JOURNAL OF BACTERIOLOGY,
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Mar. 2000, p. 1232–1242Vol. 182, No. 5
Characterization and Role of tbuX in Utilization of Toluene by
Ralstonia pickettii PKO1
HYUNG-YEEL KAHNG,1ARMANDO M. BYRNE,2† RONALD H. OLSEN,2AND JEROME J. KUKOR1,3*
Biotechnology Center for Agriculture and the Environment1and Department of Environmental Sciences,3Rutgers
University, New Brunswick, New Jersey 08901-8520, and Department of Microbiology and Immunology,
University of Michigan Medical School, Ann Arbor, Michigan 48109-06202
Received 14 September 1999/Accepted 7 December 1999
The tbu regulon of Ralstonia pickettii PKO1 encodes enzymes involved in the catabolism of toluene, benzene,
and related alkylaromatic hydrocarbons. The first operon in this regulon contains genes that encode the tbu
pathway’s initial catabolic enzyme, toluene-3-monooxygenase, as well as TbuT, the NtrC-like transcriptional
activator for the entire regulon. It has been previously shown that the organization of tbuT, which is located
immediately downstream of tbuA1UBVA2C, and the associated promoter (PtbuA1) is unique in that it results
in a cascade type of up-regulation of tbuT in response to a variety of effector compounds. In our efforts to
further characterize this unusual mode of gene regulation, we discovered another open reading frame, encoded
on the strand opposite that of tbuT, 63 bp downstream of the tbuT stop codon. The 1,374-bp open reading frame,
encoding a 458-amino-acid peptide, was designated tbuX. The predicted amino acid sequence of TbuX exhibited
significant similarity to several putative outer membrane proteins from aromatic hydrocarbon-degrading
bacteria, as well as to FadL, an outer membrane protein needed for uptake of long-chain fatty acids in
Escherichia coli. Based on sequence analysis, transcriptional and expression studies, and deletion analysis,
TbuX seems to play an important role in the catabolism of toluene in R. pickettii PKO1. In addition, the
expression of tbuX appears to be regulated in a manner such that low levels of TbuX are always present within
the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene-
3-monooxygenase. This expression pattern may relate to the possible role of TbuX as a facilitator of toluene
entry into the cell.
Ralstonia pickettii PKO1 has been investigated by our labo-
ratory for several years as a model microorganism representa-
tive of those bacteria capable of metabolizing alkylaromatic
hydrocarbons in oxygen-limited (hypoxic) aquifer environ-
ments (23, 34, 42, 43). The tbu pathway of R. pickettii PKO1,
which encodes enzymes for utilization of benzene, toluene, and
related alkylaromatic hydrocarbons as well as enabling this
strain to transform trichloroethylene (TCE), has been cloned
as a 26.5-kbp DNA fragment designated pRO1957 (41). The
genes encoding enzymes for this catabolic pathway have been
shown previously to be organized into three operons: the
tbuA1UBVA2C and tbuT operon encoding the initial toluene-
3-monooxygenase and the transcriptional activator TbuT (6),
the tbuD operon encoding phenol/cresol hydroxylase (24, 26),
and the tbuWEFGKIHJ operon encoding enzymes of the meta-
cleavage pathway for conversion of catechol and methylcat-
echols to tricarboxylic acid cycle intermediates (25). We have
previously shown through physiological analysis as well as
through transcriptional fusion analysis of promoter regions
that TbuT controls transcription of each of these operons in
response to aromatic effector compounds. Moreover, it has
been shown that the unique organization of tbuT, which is
located immediately downstream of tbuA1UBVA2C, and the
associated promoter (PtbuA1), results in a cascade type of
up-regulation of tbuT in response to a variety of effector com-
pounds, including toluene and TCE (6). In our efforts to fur-
ther characterize this unusual mode of gene regulation, we
sequenced the DNA region immediately downstream of tbuT
and identified an open reading frame whose deduced amino
acid sequence shared significant homology with a number of
putative outer membrane proteins from aromatic hydrocar-
bon-degrading bacteria as well as homology to FadL (4), an
outer membrane protein needed for uptake of long-chain fatty
acids in Escherichia coli. We report here an analysis of the
function of this gene, designated tbuX, in toluene utilization.
MATERIALS AND METHODS
Bacterial strains, plasmids and culture conditions. The bacterial strains and
plasmids used in this study are listed in Table 1. E. coli JM109 and DH5? were
used for routine maintenance and construction of plasmids and for construction
of fragments used for DNA sequence analysis. The mobilization plasmid
pRK2013 (12) carried in E. coli MM294 was used to mobilize plasmid pKRZ1
and its derivatives from E. coli DH5? into Pseudomonas aeruginosa PAO1c via
triparental matings. P. aeruginosa transconjugants resulting from triparental mat-
ings were selected on the minimal medium of Vogel and Bonner (59) supple-
mented with 0.5% glucose. E. coli cells containing recombinant plasmids were
always maintained on Luria-Bertani (LB) medium (49) supplemented with the
antibiotics ampicillin (50 ?g/ml), tetracycline (25 ?g/ml), or kanamycin (75
?g/ml) as needed. P. aeruginosa PAO1c cells containing recombinant plasmid
constructs were maintained on plate count medium (TNA ) containing car-
benicillin (500 ?g/ml), kanamycin (600 ?g/ml), or tetracycline (50 ?g/ml) as
needed. When cells were grown for enzyme assays or for high-pressure liquid
chromatography (HPLC) analyses of metabolites, all strains were routinely
grown in a basal salts medium (BM ) containing (per liter) 2.49 g of
Na2HPO4, 3.05 g of KH2PO4, 0.1995 g of MgSO4, 0.995 g of CaCl2, 0.00005 g of
FeSO4, 0.00025 g of NaMoO4, 1.0 ml of Hunter’s trace metal solution (7), 1.0 g
of (NH4)2SO4, 1.0 g of KNO3, 0.1% Casamino Acids (Difco Laboratories, De-
troit, Mich.), and 0.25% glucose. When used for enzyme induction experiments,
liquid toluene was added directly to BM to a final concentration of 2.8 mM.
Growth of liquid cultures and incubation of agar plates was carried out at 37°C
* Corresponding author. Mailing address: Biotechnology Center for
Agriculture and the Environment, Foran Hall, Cook College Campus,
Rutgers University, 59 Dudley Road, New Brunswick, NJ 08901-8520.
Phone: (732) 932-8165, ext. 318. Fax: (732) 932-0312. E-mail: kukor
† Present address: DuPont CR&D, Experimental Station, Wilming-
ton, DE 19880-0328.
except when cultures contained aromatic hydrocarbons, in which case incuba-
tions were done at 30°C.
Toluene utilization assays. Cultures were grown in 100 ml of BM with 0.1%
Casamino Acids, 0.25% glucose, and 2.8 mM toluene in tightly stoppered 500-ml
bottles at 30°C with shaking until they reached the late log phase. Cells were
harvested by centrifugation at 10,000 ? g at 4°C and were washed twice with 40
mM potassium-sodium phosphate buffer (pH 6.8). Washed cells were transferred
to 10 ml of the same buffer containing toluene (2.8 mM) to produce an A425of
1.0. These washed cell suspensions were incubated with shaking at 30°C for 24 h,
and 500-?l samples were taken every 4 h, mixed with 500 ?l of methanol in 1.5-ml
microcentrifuge tubes, and then centrifuged at 4°C for 10 min to remove cells.
The resulting supernatants were carefully transferred to autosampler vials and
were analyzed by reverse-phase HPLC. Uninoculated bottles, which served as
controls, were incubated under the same conditions, and results were corrected
for toluene losses from the controls (which were always ?5% of the initial
toluene concentration). Reverse-phase chromatography was performed with a
PhaseSep H4726 column (4.6 by 250 mm) filled with Spherisorb ODS2 (particle
diameter, 5 ?m) preceded by a Whatman CSKI guard column (6.5 by 65 mm)
coupled to a Shimadzu SCL-6B solvent delivery system and a CR501 Chro-
matopac computing integrator. A methanol-water (90:10) solvent was used at a
flow rate of 1 ml/min. Toluene was detected by monitoring at A254, and concen-
tration was calculated by comparison with a standard curve as described previ-
Deletion mutagenesis and general molecular techniques. For construction of
a DNA fragment lacking tbuX, plasmid pRO1959 (Table 1) was digested with
restriction endonucleases ClaI and BamHI. The resultant 10-kb ClaI-BamHI
DNA fragment (which when cloned into vector pRO1727 was designated
pRO1966 [Table 1]) was extracted following electrophoretic separation in a 1.2%
agarose gel, partially digested with PvuII (Fig. 1), and then ligated with ClaI- and
EcoRV-digested vector plasmid pRO1727. The ligation mixture was introduced
into P. aeruginosa PAO1c by electroporation using the method of Smith and
Iglewski (56), and electrotransformants were selected on TNA medium contain-
ing carbenicillin (500 ?g/ml). Plasmids with the expected deletion were con-
firmed by ClaI and BamHI digestion patterns of purified DNAs obtained from
selected clones. The desired tbuX deletion construct was designated pHYK1000
(Table 1). Other DNA restriction digests, ligations, and transformation proce-
dures were performed according to previously published methods (38, 40, 49).
DNA sequence analysis. Nucleotide sequencing was initially carried out man-
ually by the dideoxy-chain termination method of Sanger et al. (50), with a
Sequenase version 1.0 kit (U.S. Biochemical, Cleveland, Ohio), [?-32P]dATP,
and T7, T3, or specific synthetic oligonucleotide primers, as previously described
(6). Later sequencing efforts were carried out using an ABI 373A automated
sequencer. For PCR amplification of fragments to be sequenced, a total 10-?l
reaction mixture containing 0.2 ?g of template DNA, 1.6 pmol of primer, and 1
U of Amplitaq FS (Gibco BRL, Gaithersburg, Md.) was used for 25 cycles of 10 s
at 96°C, 5 s at 50°C, and 4 min at 60°C. Sequence analysis was done with
Lasergene software (DNA STAR, Inc., Madison, Wis.), MacVector version 4.5.3
(Oxford Molecular, Campbell, Calif.), and the Genetics Computer Group
(GCG; University of Wisconsin, Madison) software package, version 8.1.
Searches of the GenBank database and pairwise sequence comparisons were
carried out with GCG programs TFASTA and BESTFIT, respectively. Similarity
searches were also performed with the BLAST program and the National Center
for Biotechnology Information databases. Nucleotide sequence alignments were
done by using the GCG multiple sequence alignment program PILEUP or the
Lasergene program MEGALIGN.
PCR. PCRs for amplification of a DNA fragment containing the putative tbuX
promoter region were carried out in 100-?l volumes using a GeneAmp kit
(Perkin-Elmer, Branchburg, N.J.) and thermal cycler 480 (Perkin-Elmer Cetus,
Norwalk, Conn.) essentially as previously described (6. A. M. Byrne and R. H.
Olsen, Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996, abstr. Q-356, p. 447,
1996). The following oligonucleotide primers, synthesized by the DNA Core
facility at the University of Michigan, were used: 5?-GGCGCTCGAGCGAGG
GCGGGATGGGCTGG (nucleotides 455 to 437 [Fig. 1]) and 5?-AGTTCCCG
GGGGCCGCGCGAACGTAGTTGC (nucleotides 1 to 20 [Fig. 1]). The incor-
poration of an XhoI or SmaI restriction endonuclease site at the 5? ends
(underlined) of the primers allowed for directional cloning of the PCR product
into SalI-SmaI cleaved pKRZ1 vector, resulting in plasmid p454X/S (Table 1).
Protein analysis. Cells of P. aeruginosa PAO1c carrying pRO1966 or
pHYK1000 (Table 1) were grown overnight at 37°C with shaking in 50 ml of BM
with 0.1% Casamino Acids, 0.25% glucose, 500 ?g of carbenicillin per ml, and 1
mM toluene. Cells harvested by centrifugation at 4°C were washed twice in 10 ml
of 40 mM sodium-potassium phosphate buffer (pH 6.8) and then transferred in
10 ml of this buffer containing 2.8 mM toluene. The cells were incubated with
shaking at 30°C for 12 h to ensure tbu pathway gene expression. Following this
induction period, cells were harvested by centrifugation at 4°C. The harvested
cells were lysed directly in a lysis buffer consisting of 25% glycerol, 14.4 mM
?-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 60 mM Tris-HCl (pH
6.8), and 0.1% bromophenol blue. Lysis was achieved by incubation at 95°C for
5 min. The cell debris was removed by centrifugation at 14,000 ? g for 20 min,
and the supernatant was used for SDS-polyacrylamide gel electrophoresis (SDS-
PAGE) using a Mini-Protean II system (Bio-Rad, Hercules, Calif.) according to
the method of Laemmli (30). Gels were run for 3 h at 120 V through a 5%
acrylamide stacking gel and 15% acrylamide separating gel. Prestained protein
molecular weight standards (Gibco BRL) were used for molecular mass estima-
tion. Gels were stained with Coomassie brilliant blue, destained in methanol-
TABLE 1. Bacterial strains and plasmids used in this study
Strain or plasmidRelevant characteristicsa
F??80dlacZ?M15 ?(lacZYA-argF)U169 deoR recA1 endA1 phoA hsdR17 (rK
supE44 ??thi-1 gyrA96 relA1
recA1 relA1 thi ?(lac-proAB) gyrA96 hsdR17 endA1 supE44
supE44 hsdR endA1 pro thi
R. pickettii PKO1 Tol?Phl?
P. aeruginosa PAO1cPrototroph18
pBluescript II KS?(SK?)
lacZ Apr, 2.96-kb cloning vector
AprKmr; broad-host-range promoter probe vector containing lacZ as a reporter gene
TcrCbr; 3.77-kb Pseudomonas cloning vector
TcrCbr; 6-kb E. coli-Pseudomonas cloning vector
AprKmr; pKRZ1:: 352-bp XhoI-SmaI fragment; PtbuA1-lacZ fusion
AprKmr; pKRZ1:: 3.4-kbp XhoI-BglII fragment; PtbuD-lacZ fusion
AprKmr; pKRZ1:: 454-bp XhoI-SmaI fragment; PtbuX-lacZ fusion
TcsCbr; pRO1727::HindIII-BamHI (15.1-kbp) fragment of R. pickettii PKO1 containing
tbuD, tbuA1UBVA2C, tbuT, and tbuX
TcsCbr; pRO1727::ClaI-BamHI (10-kbp) fragment of pRO1959 containing
tbuA1UBVA2C, tbuT, and tbuX
TcsCbr; pRO1727::ClaI-PvuII (1.96-kbp) fragment of pRO1966 containing tbuA1UBVA2C
TcrCbspRO1614::EcoRI-PvuII (3.1-kbp) fragment of pRO1966 containing tbuT
pHYK1000 This study
aAbbreviations: Tol, toluene; Phl, phenol; Ap, ampicillin; Cb, carbenicillin; Tc, tetracycline; Km, kanamycin.
VOL. 182, 2000 CHARACTERIZATION OF tbuX FROM R. PICKETTII PKO1 1233
glacial acetic acid-water (1:1:8, vol:vol:vol), blotted onto Whatman 3-MM paper,
and dried under vacuum at 70°C.
In vivo analysis of promoter activity. Promoter activity, assessed by in vivo
transcomplementation, was determined by assaying ?-galactosidase activity in
cells of P. aeruginosa PAO1c essentially as described previously (6, 40). P.
aeruginosa PAO1c cells harboring pKRZ1 and its derivatives together with
pRO1614 and its derivatives were grown overnight in LB broth containing
kanamycin (600 ?g/ml) and tetracycline (50?g/ml) to maintain plasmids. Cells
were subsequently diluted in the same medium and grown for 8 h in the absence
or presence of toluene. ?-Galactosidase activity was assayed as described by
Miller (35) except that cells were permeabilized by addition of chloroform and
SDS. Reported ?-galactosidase activity values, which represent the average of at
least three independent experiments, are expressed in units as specified by Miller
RNA preparation and primer extension analysis. Total RNA was isolated
from toluene-induced and uninduced cells of P. aeruginosa PAO1c carrying
p454X/S in trans with pHYK1001, which has a 3.1-kb EcoRI-StuI DNA fragment
expressing tbuT carried on plasmid pRO1614 (Table 1). Cells were grown under
the conditions used for ?-galactosidase assays, described above. Total RNA was
extracted from 2 ml of the cultures using Trizol reagent (Gibco BRL) according
to the manufacturer’s recommended protocol, except that after the cells were
mixed with the Trizol reagent, the suspension was incubated at 68°C for 10 min
and then allowed to cool to room temperature. The remainder of the RNA
extraction protocol was carried out as previously described (6, 27, 40). The
concentration and purity of the RNA were estimated from the A260/A280ratio
and from visual inspection of RNA bands following electrophoretic analysis.
The 5? ends of transcripts were determined by primer extension analysis with
oligonucleotide primer 5?-GGCCGCGCGAACGTAGTTGC, which was com-
plementary to nucleotides 8 to 27 of the deduced coding sequence of tbuX (Fig.
1). The primer was labeled at its 5? end with [?-32P]ATP (ICN Biomedicals,
Costa Mesa, Calif.), using T4 polynucleotide kinase as described previously (49).
The oligonucleotides (2 ? 105cpm) were annealed with 50 ?g of RNA in 10 ?l
of hybridization buffer (50 mM Tris-HCl [pH 7.7], 100 mM KCl) at 95°C for 3
min, transferred to 65°C for 5 min, and then slowly cooled to 42°C over a 30-min
interval. The samples were chilled on ice, and 2 ?l of 5? Superscript RT buffer
(250 mM Tris-HCl [pH 8.3], 375 mM KCl, 15 mM MgCl2), 25 mM dithiothreitol,
2.5 mM each deoxynucleoside triphosphate, and 6 U of RNasin RNase inhibitor
(Promega Corp., Madison, Wis.) were added. The samples were then warmed to
42°C, and 1 ?l (25 U) of Superscript RT RNase H?reverse transcriptase (Gibco
BRL) was added. Extension reaction mixtures were incubated at 42°C for 1 h,
after which 5 ?l of stop solution containing 95% formamide, 20 mM EDTA,
0.05% bromophenol blue, and 0.05% xylene cyanol FF was added. The products
of the extension reactions were resolved on 8% polyacrylamide gels containing 8
Chemicals and reagents. All chemicals used in this study were of the highest
purity commercially available. Aromatic hydrocarbons were purchased from
Sigma Chemical Co. (St. Louis, Mo.). Components for cell growth were pur-
chased from Difco, Aldrich Chemical Co. (St. Louis, Mo.), and Gibco BRL.
Enzymes and reagents used for nucleic acid manipulations were purchased from
Promega, Gibco BRL, and Stratagene (La Jolla, Calif.).
Nucleotide sequence accession number. The sequence data in this report have
been submitted to the GenBank data library under accession no. AF100782.
Identification and nucleotide sequence analysis of the tbuX
gene of R. pickettii PKO1. Our previous studies on regulation of
the tbu pathway genes from R. pickettii PKO1 reported an
FIG. 1. Physical and genetic maps of the 9.4-kb ClaI-BamHI fragment of pRO1966. The scale bar represents 0.5 kbp. The large arrows represent the locations and
orientations of the genes (identified at the top) which comprise the toluene-3-monooxygenase operon (tbuA1UBVA2C and the positive regulator tbuT) and the tbuX
gene. The lollipop symbol between the tbuT and tbuX genes indicates the location of a putative transcriptional terminator. The location of the toluene-3-monooxygenase
promoter, PtbuA1, is also shown. Below the restriction map, the putative tbuX promoter region with relevant flanking DNA is expanded. The putative TbuT-binding
site is demarcated by the horizontal hatched bar. The ?12 and ?24 sequences of the tbuX promoter, PtbuX, are boxed and labeled accordingly. The putative
ribosome-binding site is boxed and labeled rbs. The first nine amino acids from the deduced amino acid sequence of the tbuX gene (labeled TbuX) are shown in
one-letter code beneath the corresponding DNA coding region. The underlined C residue with a ?1 designation identifies the tbuX transcriptional start site as
determined by primer extension analyses. The BclI restriction endonuclease recognition sequence is shown in italics. The location and lengths of various sets of inverted
repeats are shown by pairs of arrows.
1234KAHNG ET AL. J. BACTERIOL.
unusual organization of tbuT, the gene encoding the transcrip-
tional activator, TbuT. The unusual feature involves the orga-
nization of the toluene-3-monooxygenase genes (tbuA1UBVA2)
and the regulator tbuT with respect to PtbuA1. This organiza-
tion allows for a cascade type of regulation where the presence
of an effector molecule (an inducer) and TbuT results in in-
duced expression of tbuA1UBVA2C and simultaneously in-
creases expression of tbuT via readthrough transcription
through tbuA1UBVA2C. This further elevates expression of
tbuA1UBVA2C if additional effector molecules are present (6).
In our efforts to further characterize the overall regulation of
the tbu pathway of strain PKO1, we sequenced the region 3? of
the translational stop of tbuT and in the process identified an
incomplete open reading frame whose deduced amino acid
sequence shared significant homology with several putative
membrane proteins from other aromatic hydrocarbon-degrad-
ing bacteria. To further explore this DNA region, we deter-
mined the complete nucleotide sequence of a 2,093-bp SmaI-
EcoRI fragment of pRO1966 (Fig. 1), which is a 10-kb ClaI-
BamHI subclone of pRO1957 that contains the genes encoding
toluene-3-monooxygenase and the transcriptional activator,
TbuT (5). Analysis of the nucleotide sequence revealed a
1,374-bp open reading frame that we have designated tbuX.
The tbuX gene is encoded on the opposite DNA strand from
tbuT and is located 63 bp downstream of the tbuT stop codon
(Fig. 1). This open reading frame encodes a putative peptide of
458 amino acids with an estimated molecular mass of 47.8 kDa
and estimated pI of 8.7. Determination of the probable 5? end
of tbuX was based on the identification of a putative ribosome-
binding site (5?-AAGGAGA-3?) and the extensive homology
with genes from other organisms (as discussed below). As
found for the other genes of the tbu pathway, the tbuX coding
region is G?C rich (63.8%) and accordingly displays a prefer-
ential use of codons with either a G or a C residue in the third
Comparisons of the deduced amino acid sequence of TbuX
with translated sequences from the GenBank database re-
vealed that TbuX shares homology with a group of putative
membrane-associated proteins from several hydrocarbon-de-
grading bacteria, including PhlX from Ralstonia eutropha
JMP134 (GenBank accession no. AF065891; unpublished),
CymD from Pseudomonas putida F1 (10), PorA from Pseudo-
monas sp. strain Y2 (58), IpbH from P. putida RE204 (Gen-
Bank accession no. AF006691; unpublished), CumH from P.
fluorescens IP01 (14), TodX from P. putida F1 (60), and XylN
from P. putida PaW1 (GenBank accession no. D63341; unpub-
lished). TbuX and these related proteins all shared a low de-
gree of overall homology with FadL from E. coli (4), which is
an outer membrane protein required for binding and transport
of long-chain fatty acids. Pairwise comparisons of the deduced
amino acid sequence of TbuX with those of PhlX, CymD,
PorA, IpbH, CumH, TodX, XylN, and FadL revealed 60.3,
43.2, 37.4, 36.9, 36.5, 33.5, 31.4, and 12.8% identities, respec-
tively, and 77.1, 55.2, 47.8, 47.8, 46.6, 44.4, 40.2, and 14.3%
similarities, respectively (Fig. 2).
Analysis of the tbuX promoter and regulatory region. Exam-
ination of the nucleotide sequence upstream of the tbuX gene
revealed a promoter containing the invariant ?24GG/?12GC
sequence unique to ?54(rpoN)-dependent promoters (Fig.
3A). A putative TbuT-binding site was also identified approx-
imately 150 bp upstream of the putative tbuX promoter (Fig.
3B) based on its similarity to the promoter for tbuA1. Com-
parisons of the nucleotide sequences of both the putative tbuX
promoter and the putative TbuT-binding site upstream of tbuX
with similar sequences found within the Pu promoter of the
upper TOL operon from P. putida mt-2 (47), the Po promoter
of the dmp operon from Pseudomonas sp. strain CF600 (57),
the Ptbm promoter upstream of the toluene and benzene
monooxygenase operon from Burkholderia cepacia JS150 (20),
the PtbuD promoter upstream of the phenol/cresol hydroxylase
structural gene (40), and the PtbuA1 promoter upstream of the
toluene-3-monooxygenase structural genes (6) are shown in
Fig. 3B. Interestingly, the spacing between the islands of ho-
mology observed in the putative TbuT-binding site upstream of
tbuX is similar to that observed in the putative DmpR-binding
site upstream of its cognate promoter Po.
To ascertain whether the region upstream of tbuX contained
TbuT- and toluene-dependent promoter activity, a tbuX-lacZ
fusion, designated p454X/S, was constructed using the pro-
moter probe plasmid pKRZ1. To provide the trans-activating
function of TbuT, the compatible plasmid pHYK1001 carrying
the constitutively expressed tbuT gene was used. Plasmid
p454X/S was mobilized into P. aeruginosa PAO1c strains car-
rying pRO1614 or pHYK1001. Expression from the tbuX-lacZ
fusion was monitored by measuring ?-galactosidase levels in
these P. aeruginosa PAO1c strains grown in the presence or
absence of the effector toluene. The P. aeruginosa PAO1c
strains carrying the PtbuA1-lacZ (p352X/S) and PtbuD-lacZ
(p3.4kbX/B) fusions in trans with pRO1614 or pHYK1001 were
also used for purposes of comparison. The levels of ?-galac-
tosidase obtained are listed in Table 2. As expected, TbuT- and
toluene-dependent promoter activity was observed from
p454X/S. Surprisingly, the level of TbuT- and toluene-depen-
dent promoter activity obtained from cells carrying p352X/S
and p3.4kbX/B was two- to threefold lower than that observed
from p454X/S. In addition, a significant level of promoter ac-
tivity was observed when cells carrying p454X/S were grown in
the absence of toluene or when the cells did not carry TbuT.
These results suggest that tbuX might be expressed from either
a leaky yet strong TbuT-dependent toluene-inducible pro-
moter or perhaps from two promoters, one strong, TbuT de-
pendent, and toluene inducible and the other weak and con-
Transcriptional analysis of tbuX. A transcriptional start site
for tbuX was mapped by primer extension analysis just down-
stream of the ?54-dependent promoter sequence (Fig. 1). This
single transcript was observed only from the primer extension
reaction using total RNA extracted from toluene-induced cells
carrying p454X/S and TbuT (Fig. 4, lane 6). These results
provide evidence that the ?24/?12 promoter sequence, des-
ignated PtbuX, is the TbuT-dependent toluene-inducible pro-
moter identified in the tbuX-lacZ fusion studies, described
above. The absence of a similar transcript from RNA extracted
from uninduced cells carrying p454X/S and TbuT (Fig. 4, lane
3) suggests that the constitutive expression observed from the
tbuX-lacZ fusion studies may not be from a leaky PtbuX pro-
moter but rather from a second promoter elsewhere upstream
Analysis of PtbuX promoter activity. Given the differences
observed in ?-galactosidase levels for the tbuX-lacZ fusion in
contrast to the tbuA1- and tbuD-lacZ fusions with toluene as an
inducer (Table 2), we set out to investigate the possibility of
differential promoter response from PtbuX and PtbuA1 for a
range of effector compounds. These experiments were carried
out using P. aeruginosa PAO1c carrying either p454X/S (the
tbuX-lacZ fusion) or p352X/S (the PtbuA1-lacZ fusion) in trans
with TbuT expressed from pHYK1001, as described above.
Comparisons of promoter activity obtained from PtbuX and
PtbuA1 in response to the presence of various effector com-
pounds revealed that PtbuX is a stronger promoter (Fig. 5).
Although the differences in promoter activity (PtbuX versus
PtbuA1) in response to different effectors varied between 2-
VOL. 182, 2000 CHARACTERIZATION OF tbuX FROM R. PICKETTII PKO11235
FIG. 2. Multiple sequence alignment of TbuX from R. pickettii PKO1, PhlX from R. eutropha JMP134 (GenBank accession no. AF065891 [unpublished]), CymD from P. putida F1 (10), PorA from Pseudomonas sp.
strain Y2 (58), IpbH from P. putida RE204 (GenBank accession no. AF006691 [unpublished]), CumH from P. fluorescens IP01 (14), TodX from P. putida F1 (60), XylN from P. putida PaW1 (GenBank accession no. D63341
[unpublished]), and FadL from E. coli (4). The amino acid residues conserved in all nine sequences are indicated in boldface and marked with daggers. Residues conserved in TbuX, PhlX, CymD, PorA, IpbH, CumH,
TodX, and XylN are indicated with asterisks. Gaps are represented by dashes and were introduced to maximize the alignment. The multiple sequence alignment analysis was carried out using the Clustal method within
the MEGALIGN program of Lasergene.
1236KAHNG ET AL. J. BACTERIOL.
and 12-fold, in general the relative activation profiles from the
various effectors were maintained. The rank order for effector
activation of PtbuX was toluene ? benzene ? ethylbenzene ?
TCE ? o-xylene ? phenol ? o-cresol ? m-cresol ? m-xy-
lene ? p-xylene ? p-cresol. The rank order for PtbuA1 activa-
tion was toluene ? benzene ? ethylbenzene ? TCE ?
o-cresol ? m-cresol ? phenol ? o-xylene ? m-xylene ?
p-cresol ? p-xylene. Overall, the effector activation profile was
similar for the two promoters with the exceptions of phenol
and o-xylene. For PtbuX, o-xylene and phenol were better
effectors than o- or m-cresol, whereas for PtbuA1 o- and m-
cresol were better effectors.
Effect of tbuX deletion on expression of the tbu pathway
genes. To elucidate a possible functional role of tbuX in utili-
zation of toluene by the toluene-3-monooxygenase encoded by
tbuA1UBVA2C, a tbuX-deletion derivative of pRO1966 (Table
1) was constructed as described in Materials and Methods. The
deletion derivative, designated pHYK1000, was introduced
into P. aeruginosa PAO1c, and the cells carrying either
pHYK1000 or pRO1966 were then analyzed for toluene utili-
zation and protein synthesis.
Analysis of toluene utilization and product formation by
reverse-phase HPLC showed that cells carrying pHYK1000,
the construct containing tbuA1UBVA2C and tbuT but lacking
tbuX, were significantly impaired in the ability to utilize tolu-
ene (Fig. 6A). In addition, m-cresol, the first intermediate in
the toluene degradation pathway of R. pickettii PKO1, was also
not detected for this construct, whereas cells of P. aeruginosa
FIG. 3. DNA sequence similarities among regions within and upstream of the PtbuX promoter sequence; promoters and UASs with homology to PtbuX. Nucleotide
sequence identities among the compared sequences are indicated in boldface. The consensus sequences established from comparison of the region within and upstream
of the PtbuX, PtbuD, Po, Ptbm, Pu, and PtbuA1 sequences are displayed below each alignment. (A) DNA sequence alignment of the ?54-dependent promoter sequences
of PtbuX, PtbuA1, PtbuD, Pu, and Po. The ?24 and ?12 sequences within the consensus sequence are labeled accordingly. (B) DNA sequence alignment of the region
upstream of tbuX with palindromic regions identified as the XylR-binding site upstream of Pu and those proposed for DmpR binding upstream of Po, TbuT binding
upstream of tbuD and tbuA1, and TbmR binding upstream of tbmA. Gaps, indicated by dashes, were introduced to maximize homology.
TABLE 2. Expression of putative tbuX promoter regiona
aP. aeruginosa PAO1c cells carrying plasmids were grown overnight in LB
medium containing kanamycin (600 ?g/ml) and tetracycline (50 ?g/ml) to main-
tain plasmids. Cells were subsequently diluted (1:50) in the same medium and
grown for 8 h in the absence or presence of toluene.
b?, presence of pHYK1001 in trans with the listed plasmid; ?, pRO1614 is in
cDetermined as described by Miller (35). Data are averages of three inde-
pendent determinations, each conducted in duplicate samples. The variability
between values did not exceed 10%.
dRatio of ?-galactosidase activities under uninduced (none) versus induced
FIG. 4. Determination of the 5? end of the tbuX transcript by primer exten-
sion analysis. RNA was isolated from P. aeruginosa PAO1c(pKRZ, pHYK1001),
P. aeruginosa PAO1c(p454X/S, pRO1614), and P. aeruginosa PAO1c(p454X/S,
pHYK1001) grown in the absence (lanes 1 to 3, respectively) and presence (lanes
4 to 6, respectively) of toluene. A sequence ladder using the same primer and
p454X/S is also shown (A, T, C, and G). To the left, an expanded view of the
nucleotide sequence surrounding the transcriptional start site (marked with an
asterisk) is shown.
VOL. 182, 2000 CHARACTERIZATION OF tbuX FROM R. PICKETTII PKO11237
PAO1c carrying pRO1966, which contains tbuA1UBVA2C,
tbuT, and tbuX, were able to produce significant amounts of
m-cresol from toluene (Fig. 6B).
From one-dimensional SDS-PAGE analysis of the soluble
peptides produced from P. aeruginosa PAO1c cells containing
either pHYK1000 or pRO1966, it was clearly evident that
toluene-grown cells carrying pRO1966 synthesized novel peptides
of the sizes expected for components of the tbuA1UBVA2C
and tbuT operon, whereas cells carrying pHYK1000 produced
only barely detectable amounts of these peptides (Fig. 7).
In this study, we have identified and characterized a new
gene, tbuX, associated with the tbu pathway for toluene and
benzene utilization in R. pickettii PKO1. The tbuX gene was
identified downstream of and on the DNA strand opposite that
of tbuT, the NtrC-like positive regulator that controls tran-
scription of the tbu regulon (6). Analysis of the deduced amino
acid sequence of TbuX demonstrated that this protein shared
significant identity with a group of proteins associated with
pathways for hydrocarbon degradation in a variety of gram-
negative bacteria. These pathways included styrene utilization
in Pseudomonas sp. strain Y2; isopropylbenzene utilization in
P. putida RE204 and P. fluorescens IP01; toluene utilization in
P. putida PaW1 which carries the TOL plasmid pWW0; two
FIG. 5. Activation of the PtbuX and PtbuA1 promoters by TbuT in response
to the presence of effectors. P. aeruginosa PAO1c cells containing pHYK1001
and either the PtbuX-lacZ transcriptional fusion plasmid p454X/S or the PtbuA1-
lacZ transcriptional fusion plasmid p352X/S were grown and treated as described
in Materials and Methods. The histogram represents the accumulation of ?-ga-
lactosidase after 8 h of exposure of the cultures to the different effectors. Values
are averages of duplicate determinations from three independent experiments.
The variability between triplicate values did not exceed 10%. Abbreviations for
effector compounds: Ben, benzene; Tol, toluene; Eth, ethylbenzene; oXyl, o-
xylene; mXyl, m-xylene; pXyl, p-xylene; TCE, trichloroethylene; Phl, phenol;
oCr, o-cresol; mCr, m-cresol; pCr, p-cresol.
FIG. 6. Toluene utilization (A) and m-cresol production (B) for cells of P. aeruginosa PAO1c carrying pRO1966 (tbuX?) or pHYK1000 (tbuX).
FIG. 7. SDS-PAGE profile of soluble cellular proteins of P. aeruginosa
PAO1c carrying pRO1966 (lanes 2 and 4) or pHYK1000 (lanes 3 and 5), grown
on 0.1% Casamino Acids and 0.25% glucose (lanes 2 and 3) or 0.1% Casamino
Acids, 0.25% glucose, and 1 mM toluene (lanes 4 and 5). Sizes of molecular
weight markers are shown to the left; arrows indicate the positions of
TbuA1UBVA2C and TbuT components, as described in the text.
1238KAHNG ET AL.J. BACTERIOL.
separate pathways in P. putida F1, one for toluene and one for
p-cymene utilization; and phenol utilization in R. eutropha
JMP134. The group of homologous proteins found in these
organisms all shared a low but significant level of similarity to
FadL, an outer membrane protein required for binding and
transport of long-chain fatty acids in E. coli. Except for FadL,
none of the members of this group has been analyzed in detail
with regard to subcellular location. However, the provisional
identification of TbuX as an outer membrane protein in R.
pickettii PKO1 appears to be reasonable given the following
observations derived from comparative sequence analysis: (i)
the presence of a putative signal sequence (37) (residues 1 to
charged amino acid (K, position 2) followed by a mostly hy-
drophobic segment and a peptidase cleavage site adjacent to
three conserved residues (ARA, positions 21 to 23); (ii) the
absence of cysteinyl residues in the putative mature peptide;
and (iii) an abundance of hydrophobic amino acids (37.4% by
frequency) in the putative mature peptide (16). In addition,
comparative hydropathy analyses of TbuX, PhlX, CymD,
PorA, IpbH, CumH, TodX, and XylN using the algorithms of
Hopp and Woods (data not shown) revealed the presence of
similar regions of hydrophobicity in each of these proteins, as
previously demonstrated for FadL (4, 28). It is noteworthy that
the putative outer membrane protein with which TbuX shares
the greatest overall similarity is PhlX, which is associated with
a catabolic pathway for phenol degradation in R. eutropha
JMP134 (22). The tbu pathway of R. pickettii PKO1 can ac-
commodate both hydroxylated aromatic compounds such as
phenol and cresols as well as unactivated aromatic hydrocar-
bons such as benzene and toluene as both effectors and sub-
strates (5, 24, 25, 41). It is possible that the similarities between
PhlX and TbuX relate, in part, to their potential roles in
phenol utilization in these two strains, in contrast to the other
members of this protein group (CymD, PorA, IpbH, CumH,
TodX, and XylN) which are all associated with pathways for
utilization of unactivated aromatic hydrocarbons. In this re-
gard, it would also be of interest to determine whether PhlX
and the associated phl pathway of strain JMP134 can accom-
modate toluene and benzene as effectors and as substrates.
Deletion of TbuX severely affected toluene utilization in
cells of P. aeruginosa PAO1c carrying tbu pathway genes (Fig.
6). This result can be explained by a model in which TbuX
plays a role in the entry of toluene into the cell. A possible
scenario might involve the facilitated passage of toluene
through the outer membrane as a consequence of interaction
with the TbuX protein. Such facilitated entry could provide
toluene to TbuT, which in turn would result in activation of the
various tbu pathway promoters. Wang et al. (60) have sug-
gested that TodX, a homolog of TbuX that occurs in P. putida
F1, may function as a facilitator of toluene entry into this
strain. A necessary feature of this model is the constant pres-
ence of the TbuX protein in the cell’s outer membrane as a
consequence of a low basal level of expression. This prediction
is consistent with our observations on the behavior of the
PtbuX promoter, namely that a significant level of PtbuX pro-
moter activity was observed when cells were grown in the
absence of the effector, toluene, or when the cells did not carry
the transcriptional activator, TbuT (Table 2), suggesting that
there may be partially constitutive expression of tbuX. Our
transcriptional analysis using primer extension did not reveal
any message initiating from the major TbuT-dependent and
toluene-responsive tbuX promoter, PtbuX, when analyzed in
the absence of TbuT or toluene (Fig. 4, lanes 2 and 3); there-
fore, it is possible that the low basal level of tbuX expression is
dependent on a second weakly constitutive promoter located
further upstream and that was not contained in p454X/S. In-
spection of the DNA sequence 5? of the putative translational
start of TbuX to the BamHI site at the right-hand terminus of
the pRO1966 clone (Fig. 1) did not reveal any obvious ?70-like
promoter (17), which might be expected to allow for partially
constitutive expression; therefore, additional research will be
required to further elucidate this.
In addition to affecting toluene utilization, deletion of tbuX
also affected expression of peptides necessary for toluene uti-
lization (Fig. 7). Analysis of the expression pattern for soluble
proteins obtained from toluene-grown cells of P. aeruginosa
PAO1c carrying pHYK1000, which has been deleted for tbuX,
shows (Fig. 7, lane 5) the absence of bands at estimated mo-
lecular masses of 62, 57, and 37 kDa. In contrast, protein
pattern analysis for toluene-grown P. aeruginosa PAO1c car-
rying pRO1966, which contains tbuA1UBVA2C, tbuT, and tbuX
(Fig. 1), clearly shows (Fig. 7, lane 4) the presence of novel
peptide bands with these estimated molecular masses. Peptides
of these sizes would correspond to the predicted molecular
masses for TbuT (65.2 kDa) and TbuA1 (57.6 kDa) and for
TbuA2 (37.5 kDa) and TbuC (36.1 kDa), which are similar to
one another in size. The other peptides that would be ex-
pressed from pRO1966, TbuB, TbuU, and TbuV, would not be
expected to be detectable in this analysis since the molecular
masses of these components are 12.3, 9.6, and 11.7 kDa, re-
spectively. These results are consistent with the toluene-3-
monooxygenase peptide expression patterns that we have pre-
viously determined from related work on regulation of the tbu
pathway (5, 24, 40, 41), and they support the model that dele-
tion of tbuX impaired the ability of toluene to enter the cell,
which in turn negatively affected the expression of the toluene-
3-monooxygenase structural genes as a result of the lack of
effector binding to the transcriptional activator, TbuT. There-
fore, Pseudomonas cells harboring pHYK1000 could not utilize
toluene even though the toluene-3-monooxygenase structural
genes and the transcriptional regulatory gene were present.
PtbuX is a stronger TbuT-dependent and toluene-responsive
promoter than is PtbuA1. This finding is also consistent with
our overall model of TbuX as a participant in facilitated entry
of toluene into the cell, inasmuch as rapid and high-level syn-
thesis of TbuX from a strong promoter would allow for more
toluene entry and hence would provide more substrate for
toluene-3-monooxygenase which is transcribed from the com-
paratively weaker PtbuA1 promoter. Such a regulatory scheme
might be part of a general mechanism by which carbon flow
could be controlled during toluene utilization by strain PKO1.
In this regard, the tbu regulon appears to be unique among
bacterial regulatory systems in that the binding of a single
transcriptional activator, TbuT, to multiple promoter-up-
stream regions of different architecture (Fig. 3B) appears to
allow for differential promoter response. The catabolic path-
ways that have been studied in the greatest detail and with
which the tbu pathway shares the greatest homology with re-
gard to transcriptional activators are the xyl pathway of P.
putida PaW1 carrying the TOL plasmid, pWW0 (1, 2, 9, 19),
and dmp pathway of Pseudomonas sp. strain CF600 (55). These
pathways, although they show some similarity in overall mode
of regulation to the tbu pathway, are substantially different in
the detailed mechanisms by which the transcriptional units are
controlled. The xyl regulatory system is controlled by two sep-
arate transcriptional activators, XylR (an NtrC-like activator)
and XylS (a member of the AraC family of activators), that
respond to separate effectors and that bind to separate and
distinct promoter-upstream regions for each operon (48). In
contrast, the dmp pathway is controlled by a single NtrC-like
VOL. 182, 2000 CHARACTERIZATION OF tbuX FROM R. PICKETTII PKO11239
activator, DmpR, that binds to a single promoter-upstream
region that controls the single dmp operon (55).
There seems to be some similarity in regulatory organization
between the tbu pathway of strain PKO1 and the tbm pathway
of B. cepacia JS150, inasmuch as the tbm system appears to
controlled by one transcriptional activator, TbmR, that regu-
lates expression of two different monooxygenases expressed
from divergent promoters (20, 21), but this regulatory system
has not yet been investigated in sufficient detail to determine
the extent of its similarity to the tbu model system. Other
catabolic pathways that have significant homology to the tbu
pathway with respect to the structural genes, such as the path-
ways for toluene utilization in Pseudomonas mendocina KR1
(61) or in B. cepacia G4 (51–53), have not yet been investigated
with regard to operonic organization or gene regulation.
Therefore, it is not clear what regulatory paradigm would be
expected to occur in these organisms, although data from an
analysis of the kinetics of toluene degradation indicate that
there are similarities between strains G4 and PKO1 with re-
spect to the effect of oxygen availability on the rate of toluene
degradation for the two strains (32). This suggests that these
strains might share similar features for regulation of their
toluene biodegradative pathways. The overall picture that is
emerging from comparative analysis of these regulatory sys-
tems suggests that regulatory units have evolved independently
of catabolic genes (11) and that regulatory units can be re-
cruited in a modular fashion. Further evidence for the latter
observation comes from our recent work on comparative or-
ganization of the regulatory systems controlling the phenol
hydroxylase in R. pickettii PKO1 and the toluene/benzene-2-
monooxygenase in Burkholderia sp. strain JS150, in which it
appears that a regulatory module and associated promoter
structure has been recruited, in the case of strain PKO1, to be
associated with a unit peptide phenol hydroxylase, whereas in
strain JS150 a similar regulatory module is associated with a
multicomponent toluene/benzene-2-monooxygenase (40).
A central part of our studies on tbuX have focused on reg-
ulation of this operon as part of the overall tbu regulon in
strain PKO1. Consistent with previous observations, we have
shown that expression from PtbuX is dependent on the pres-
ence of the regulatory protein, TbuT, and appropriate effector
compounds. Sequence analyses have suggested that the PtbuX
promoter is dependent on the alternative sigma factor, ?54,
and that activation of PtbuX is dependent on a DNA region
178 bp upstream of the transcription start site. Taken together,
these findings indicate that the organization and regulation of
the tbuX operon are similar to what has been found previously
for the other operons in the tbu regulon (5, 6, 25–27, 40). The
tbu pathway promoters are regulated by TbuT, which is an
NtrC-like transcriptional activator that belongs to the large
family of ?54-dependent activators (29, 33, 36, 54). These tran-
scriptional activators function by binding to DNA upstream
activation sequences (UAS) located 100 to 200 bp upstream of
the promoters that they regulate. For the tbu pathway UAS
shown in Fig. 3B, these sequences are found approximately 150
to 200 bp upstream of the transcriptional start site for each
promoter. The tbu UAS regions have significant sequence ho-
mology to the palindromic regions upstream of Pu, the upper
pathway promoter from the TOL plasmid, to which the tran-
scriptional activator XylR has been shown to bind (9, 19). The
sequences recognized by XylR in the Pu UAS region include
the motif 5?-TTGANCAAATC-3? (9). A sequence highly sim-
ilar to this is present within each arm (Fig. 3B) of the inverted
repeat upstream of PtbuX; thus, it is likely that this is the target
site for TbuT binding for the PtbuX promoter. It is of interest
that the spacing between the conserved inverted repeats in the
PtbuX UAS region is similar to that found for the UAS region
of the PtbuD promoter and not the PtbuA1 promoter (Fig. 3B).
This difference in UAS palindrome architecture is correlated
with promoter activity; i.e., greater promoter activity is ob-
served when tbuT is in trans with promoter/UAS ?lacZ fusions
of PtbuX or PtbuD than when it is in trans with fusions of
PtbuA1. This suggests that promoter function can be affected
by the structure of the site to which the transcriptional activa-
tor binds. Such an effect was seen previously by Leahy and
coworkers (31) for cross-regulation of the PtbuA1 promoter of
R. pickettii PKO1 and the PtbmA promoter of B. cepacia JS150
by either TbuT or TbmR. These researchers observed that the
cognate and noncognate activators were capable of transcrip-
tionally activating each promoter in response to the same set of
effectors, but that the magnitude of the transcriptional re-
sponse was greater for PtbmA than for PtbuA1 for each of the
effectors tested. As shown in Fig. 3B, PtbmA is similar in
architecture to PtbuX and not to PtbuA1.
As indicated above, members of the ?54-dependent family of
promoters require an interaction between the transcriptional
activator bound at the UAS and RNA polymerase bound at the
?24/?12 region in order for an open transcriptional complex
to form. This process necessarily requires DNA bending in
order to achieve close physical contact between the UAS-
bound activator and the promoter-bound ?54-RNA polymer-
ase (45). In some ?54-dependent promoters, the required DNA
bending is mediated by specific DNA-bending proteins such as
integration host factor (IHF), which recognizes and binds to
specific DNA sequences and induces sharp bends in the DNA.
IHF-dependent DNA bending has been shown to be essential
for promoter function for the XylR-dependent Pu promoter
(1, 9, 46) as well as the DmpR-dependent Po promoter (55,
57). Moreover, in the case of the Pu promoter, IHF binding has
been shown to specifically stimulate recruitment of RNA poly-
merase to the ?24/?12 promoter region as a consequence of
interaction of the carboxy-terminal domain of the ? subunit of
RNA polymerase with specific sequences upstream of the IHF-
binding site (3). Inspection of the DNA sequence between the
putative TbuT-binding site and the ?24/?12 promoter region
of the PtbuX promoter did not reveal a consensus IHF-binding
site; therefore, it seems unlikely that IHF could contribute to
the necessary promoter architecture for PtbuX. However, pro-
tein-assisted DNA bending necessary for ?54promoter func-
tion can be achieved by mechanisms that are independent of
IHF, as has been shown for the XylR-dependent Ps promoter
of the TOL plasmid of P. putida (13, 44). With this promoter,
the combined effects of intrinsically curved DNA and HU
protein-mediated DNA bending have been shown to be nec-
essary to achieve full transcriptional activity. Since the DNA
region between the putative TbuT-binding site and the ?24/
?12 promoter region of the PtbuX promoter contains a greater
number of dT stretches (Fig. 1) than found in the rest of the
tbuX operon, it is possible that PtbuX is similar to Ps, relying on
both the intrinsic curvature of promoter DNA and a nonspe-
cific DNA-bending protein, such as HU, to achieve the pro-
moter architecture required for transcription.
This research was supported by the National Institute of Environ-
mental Health Sciences through Superfund Basic Research Program
grant P42-ES-04911. Some DNA sequence analyses were supported in
part by the General Clinical Research Center at the University of
Michigan, funded by grant M01RR00042 from the National Center for
Research Resources, National Institutes of Health.
We thank Don Clewell for providing E. coli MM294(pRK2013) and
Ananda Chakrabarty for providing plasmid pKRZ1. Technical assis-
1240 KAHNG ET AL. J. BACTERIOL.
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