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Journal of Bacteriology, May 2001, p. 3256-3260, Vol. 183, No. 10
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.3256-3260.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Effects of Mutations in the Pseudomonas
putida miaA Gene: Regulation of the trpE and
trpGDC Operons in P. putida by
Attenuation
Igor
Olekhnovich1,2,
and
Gary N.
Gussin2,*
Department of Microbiology, Belarus State
University, Minsk 220050, Belarus,1 and
Department of Biological Sciences, University of Iowa, Iowa
City, Iowa 522422
Received 30 October 2000/Accepted 23 February 2001
 |
ABSTRACT |
Tn5 insertion mutants defective in regulation of the
Pseudomonas putida trpE and trpGDC
operons by tryptophan were found to contain insertions in the P.
putida miaA gene, whose product (in Escherichia
coli) modifies tRNATrp and is required for
attenuation. Nucleotide sequences upstream of trpE and
trpG encode putative leader peptides similar in sequence to leader peptides found in other bacterial species, and the phenotypes of the mutants strongly suggest that transcription of these operons is
regulated solely by attenuation.
| |
TEXT |
The arrangements of
trp genes and their patterns of regulation differ widely
among prokaryotes (8). The Escherichia coli trp
operon, which consists of five contiguous genes
trpE,
trp(G)D, trpC(F), trpB, and
trpAencoding seven enzymatic activities, is negatively
regulated by TrpR in the presence of tryptophan and by the
trp attenuator in the presence of acylated
tRNATrp (28). However, in
Pseudomonas putida, Pseudomonas aeruginosa, and
Pseudomonas syringae, the trpB and
trpA genes, which encode the subunits of tryptophan
synthase, comprise a separate operon that is positively regulated. In
the presence of indoleglycerol phosphate (InGP), a substrate for
tryptophan synthase, the trpBA operon is activated by the
product of a gene, trpI, that is unique to these three
species (1, 6, 19). Furthermore, trpE and trpGDC are transcribed independently (13, 14).
Isolation of unlinked mutations causing constitutive expression of
P. putida and P. aeruginosa trpE,
trpG, trpD, and trpC suggested that
these genes, like their E. coli counterparts, were regulated
by a TrpR-like repressor (5, 20). However, we have isolated phenotypically similar mutants induced by Tn5
insertion; the mutations all disrupt the P. putida miaA
homolog, whose product (in E. coli) modifies
tRNATrp and is required for attenuation
(12, 18, 30). Assays of trp enzyme synthesis in
the mutant strains strongly suggest that transcription of the
trpE and trpGDC operons is regulated by
attenuation and not by repression.
Selection of P. putida 5-MTr
mutants.
P. putida strain M (22) was
mutated by transposition of a mini-Tn5 lacZ1
transposon from the suicide plasmid pUT/mini-Tn5lacZ1, which
encodes kanamycin resistance (11). Mutants were selected at 30°C on M9 minimal medium containing 200 µg of
5-methyl-DL-tryptophan (5-MT)/ml, 20 µg of
kanamycin/ml, and 100 µg of rifampin (to which P. putida,
but not E. coli, is resistant)/ml. Five
Kanr 5-MTr mutants
(MTR1 through MTR5), chosen randomly from among 19 mutants that
remained after a preliminary screening of more than 100 original isolates from several experiments, were assayed for expression of
anthranilate synthase II (AS II) (trpG gene product); in the presence of exogenous tryptophan, all of the mutants were derepressed (data not shown). Strains MTR1 through MTR5 were also defective in
utilization of phenylalanine (0.1%) as the sole carbon and energy
source. Thus, the insertions appeared to cause defects in one or more
general regulatory functions, rather than in a single function specific
for trp gene expression, but the defect in phenylalanine
utilization was not explored further.
Strains MTR1 through MTR5 contain Tn5 insertions in
the P. putida miaA gene.
PstI-cut DNA
from strains MTR1 through MTR5 was ligated into pUC18
(25), and plasmids containing the Tn5 insertion
were identified. Restriction site analysis revealed that all five
strains contained inserts at either of two sites within a single
chromosomal 1.49-kb PstI fragment (data not shown). Southern
hybridization to a mutant PstI fragment was used to screen a
plasmid library containing PstI fragments isolated from
wild-type cells; the screen identified two plasmids, pTR301 and pTR302,
that contained the wild-type PstI fragments in opposite
orientations. The nucleotide sequences of the pTR301 and pT302 inserts
and the corresponding MTR1 and MTR4 PstI fragments (GenBank
accession no. AF016312) were identical except for the Tn5
insertion. An open reading frame that spans the region delineated by
the mini-Tn5 insertions in MTR1 through MTR5 is 65%
identical in nucleotide sequence and 62% identical in amino acid
sequence to the E. coli miaA gene and its product,
respectively (7). The P. putida open reading frame is homologous to the Agrobacterium tumefaciens miaA
gene and the Saccharomyces cerevisiae mod5 gene
(16, 21), each of which encodes a protein with a known
tRNA-isopentenyladenine transferase activity. In E. coli,
absence of the MiaA-mediated modification of
tRNATrp prevents attenuation in the
trp operon and promotes attenuation in the tna
(tryptophanase) operon, in each case by affecting the translational
efficiency of the tRNA (15, 30). The presence of the
miaA gene in pTR301 and pTR302 was confirmed by
complementation in E. coli DEV15 lacZ
(UGA) (23); formation of red colonies on MacConkey
agar-lactose plates showed that transformants containing pTR301 and
pTR302 were MiaA+.
Effects of miaA insertions on trp
gene expression.
Enzyme assays were used to investigate the
effects of P. putida miaA mutations on the production of
phosphoribosyltransferase (PRT) (trpD gene product) and InGP
synthase (InGPS) (trpC gene product), which are encoded in
the trpGDC operon, and AS I, the product of the
trpE gene, which is transcribed separately. In trp+ miaA+
P. putida (Table 1, line 1),
enzyme levels are low in the absence of tryptophan and are not
repressed even by 500 µg of tryptophan/ml. The absence of repression
of trp gene expression in miaA+
trp prototrophs, which has been observed previously
(9, 20), is due to the inability to starve for
tryptophan sufficiently to relieve attenuation (17).
However, in the presence of tryptophan, miaA mutations
increase expression of trpE between 10- and 30-fold and of
trpGDC between 3- and 4-fold (Table 1, lines 2 and 3). Furthermore, in the two miaA mutant strains, trp
gene expression is roughly the same in the presence and in the absence
of tryptophan. The unexpectedly high level of AS I activity in the
presence of tryptophan in the miaA1 mutant most likely
reflects the extreme variability of the assay. If we assume that the
phenotype of the miaA mutants is evidence for attenuation,
these data suggest that trpE and trpGDC
transcription is regulated only by attenuation, since AS I, PRT, and
InGPS levels are either unaffected or decreased by at most 40% when
miaA mutant cultures are incubated in the presence of
tryptophan. This small decrease could be due to differences in culture
conditions caused by pleiotropic effects of the mutations or to
residual trp-tRNA activity (and translation of the leader peptide) in the absence of MiaA. In any case, it is insignificant compared to the 10- to 100-fold effects of trpR in E. coli
(29).
Enzyme activity was also assayed in a
trp auxotroph,
trpB9 (
22), in which endogenous tryptophan
levels are lower, making
cells more sensitive to the concentration of
tryptophan in the
culture medium (Table
1, line 4). As expected, excess
tryptophan
reduces expression of
trpE by approximately
20-fold and of
trpGDC by 2- or 7-fold. The
miaA1
insertion (Table
1, line 5) completely
relieves tryptophan-mediated
regulation and increases expression
of
trpE,
trpD, and
trpC in the presence of excess
tryptophan to
approximately the levels attained in the
miaA+ strain in medium containing limiting
tryptophan. These results
indicate that tryptophan-mediated repression
can be completely
relieved by the
miaA insertion and
therefore that the
trpE and
trpGDC operons are
regulated solely by attenuation (or by some
other mechanism requiring
the translational activity of tRNA
Trp).
Effects of miaA1 on transcription of
trpE.
Published nucleotide sequences (10, 13,
14) reveal highly conserved sequences that may encode
14-amino-acid polypeptides upstream of trpE in P. putida, P. aeruginosa, and P. syringae and
upstream of trpG in P. putida (Table
2). There is a putative leader peptide,
but no methionine initiator codon, upstream of trpG in
P. aeruginosa, which suggests loss of the ability to be regulated by attenuation. A comparison to leader peptides from other
species (Table 2) reveals limited sequence similarity between groups of
related sequences. The Pseudomonas sequences are highly positively charged relative to the others. An unusual feature of the
upstream region of Pseudomonas trpE is that the
putative leader peptide is the carboxy terminus of a reading frame
homologous to phosphoglycolase phosphatase (Gph) of E. coli
(13, 14; GenBank accession no. AB030825), although
in the E. coli genome gph is not upstream of
trpE (4).
Possible promoters for
P. putida and
P. aeruginosa
trpE and
trpG were identified upstream of the putative
leader sequences
(Fig.
1). Reverse
transcriptase mapping using a primer complementary
to the
P. putida trpE coding region confirmed both in vivo and
in vitro the
location of the transcription start site schematized
in Fig.
1 (Fig.
2). Synthesis of this transcript in vivo
is regulated
by tryptophan in a
trpB9 miaA+
strain but not in a
trpB9 miaA strain (Fig.
2, lanes 6 to
9),
although the ratio of the observed
trpE RNA level in the
absence
of tryptophan to that in the presence of tryptophan in the
miA+ strain was only 6.6, which was
substantially lower than the corresponding
ratio of enzyme activities
(Table
1). We were unable to detect
trpE RNA by reverse
transcriptase mapping using a primer complementary
to the putative
leader transcript, possibly because of a secondary
structure in the
leader. We also attempted to identify a terminated
transcript in vitro.
Because the
trpE promoter is weak, we mutagenized
the
presumed
10 region (TAACGT) to TATAAT in order to increase
the
level of transcription. Although the phenotype of the mutant
in vitro
confirmed the location of the promoter (data not shown),
we did not
detect a terminated transcript.
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FIG. 1.
Nucleotide sequences upstream of the
Pseudomonas trpE and trpG
genes. Putative 35 and 10 regions, leader peptide initiation
(met) and termination (stop) codons, and
transcription termination signals (term) were
deduced from published sequences (10, 13, 14). The
transcription initiation site (indicated by an arrow in each sequence)
was confirmed experimentally for P. putida trpE (Fig.
2). Abbreviations: P.p., P. putida;
P.a., P. aeruginosa; P.s.,
P. syringae.
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FIG. 2.
Reverse transcriptase mapping of the P. putida
trpE transcription start site. (Lanes 1 to 5) Mapping of the in
vitro transcript. A 412-bp NcoI-XhoI DNA
fragment containing the wild-type P. putida trpE
promoter was used as a template for in vitro transcription by methods
outlined in reference 1. The primer for reverse
transcriptase mapping was complementary to bases 830 to 859 (13), which encode amino acids 2 to 11 of
trpE and correspond to positions +174 to +203 relative
to the transcription start site for the trpE promoter.
The substrate for Moloney murine leukemia virus reverse transcriptase
(Promega) (200 U per reaction) was the total unlabeled RNA product
extracted from the reaction mixture with phenol-chloroform (1:1)
following treatment with DNase I. The same primer was used to generate
a sequence ladder (lanes 2 to 5) from the DNA template. Following
electrophoresis, samples were fractionated in 7 M urea on a 5%
polyacrylamide gel and exposed to X-ray film. The arrow indicates the
start site (lane 1), which is the initiating A in the sequence shown in
Fig. 1. (Lanes 6 to 13) Mapping of the in vivo transcript. The primer
and enzyme for reverse transcriptase mapping were the same as those
used for mapping of the in vitro transcript. The indicated strains
(trpB9 miaA+ or trpB9
miaA1::Tn5) were grown to mid-log
phase in M9 medium plus tryptophan at 50 µg/ml (+) or 2 µg/ml ().
The substrate for reverse transcriptase was 19 µg of total cellular
RNA extracted from each culture. Following electrophoresis, samples
were fractionated on a 5% polyacrylamide gel and exposed to a
PhosphorImager screen (Molecular Dynamics). In the experiment whose
results are shown, the ratios of DNA products, determined by analysis
of PhosphorImager data, in the presence and absence of tryptophan were
6.6 (trpB9 miaA+) and 1.3 (trpB9
miaA1::Tn5), respectively. Because of
long exposure times, the visual separation between lanes 6 to 9 is
poor; however, quantitative analysis was facilitated by using the
entire gel, only a portion of which appears in the figure.
|
|
Regulation of trp genes in fluorescent
pseudomonads.
Five 5-MTr mutants contained
Tn5 insertions in the P. putida miaA homolog, and
no insertion was detected in a trpR-like gene. The
phenotypes of miaA insertion mutants strongly suggest that the trp genes are regulated by attenuation rather than by
repression, since trp enzyme levels in the mutants were
approximately the same as the "derepressed" levels in
miaA+ strains. Furthermore, the pattern of
regulation of the trpG and trpEDC operons in
fluorescent pseudomonads (5, 9) resembles that described
for the E. coli trp operon in that regulation of attenuation, in contrast to repression, by tryptophan is detected only
in trp auxotrophs or, in E. coli, under
conditions of extreme starvation (29). Regions upstream of
P. putida and P. aeruginosa trpE and
trpGDC (13, 14) and P. syringae trpE
(10) contain potential leader sequences that may encode a
highly conserved polypeptide containing three tryptophan residues (Fig.
1), but there is no operator-like sequence overlapping the promoters
for the two operons. Furthermore, during preparation of this article, the entire sequence of P. aeruginosa was published
(24); that sequence contains no trpR homolog.
Based on computerized and manual trial-and-error sequence analyses,
several possible attenuator-like RNA secondary structures
exist (not
shown), but putative terminator stem-loops are much
less stable than
those identified in known attenuators (
17).
Several patterns of
trp gene regulation in gram-negative
bacteria have been reported. First, the entire
E. coli trp
operon
is subject to both repression and attenuation. Second, in
P. putida,
P. aeruginosa, and
P. syringae, the
trpG and
trpEDC transcripts
are distinct and appear to be subject only to attenuation while
the
trpBA operon is activated by the TrpI protein, which is
unique
to these three species. And third, in
Rhizobium
meliloti (
3),
only the
trpE(
G)
gene is regulated (by attenuation). TrpI-mediated
regulation is
logically similar to regulation in species in which
trpB and
trpA are repressed by TrpR. InGP is produced and the
trpPB promoter is activated when
trp genes whose products function
earlier in the pathway are
expressed. In a prototroph, endogenous
tryptophan is sufficient to
reduce expression of both
trpE and
trpGDC nearly
to basal levels. Thus, addition of tryptophan causes
only a small
decrease in expression of these genes. Exogenous
tryptophan
down-regulates
trpBA transcription to a much greater
extent
(~10-fold), most likely through feedback inhibition of
AS I, since
inhibiting the conversion of chorismate to anthranilate
would prevent
synthesis of
InGP.
Gram-positive bacteria have evolved very different mechanisms for
achieving similar results. In
Bacillus subtilis, attenuation
is mediated by an RNA-binding protein (TRAP), and an operon containing
6 of the 7
trp genes (not including
trpG) is part
of a 13-gene
"supra-operon" whose transcription can be initiated at
the upstream
aroF promoter as well as at the
trpE
promoter (for a review, see
reference
27).
| |
ACKNOWLEDGMENTS |
This work was supported in part by NIH grant GM50577 to G.N.G.
We thank Susan Brown for technical assistance and S. Plattner for help
in preparing Fig. 2.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Iowa, Iowa City, Iowa 52242. Phone: (319) 335-1113. Fax: (319) 335-1069. E-mail:
gary-gussin{at}uiowa.edu.
Present address: Department of Microbiology, University of
Virginia, Charlottesville, Virginia 22908.
| |
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Journal of Bacteriology, May 2001, p. 3256-3260, Vol. 183, No. 10
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.3256-3260.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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