table 4), as well as for ASase activity in response to tryptophan (Table 3) . Hence, there does not appear to be any translational control of trpE in B . halodurans .

Together, these results indicate that B . halodurans and B . subtilis use very similar TRAPs to regulate transcription of the trp operon but that the range of regulation is far less in B . halodurans and that unlike B . subtilis, B . halodurans does not regulate the translation of these genes . These differences likely reflect the different environments and lifestyles of these two bacilli . While there are clear differences in the means and degrees to which these two bacilli regulate the expression of the trp operon, regulation of pabA is rather similar in the two bacteria . This similarity may reflect the roles that pabA plays in the biosynthesis of both tryptophan and folic acid in both organisms .

 
We thank Paul Babitzke for critical reading of the manuscript . We also thank Min Yang for the use of his pabA'-'lacZ fusion-containing B . subtilis strains .

This work was supported by grants GM62750 from the National Institutes of Health and MCB 9982652 from the National Science Foundation .

 
* Corresponding author . Mailing address: Department of Biological Sciences, State University of New York, Buffalo, NY 14260 . Phone: (716) 645-2363, ext . 189 . Fax: (716) 645-2975 . E-mail: gollnick@acsu.buffalo.edu.

Present address: Department of Dermatology, Medical University of Pecs, Kodaly u . 20, Hungary .

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