JOURNAL OF BACTERIOLOGY, Mar. 2010, p. 1518–1526
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 192, No. 6
Positions of Trp Codons in the Leader Peptide-Coding Region of the
at Operon Influence Anti-Trap Synthesis and trp Operon
Expression in Bacillus licheniformis?
Anastasia Levitin§ and Charles Yanofsky*
Department of Biology, Stanford University, Stanford, California 94305-5020
Received 29 October 2009/Accepted 18 December 2009
Tryptophan, phenylalanine, tyrosine, and several other metabolites are all synthesized from a common
precursor, chorismic acid. Since tryptophan is a product of an energetically expensive biosynthetic pathway,
bacteria have developed sensing mechanisms to downregulate synthesis of the enzymes of tryptophan forma-
tion when synthesis of the amino acid is not needed. In Bacillus subtilis and some other Gram-positive bacteria,
trp operon expression is regulated by two proteins, TRAP (the tryptophan-activated RNA binding protein) and
AT (the anti-TRAP protein). TRAP is activated by bound tryptophan, and AT synthesis is increased upon
accumulation of uncharged tRNATrp. Tryptophan-activated TRAP binds to trp operon leader RNA, generating
a terminator structure that promotes transcription termination. AT binds to tryptophan-activated TRAP,
inhibiting its RNA binding ability. In B. subtilis, AT synthesis is upregulated both transcriptionally and
translationally in response to the accumulation of uncharged tRNATrp. In this paper, we focus on explaining
the differences in organization and regulatory functions of the at operon’s leader peptide-coding region, rtpLP,
of B. subtilis and Bacillus licheniformis. Our objective was to correlate the greater growth sensitivity of B.
licheniformis to tryptophan starvation with the spacing of the three Trp codons in its at operon leader
peptide-coding region. Our findings suggest that the Trp codon location in rtpLP of B. licheniformis is designed
to allow a mild charged-tRNATrpdeficiency to expose the Shine-Dalgarno sequence and start codon for the AT
protein, leading to increased AT synthesis.
Bacteria regulate the expression of their tryptophan (trp)
biosynthetic genes and operons by using various strategies that
sense the levels of free tryptophan (Trp) and/or uncharged
tRNATrp(29). Trp is synthesized from chorismic acid, which is
also the precursor of phenylalanine, tyrosine, p-aminobenzoic
acid (PABA), and several other metabolites (50). Trp biosyn-
thesis involves catalysis by the protein products of seven genes
or genetic segments. Many bacilli have all seven trp genes in
one operon, and this operon is regulated transcriptionally by
an uncharged-tRNATrp-sensing T-box sequence or by tandem
T-box sequences (23, 29). In some bacilli, including Bacillus
subtilis and Bacillus licheniformis, the trp operon is organized
differently. A cluster of six of the seven trp genes, trpEDCFBA,
is located as a trp suboperon within a larger aromatic (aro)
supraoperon (19). Initiation of transcription of the trp sub-
operon occurs at two promoters, one at the beginning of the
aro supraoperon and the second preceding trpE of the trp
suboperon (50). The seventh trp gene, trpG-pabA, specifying a
bifunctional protein involved in both Trp and PABA synthesis,
is located in the unlinked folate operon (19, 21, 29). Expression
of trpG-pabA is also regulated in response to the availability of
Trp (17, 46).
Prior studies (10, 20, 21, 24) identified many genes, operons,
and regulatory molecules and events that are involved in Trp
biosynthesis and its regulation in B. subtilis (Table 1). These
same genes and other cell components appear to be present in
B. licheniformis, a closely related bacterium (29). However, the
aro supraoperon, containing a trp suboperon, is a relatively
uncommon organizational strategy in the bacilli (29) or in
other bacteria, and the presence of a regulatory at operon,
responding to uncharged tRNATrpas a regulatory signal, is
even rarer (29).
Transcription of the trp suboperons of B. subtilis and of B.
licheniformis, initiated at either the aro or trp suboperon pro-
moter, is regulated by transcription attenuation (termination)
in the region immediately preceding trpE. The principal regu-
lator is the Trp-activated RNA-binding attenuation protein,
TRAP (15, 19–21, 46). When free Trp is plentiful and avail-
able, it binds to—and activates—TRAP. The TRAP protein
contains 11 identical protein subunits, 11 Trp binding sites, and
11 Lys-Lys-Arg motifs on the periphery of the protein complex
(2, 3, 13, 49). When activated by Trp, each Lys-Lys-Arg motif
is capable of binding to a (G/U)AG repeat in a target tran-
script, resulting in the RNA being wrapped around TRAP’s
perimeter (8, 9, 30). This prevents the formation of an RNA
antiterminator structure, thereby promoting the formation of
an RNA terminator structure that causes transcription termi-
nation (8, 9, 30). Activated TRAP also binds to (G/U)AG
repeats in mRNA segments of other genes and inhibits initia-
tion of their translation. This occurs in transcripts of the fol-
lowing genes: trpE (16), trpG-pabA (17, 46); trpP-yhaG, encod-
ing a putative Trp import protein (32); and ycbK, encoding a
putative Trp efflux protein (32). The ycbK gene is located
within the rtpA-ycbK (at) operon (45, 47). The rtpA gene en-
codes the anti-TRAP protein, AT, which is capable of binding
* Corresponding author. Mailing address: Department of Biology,
Stanford University, Stanford, CA 94305. Phone: (650) 725-1835. Fax:
(650) 725-8221. E-mail: firstname.lastname@example.org.
§ Present address: Department of Genetics, Stanford University
School of Medicine, Stanford, CA 94305.
?Published ahead of print on 8 January 2010.
to Trp-activated TRAP at its RNA-binding surface (35, 36, 43)
and preventing TRAP from binding to its target RNAs. Thus,
by binding to TRAP, AT can regulate transcription or trans-
lation of all the operons regulated by TRAP (39, 40). AT
synthesis is highly regulated itself, both transcriptionally and
translationally, in response to the accumulation of uncharged
In a number of bacilli, AT-dependent mechanisms for reg-
ulating trp operon expression appear to exist. They include B.
subtilis, B. licheniformis, Bacillus amyliquefaciens, Bacillus mo-
javensis, and Bacillus spizizenii (14). Most studies of AT and
TRAP synthesis and function have been performed with B.
subtilis (15, 19, 39–41, 44, 49). In that organism, AT synthesis
is regulated both transcriptionally and translationally by sens-
ing the accumulation of uncharged tRNATrp. Transcription
regulation of at mRNA synthesis is achieved in one segment of
the operon’s leader region by an uncharged-tRNATrp-sensing
T-box transcription antitermination mechanism (23, 32). In B.
subtilis, AT synthesis is also regulated translationally at a 10-
residue leader peptide-coding region, rtpLP, located immedi-
ately upstream of rtpA, the structural gene for the AT protein.
The rtpLP-coding region of B. subtilis contains three adjacent
Trp codons, and its stop codon is located 6 nucleotides pre-
ceding the rtpA Shine-Dalgarno (SD) sequence (Fig. 1) (12).
Completion of the translation of rtpLP mRNA of B. subtilis
inhibits initiation of AT synthesis, presumably by ribosome
blockage of the rtpA SD sequence. However, when there is a
cellular charged-tRNATrpdeficiency, the ribosome translating
TABLE 1. Operons, product functions, regulating signals, regulatory products, and transcription and translation regulators involved in Trp
biosynthesis and its regulation in B. subtilis and B. licheniformis
Operon Product functionRegulating signal
TRAP, ATTRAP, AT
FIG. 1. Comparison of the at operon’s leader peptide nucleotide sequence (rtpLP) and amino acid sequence (LP) and neighboring nucleotide
regions of B. subtilis and B. licheniformis. The at operons of both B. subtilis and B. licheniformis contain two structural genes, rtpA and ycbK,
preceded by a short leader peptide-coding region, rtpLP. The rtpLP, rtpA, and ycbK coding regions of both organisms are transcriptionally regulated
by a T-box leader RNA region that is responsive to uncharged versus charged tRNATrp. The T-box region is followed by the leader peptide-coding
region, rtpLP. (A) The B. subtilis leader peptide (LP) consists of 10 amino acids, with three Trp residues arranged in tandem. The Shine-Dalgarno
sequence for rtpA of B. subtilis is in an untranslated region preceding the leader peptide-coding region. (B) The B. licheniformis LP contains 22
amino acids. It also has three Trp residues; however, these are spaced throughout the leader peptide. In B. licheniformis, the rtpA Shine-Dalgarno
sequence and the rtpA start codon are located within the 3? segment of the rtpLP leader peptide-coding region. The start codon of rtpA is out of
frame with the third Trp codon of the rtpLP leader peptide-coding region and is followed by an out-of-phase stop codon for rtpLP.
VOL. 192, 2010 B. LICHENIFORMIS at OPERON LEADER PEPTIDE1519
rtpLP mRNA presumably stalls at any one of its three Trp
codons, exposing the SD region of rtpLP mRNA, allowing rtpA
mRNA translation and AT synthesis (11). In B. licheniformis,
AT synthesis is also regulated transcriptionally by the T-box
mechanism and translationally by rtpLP, the at operon’s leader
peptide-coding region. However, in B. licheniformis, rtpLP is a
22-residue-coding region rather than a 10-residue-coding re-
gion, as it is in B. subtilis. In addition, in B. licheniformis, the
rtpLP coding region includes the SD sequence, as well as the
start codon of rtpA, the structural gene for the AT protein (Fig.
1). Most importantly, the three Trp codons of the rtpLP
mRNA of B. licheniformis are dispersed throughout this coding
region, rather than adjacent to one another as they are in rtpLP
of B. subtilis. In the studies described in this article, we ana-
lyzed the significance of the different Trp codon locations
within the rtpLP leader peptide-coding region of B. lichenifor-
mis. We focus on explaining the differences in organization and
function of this rtpLP coding region relative to rtpLP of B.
subtilis. Our findings suggest that the Trp codon location and
other features of the rtpLP leader mRNA of B. licheniformis
are designed to allow the organism to respond predominantly
to conditions leading to the accumulation of low levels of
MATERIALS AND METHODS
Bacterial strains, plasmids, and transformations. The strains used in this
study are listed in Table 1. CYBS400 is a B. subtilis prototroph, Bl54A is a B.
licheniformis prototroph, and CYBS318 [CYBS400 ?(rtpA-ycbK)::Spr] is a B.
subtilis strain lacking its AT-coding region and the region encoding the 5? end of
the ycbK open reading frame (ORF); it does not produce either the AT or the
YcbK protein. The strain was constructed by replacing a 614-bp chromosomal
segment of the rtpA-ycbK region with a gene conferring spectinomycin resistance
(32). The plasmid Pat-LR-?T-rtpLP-rtpA-ycbK?-lacZ was used to construct
CYBL derivative strains (Table 2). It contained a 736-bp leader region encom-
passing the rtpA-ycbK promoter, the leader region, the rtpA ORF, the intergenic
region (33), and 9 nucleotides of the ycbK coding region, followed by the lacZ
gene. Part of the terminator region was deleted to disrupt terminator function,
as described by Sarsero et al. (33). Strain CYBL has the B. subtilis rtpA-ycbK
promoter with the terminator region deleted (33), followed by B. licheniformis
rtpLR replacing B. subtilis rtpLP, followed by B. subtilis rtpA-ycbK?-lacZ. In other
constructs, each of the Trp codons of B. licheniformis rtpLP in the hybrid plasmid
was replaced with an arginine codon (Trp13Arg or Trp23Arg) or with a
cysteine codon (Trp33Cys), thereby preserving the predicted rtpLP RNA sec-
ondary structure and the normal location of the rtpA ATG start codon (strains
CYBL1, CYBL2, and CYBL3). A 15-nucleotide spacer (5 GAT repeats) intro-
ducing repeat UGAs in the coding region was also inserted at nucleotide position
15 of B. licheniformis rtpLP (strain CYBL1 spacer) (see Fig. 4).
Transformations were carried out by using natural competence (1). Gene
fusions and cloned DNA fragments were integrated into the chromosomal amyE
locus by homologous recombination after being introduced into the integration
vector ptrpBG1-PLK (33). Mutant strains were isolated following transformation
by selecting for chloramphenicol resistance, and disruption of amyE was con-
firmed by the absence of amylase production as shown by iodine staining (34).
Growth curves. All strains were grown in Vogel-Bonner minimal medium (42)
supplemented with 0.5% glucose and trace elements at 37°C (10). Where indi-
cated, the following supplements were included: various concentrations of indole
acrylic acid (IA), 100 ?g/ml phenylalanine, 100 ?g/ml tryptophan, 100 ?g/ml
tyrosine, or 100 ?g/ml PABA. Growth rates were determined by measuring cell
density using a Klett-Summerson colorimeter equipped with a 660-nm filter.
RNA extraction. Ten milliliters of cultures grown to 150-Klett-unit densities
were harvested by centrifugation. RNA extraction was performed as described
Real-time PCR. cDNA synthesis was carried out with a SuperScript III First
Strand Synthesis System for RT-PCR from Invitrogen (catalog no. 180980-051)
using 1 ?g of RNA. One-tenth of the total cDNA reaction mixture was used for
real-time PCR analyses. Gene-specific primers were designed to amplify 100-
nucleotide fragments of target genes (Tables 3 and 4). Each reaction was carried
out in a 15-?l volume with 50% SYBR green mixture, according to the manu-
facturer’s protocol (Bio-Rad IQ SYBR green Supermix; catalog no. 170-8882).
Reactions were performed in a MyiQ Single-Color Real-Time PCR Detection
System (catalog no. 170-9770) with the cycling conditions 72°C for 5 min and
95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 52°C for 15 s, and 72°C for
20 s, plus 5 min at 72°C as a final step. The expression of each gene analyzed was
normalized against rpoB gene expression, which served as the internal control
(31). Relative mRNA levels were subsequently calculated using the 2???CT
threshold cycle method (26).
Western blot analysis. Cells were harvested by centrifugation and resuspended
in 300 ?l of 100 mM Tris-HCl, pH 8.0, and 50 mM NaCl. Samples were disrupted
by sonic oscillation, and cell debris was removed by centrifugation. SDS Tris-
tricine (300 ?l) buffer was added, and protein extracts were boiled for 5 min. The
Quick Start Bradford protein assay (Bio-Rad catalog no. 500-0201) was per-
formed on the final samples, and equal amounts of protein were loaded in each
lane. The samples were electrophoresed on SDS-15% polyacrylamide gels in
Tris-tricine buffer and were electrophoretically transferred onto a Trans-Blot
Transfer Medium nitrocellulose membrane (Bio-Rad catalog no. 162-0115). Im-
munoblotting was performed using rabbit polyclonal antibodies against B. subtilis
AT prepared by the Covance Company and peroxidase-conjugated affinity-pu-
rified anti-rabbit antibodies from Rockland (catalog no. 611-1302). The bound
antibodies were visualized using Super Signal WestPico chemiluminescent sub-
strate from ThermoScientific (catalog no. 34077). Their levels were quantitated
by the Adobe Photoshop program, version 7.0.
Real-time PCR comparisons of transcription of the trp
operon and other operons of B. subtilis and B. licheniformis. To
compare trp operon sensitivity to Trp starvation in B. subtilis
and B. licheniformis, we analyzed relevant mRNA levels in
cultures grown in minimal medium with or without excess
Trp and in minimal medium containing different levels of
IA, a structural homolog of Trp that is an inhibitor of
tryptophanyl-tRNA synthetase activity (27). The presence
of IA reduces tRNATrpcharging and therefore the availabil-
ity of Trp-tRNATrpfor new protein synthesis.
Gene expression (mRNA) levels were related to expression
TABLE 2. Strains and constructs used in the studies
B. subtilis wild type
B. licheniformis wild type
CYBS318 amyE::?Pat-LR-?T-Bl rtpLP-rtpA-ycbK?-lacZ?
CYBS318 amyE::?Pat-LR-?T-Bl rtpLP-rtpA-ycbK?-lacZ?
CYBS318 amyE::?Pat-LR-?T-Bl rtpLP-rtpA-ycbK?-lacZ?
CYBS318 amyE::?Pat-LR-?T-Bl rtpLP-rtpA-ycbK?-lacZ?
CYBS318 amyE::?Pat-LR-?T-Bl rtpLP-rtpA-ycbK?-lacZ?
No AT, no YcbK
?T Bl rtpLP
?T Bl rtpLP W5R
?T Bl rtpLP W5R; 15-nucleotide spacer
?T Bl rtpLP W8R
?T Bl rtpLP W22C
1520LEVITIN AND YANOFSKYJ. BACTERIOL.
levels for the rpoB gene, which served as the internal control
(see Materials and Methods). The mRNAs analyzed (Table 5)
were transcribed from the following genes: rtpA, encoding the
AT protein (38, 39); mtrB, encoding the TRAP protein (20,
21); aroF, the first gene in the aromatic supraoperon (21, 50);
trpE, the first gene of the trp suboperon (21); and trpS, the gene
encoding tryptophanyl-tRNA synthetase, the enzyme respon-
sible for charging of tRNATrpwith Trp (37). Our RT-PCR
analyses showed that in B. licheniformis, expression of rtpA and
trpE is upregulated in response to IA addition, as it is in B.
subtilis. However, most noticeably, B. licheniformis produces
substantially higher relative trp operon (trpE) mRNA levels
than B. subtilis when grown in the presence of IA (Table 5).
One possible explanation for this response is that B. licheni-
formis may need to produce more of the trp operon enzymes
under these conditions if it is to provide sufficient Trp for
overall protein synthesis and near-normal growth rates (Ta-
Growth sensitivities of B. subtilis and B. licheniformis to Trp
starvation. Our real-time PCR results suggested that Trp star-
vation caused by IA addition is more pronounced in B. licheni-
formis than in B. subtilis. Thus, the growth of B. licheniformis
should be more sensitive to Trp starvation. To test this hypoth-
esis, we grew cultures of both organisms under different mild
Trp starvation conditions: low levels of IA. We observed that
B. licheniformis was in fact more sensitive to growth inhibition
by IA, an inhibitor of tryptophanyl-tRNA synthetase charging
(Fig. 2). B. licheniformis’ metabolism presumably is more sen-
TABLE 3. Primers used to create the constructs analyzed
Primer Sequence Remarks
B. licheniformis rtpLP
B. licheniformis rtpLP
B. licheniformis rtpLP
B. licheniformis rtpLP
Introduce spacer to
Introduce spacer to
BlLP W5R spacer-5?
BlLP W5R spacer-3?
TABLE 4. RT-PCR primers used in determining relative mRNA levels
Gene5? Sequence3? Sequence
VOL. 192, 2010B. LICHENIFORMIS at OPERON LEADER PEPTIDE 1521
sitive to IA addition because it does not produce enough
charged tRNATrpunder these conditions.
Growth sensitivity of B. licheniformis to IA addition in the
presence or absence of tryptophan, phenylalanine, tyrosine,
and p-aminobenzoic acid. To obtain additional understanding
of the consequences of Trp starvation for B. licheniformis
growth, we also analyzed the growth rates of IA-treated Trp-
starved cells grown in the presence of different products of the
aromatic biosynthetic pathway that have chorismic acid as a
common precursor (50). Thus, we examined the effects of
added phenylalanine, tyrosine, PABA, and Trp on IA-pro-
duced growth inhibition (Fig. 3). The addition of phenyl-
alanine, tyrosine, and PABA did not reverse the IA-produced
growth inhibition of B. licheniformis, whereas added Trp did, in
the absence or presence of phenylalanine, tyrosine, and PABA
(Fig. 3). Thus, IA addition appears to create a charged-
tRNATrpdeficiency that can be reversed by Trp addition.
Effects of replacing individual rtpLP Trp codons on AT pro-
duction in B. subtilis strains with rtpLP of B. licheniformis. We
assume that synthesis of the AT protein is regulated both
transcriptionally and translationally and that the locations of
the Trp codons in rtpLP play a role in regulating AT synthesis.
With these possibilities under consideration, experiments were
performed (see Fig. 5) to examine the effects on AT synthesis
of replacing each of the Trp codons of the leader peptide-
coding region with some other codon. As previously described
in studies with B. subtilis, translation of the entire rtpLP coding
region inhibits AT synthesis, whereas ribosome stalling at the
rtpLP Trp codon cluster increases AT synthesis (12). Presum-
ably, the ribosome reaching the rtpLP stop codon of B. subtilis
masks the rtpA SD sequence, reducing initiation of AT synthe-
sis, whereas a ribosome stalled at any one of the three rtpLP
Trp codons should expose this SD sequence, allowing efficient
initiation of AT synthesis (12). These experiments (see Fig. 5)
were performed to determine the regulatory significance of
ribosome stalling at each of the 3 dispersed Trp codons in the
B. licheniformis rtpLP sequence for the level of AT synthesized.
Strains were constructed in which a hybrid plasmid bearing a
modified at operon of B. subtilis containing rtpLP of B. licheni-
formis was integrated into the chromosome of a B. subtilis
strain bearing a deletion of the resident at operon. In this
plasmid, rtpLP of B. licheniformis replaced rtpLP of B. subtilis
and the B. subtilis T-box terminator was deleted. Thus, at
operon mRNA would be synthesized and rtpLP translational
control of AT synthesis would determine the level of AT pro-
tein that was produced. In the 3 specific constructs, each of the
three Trp codons of B. licheniformis rtpLP was replaced by a
codon specifying another amino acid. Thus, the first Trp codon
(UGG) was replaced by an Arg codon (AGG) in one construct,
the second Trp codon (UGG) was replaced by an Arg codon
(AGG) in a second construct, and the third Trp codon (UGG)
was replaced by a Cys codon (UGU) in a third construct. These
changes did not alter the predicted important RNA secondary
TABLE 5. Relative real-time PCR gene expression (mRNA) levels
in B. subtilis versus B. licheniformis grown under the
Ratio of relative gene expressionb
aRNA was extracted from cultures grown in minimal medium with the sup-
plements indicated (see Materials and Methods).
bmm, minimal medium; Trp, minimal medium supplemented with 100 ?g/ml
of L-tryptophan; IA, minimal medium supplemented with IA. All mRNA levels
were normalized against the rpoB mRNA level in the same extract as an internal
control. The relative mRNA levels were subsequently calculated using the
2???CTthreshold cycle method. IA2, IA5, IA40, and IA80 correspond to the
various levels of IA (?g/ml) added to the cultures.
FIG. 2. Comparative growth sensitivities of B. subtilis and B. licheniformis to different levels of IA (?g IA/ml) added to minimal medium (mm).
Wild-type cultures of B. subtilis CYBS400 (A) and B. licheniformis Bl54A (B) were grown in Vogel-Bonner minimal medium (40) supplemented
with 0.5% glucose and trace elements at 37°C. Various amounts of IA were added to selected cultures. The cell density was determined hourly,
using a Klett-Summerson colorimeter (660-nm filter). The Klett units were plotted versus time to obtain the growth curves shown.
1522LEVITIN AND YANOFSKY J. BACTERIOL.
structure of B. licheniformis rtpLP RNA (Fig. 4). Each plasmid
construct was integrated into the amyE locus of a B. subtilis
strain lacking the at operon, and thus, production of B. subtilis
AT in these strains would be expected to be based on the
organism’s ability to translate the modified B. licheniformis
rtpLP mRNA coding region (Fig. 1). The strains were grown
both in minimal medium minus inducer and in minimal me-
dium plus inducer (IA). The results obtained in Western blot-
ting AT measurements performed with these strains are shown
in Fig. 5. Clearly, replacing the first Trp codon (Trp1) of B.
licheniformis rtpLP had the greatest effect on increasing AT
production. In this strain, the Trp1-to-Arg replacement would
presumably allow the translating ribosome to reach the second
Trp codon, and to stall there, when there is a deficiency of
charged tRNATrp(Fig. 5). Ribosome stalling at this codon
would presumably disrupt the RNA secondary structure that
blocks the SD sequence needed for AT synthesis, and AT
synthesis would be elevated. Apparently, even during growth in
minimal medium (mild Trp starvation) there must be some
increased stalling at Trp2 with this strain, since AT production
in minimal medium was also higher in the Trp1 mutant than in
the wild-type construct. When the wild-type rtpLP construct
was grown with IA (severe Trp starvation), the AT level was
also elevated, but it was not as high as with the Trp1 mutant.
This result indicates that the presence of the Trp1 codon can
prevent the large increase in AT production observed with the
construct lacking Trp1 (Fig. 5). Therefore, in the wild-type
construct, there must be little pausing at the Trp2 codon under
the conditions tested. In the construct with the Trp2 codon
replaced by an Arg codon, AT synthesis was comparable to
that of the wild-type control culture in cultures grown in min-
imal medium and with IA (Fig. 5). Thus, there must be little or
no ribosome stalling at the Trp2 codon in the wild type under
these growth conditions. Clearly, the existence of Trp codon 2
is not solely responsible for the increased AT production as-
sociated with a charged-tRNATrpdeficiency. Replacing the
Trp3 codon with a Cys codon did not result in a significant
change in AT production (Fig. 5). We suspect that a ribosome
reaching this position or stalled at this codon would block
initiation of AT synthesis. Additional experiments are required
to explain the significance, if any, of the location of the Trp3
To confirm the significance of the close proximity of the
second Trp codon of rtpLP to the presumed downstream rtpLP
mRNA secondary structure that sequesters the SD sequence,
we introduced a 15-nucleotide spacer downstream from the
Trp2 codon in the context of the Trp1 codon mutant (Fig. 4).
This spacer should prevent a ribosome stalled at the Trp2
codon from disrupting the RNA secondary structure that pre-
sumably limits AT production. Western blot analyses showed
that, indeed, introduction of the spacer sequence reduced AT
production to the same level observed with the wild-type rtpLP
construct (Fig. 5). Complicating interpretation of these find-
ings is the predicted formation of different RNA secondary
structures in the various transcripts. However, these findings
do suggest that in the rtpLP leader peptide-coding region of B.
licheniformis the location of the Trp2 codon is critical in ob-
taining elevated AT protein production under certain Trp star-
vation conditions. The existence of the Trp1 codon at its loca-
tion may reduce this elevation whenever a translating ribosome
stalls at this codon and does not reach the Trp2 codon.
The purpose of this study was to examine trp operon regu-
lation in two closely related species, B. licheniformis and B.
subtilis, and to determine the significance of the organizational
differences in Trp codon location in their respective at operon
leader peptide-coding regions, rtpLP (Fig. 1). In both B. subtilis
and B. licheniformis, the trp operon is located within an aro
supraoperon, which is regulated by sensing the levels of Trp
and uncharged tRNATrpand by the actions of the TRAP and
AT proteins. These two signal molecules and two regulatory
proteins influence both transcription and translation. In addi-
tion, expression of trpS, the gene encoding tryptophanyl-tRNA
synthetase, is regulated in both organisms by the T-box mech-
anism in response to uncharged-tRNATrpaccumulation (21).
The tryptophanyl-tRNA synthetase level is a major determi-
nant of how much charged tRNATrpwill be produced and
maintained per cell. However, the free-Trp level is also rate
limiting for tRNATrpcharging. In the studies described in this
paper, we showed that the growth of B. licheniformis is more
sensitive to the addition of IA, an inhibitor of tryptophanyl-
tRNA synthetase activity, than is B. subtilis growth (Fig. 2). B.
FIG. 3. Comparative growth sensitivities of B. licheniformis to IA in the presence or absence of Trp and/or Phe, Tyr, and PABA. Wild-type
cultures of B. licheniformis Bl54A were grown in Vogel-Bonner minimal medium with and without 5 ?g/ml (A) or 10 ?g/ml (B) IA, with the various
additional supplements indicated, plus 0.5% glucose and trace elements at 37°C. The cell density was determined hourly, using a Klett-Summerson
colorimeter (660-nm filter). The Klett units were plotted versus time to obtain the growth curves shown.
VOL. 192, 2010B. LICHENIFORMIS at OPERON LEADER PEPTIDE1523
licheniformis responds to IA addition by producing higher trp
operon mRNA levels (and presumably trp operon protein lev-
els) than B. subtilis (Table 5). Furthermore, the lower aroF
mRNA level (aroF is the first gene in the aro supraoperon) in
B. licheniformis may indicate that the organism synthesizes less
chorismate than B. subtilis. Therefore, it presumably must syn-
thesize higher trp enzyme levels for the Trp pathway to com-
pete effectively with the Phe and Tyr pathways for chorismate,
their common precursor. Possibly, B. licheniformis must form
higher levels of each of the Trp pathway enzymes in order to
provide sufficient Trp to support growth. However, the relative
contribution of Trp versus uncharged tRNATrpas a regulatory
signal molecule and potential differences in Trp, Phe, and Tyr
biosynthesis must be further analyzed in these two organisms
FIG. 4. (A) Predicted secondary structures and their stabilities in B. licheniformis at operon leader RNA. Trp codons are shaded in gray, and
the SD sequences of rtpLP and rtpA are in boldface. The start codon of rtpA overlaps the third Trp codon of rtpLP, followed by the rtpLP stop
codon. The 3? end of the segment of nucleotides of rtpLP mRNA that are presumably masked by a ribosome stalling at the Trp1 codon or the Trp2
codon are indicated by three-sided boxes. The vertical arrow indicates the site of insertion of a 15-nucleotide spacer in the rtpLP coding region.
(B to D) Predicted secondary structures and their stabilities upon ribosome stalling in B. licheniformis at operon leader RNA in strains in which
rtpLP Trp1 and -2 codons are replaced (B and C, respectively) and a strain in which a nucleotide spacer is inserted into the strain in which rtpLP
Trp1 is replaced (D).
1524 LEVITIN AND YANOFSKY J. BACTERIOL.
before an explanation can be given for their differences in
sensitivity to IA.
Some organisms that have their trp operons within an aro
supraoperon contain an at operon, providing the anti-TRAP
protein AT and allowing a regulatory response to a charged
tRNATrpdeficiency (29). They include B. subtilis, B. lichenifor-
mis, B. amyloliquefaciens, B. mojavensis, and B. spizizenii (14).
In each of these organisms, the Trp-activated regulatory pro-
tein, TRAP, is primarily responsible for regulating trp operon
transcription (5–7, 9, 15, 21, 46). Depending on how many
TRAP molecules per cell are Trp activated and AT free,
TRAP will be partially or fully active. Thus, TRAP can bind
up to 11 molecules of Trp, to some extent cooperatively (4, 25,
36, 43), and each bound Trp molecule can contribute to
TRAP’s RNA-binding ability. However, even when TRAP is
fully activated, AT can bind to TRAP and block TRAP’s RNA-
binding ability. AT synthesis is regulated both transcriptionally
and translationally in response to the accumulation of un-
charged tRNATrp. The existence of the at operon in B. subtilis
and B. licheniformis allows posttranscriptional decisions to in-
fluence the regulation of the synthesis of the enzymes of the
Trp biosynthetic pathway.
The leader peptide-coding regions of the at operons of B.
licheniformis and B. subtilis are organized differently, and these
differences are presumably designed to allow each organism to
respond appropriately to a charged-tRNATrpdeficiency. Both
B. subtilis and B. licheniformis have three Trp codons in their
rtpLP leader mRNA sequences. However, in B. subtilis, the
three Trp codons are adjacent, and the SD sequence and start
codon of AT are located downstream from the rtpLP stop
codon (Fig. 4A). Pausing at any one of these three codons
would be expected to have a nearly equivalent severe effect,
promoting maximal AT production. In B. licheniformis, its
three Trp codons are spaced throughout the sequence, with the
third Trp codon located just upstream of the stop codon, over-
lapping the SD sequence and start codon for AT (Fig. 4A). The
locations of the Trp codons in the rtpLP leader mRNA se-
quence are designed to allow the organism to be particularly
sensitive to a slight reduction in the level of charged tRNATrp
and to be less sensitive to a severe charged-tRNATrpdeficiency
Ribosome stalling at the first Trp codon of rtpLP leader
RNA (under severe Trp starvation conditions) of B. lichenifor-
mis should reduce leader peptide synthesis by allowing the
rtpPL RNA secondary structure to form, reducing translation
initiation at the rtpA (AT) SD sequence and start codon (Fig.
4B). Stalling at the second rtpLP Trp codon, upon a mild
charged-tRNATrpdeficiency, should eliminate this secondary
structure, allowing efficient ribosome binding and translation
initiation at the rtpA SD sequence and start codon (Fig. 4C).
However, ribosome stalling at the Trp2 codon would allow a
second potential RNA secondary structure to form, but pre-
sumably it is less effective in inhibiting ribosome binding at the
rtpA SD sequence and start codon region. Thus, depending
upon the severity of Trp starvation, the accumulation of un-
charged tRNATrp, and the timing of charged-tRNATrpavail-
ability, the ribosome translating the rtpLP coding region could
stall at either Trp codon, Trp1 or Trp2, and determine the
efficiency of translation initiation at the rtpA start codon. Per-
haps the objective of this design is to allow appreciable AT
synthesis only when there is a slight charged-tRNATrpdefi-
ciency and to prevent additional AT synthesis when most of the
tRNATrpin the cell is uncharged. This would be advantageous
if under severe starvation conditions there was insufficient Trp
to activate TRAP. Therefore, there would be no need to pro-
duce additional AT protein to inactivate TRAP. When a trans-
lating ribosome reached the third Trp codon of rtpLP mRNA,
it would block AT synthesis until translation of rtpLP was
completed. To explain the purpose of each of the three Trp
codons in the rtpLP coding region of B. licheniformis, addi-
tional experiments are needed in which stalling at each of these
Trp codons is related to the level of uncharged tRNATrpin the
cell and the level of AT protein that is produced.
We are indebted to Paul Babitzke and Paul Gollnick for their ex-
cellent comments on the manuscript. We also thank Luis Cruz-Vera
for help with the figures. This paper is the last publication based on
research performed in my laboratory (C.Y.). I express my heartfelt
appreciation to the many undergraduates, graduate students, postdoc-
toral students, visiting fellows, and other investigators who have con-
tributed to our investigations. I have enjoyed every minute!
The studies described in this paper were performed with the support
of the National Science Foundation (MCB-0615390).
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