Department of Molecular Biosciences, University of Kansas,
Lawrence, Kansas 660451; Department of
Biological Sciences, University of Maryland Baltimore County,
Baltimore, Maryland 212502; and
Laboratory of Molecular Biology, National Institute of Diabetes
and Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, Maryland 20892-05603; and
Consejo Superior de Investigaciones Cientificas, Estación
Experimental del Zaidín, Department of Plant Biochemistry
and Molecular and Cellular Biology of Plants, Granada,
Spain4
 |
TEXT |
The AraC/XylS family is a large
family of transcription regulators, many of whose members activate
virulence factors in bacterial pathogens and hence are of interest as
potential targets of antibacterial agents (9). Virtually all
AraC/XylS family members are capable of transcription activation,
and thus it is likely the mechanisms used by these proteins to activate
transcription have been conserved, although subsets of family
members may use different mechanisms. A variety of
AraC/XylS family members have been shown to require the
RNA polymerase 
subunit C-terminal domain (-CTD) and the C-terminal end of the 
subunit for full activation (4,
12-19; S. M. Egan and C. C. Holcroft, unpublished
results; R. Ruiz, J. L. Ramos, and S. M. Egan, unpublished
results). However, for several family members it is believed that one
or more additional activation targets have yet to be identified. For
example, SoxS has been shown to be capable of activating in vitro
transcription of a class II promoter when the reconstituted RNA
polymerase lacks both the -CTD and the C-terminal residues of
70 (K.-W. Jair and R. E. Wolf, Jr., unpublished
results). There is also evidence that additional activation targets may
exist in the cases of MarA, RhaS and RhaR (4, 12, 13)
(Holcroft and Egan, unpublished results; R. Martin, personal communication).
Effect of -NTD derivatives on activation by RhaS, RhaR, XylS,
MarA and SoxS.
Niu et al. (24) have demonstrated that
residues 162 to 165 of the RNA polymerase subunit N-terminal domain
(-NTD) are required for transcription activation at class II cyclic
AMP (cAMP) receptor protein (CRP)-dependent promoters where the CRP
binding site overlaps the promoter 
35 hexamer. To test whether
-NTD plays a role in transcription activation by a variety of
AraC/XylS family activators, we transformed strains carrying a
wild-type chromosomal copy of rpoA with plasmids
overexpressing either wild-type or previously described alanine
substitution derivatives of (24).
Activation of the rhaBAD promoter requires RhaS bound
to a site that overlaps the 35 hexamer, CRP bound at
92.5, and -CTD (6, 7, 12). Activation of the divergent
rhaSR operon requires RhaR bound to a site that
overlaps the 35 hexamer, CRP bound at 111.5 and -CTD (29,
30) (Holcroft and Egan, unpublished). We grew strains carrying
the -NTD derivatives and rhaB-lacZ or rhaS-lacZ fusions in MOPS (morpholinepropanesulfonic acid)
minimal medium with 0.4% glycerol, 0.2% L-rhamnose, and
125 µg of ampicillin per ml and then assayed for 
-galactosidase
expression as previously described (3). We found that none
of the substitutions produced a significant defect in activation (Table
1).
In the presence of an effector such as 3-methylbenzoate, XylS
activates expression of the Pseudomonas putida TOL
plasmid meta promoter, Pm, from a site that
overlaps the 35 hexamer (10). The Pm promoter system was
reconstituted in Escherichia coli MC4100 (28) by transformation with pERD100, which carries
(Pm-'lacZ) (1), a derivative of pLOW2
(11) encoding xylS, and the plasmid encoding
wild-type or alanine substitution derivatives. These strains were
grown in Luria-Bertani (LB) medium with 100 µg of ampicillin, 25 µg of kanamycin, and 10 µg of tetracycline per ml,
and -galactosidase assays were performed as previously
described (23, 26). We found that expression of the -NTD
derivatives had no significant effects on activation by XylS
(Table 2) or the related activator
(25) XylS1 (data not shown).
The structure of the single domain MarA protein has been determined in
complex with DNA (27). MarA is capable of activating transcription of a large variety of promoters (2), in some cases from a site that overlaps the 35 hexamer (class II), and in
other cases from a site further upstream (class I) (20). We
tested the effect of the -NTD derivatives on MarA-dependent activation at lacZ fusions to two class I (fpr
and zwf, data not shown) and three class II
(inaA, fumC, and micF) promoters
(Table 3). Cultures were grown in LB
medium-ampicillin (100 µg/ml) and induced with 5 mM salicylate for
1 h, and -galactosidase activity was assayed as described
previously (22, 23). We found no significant defects at any
of the MarA-dependent promoters.
Similar to MarA, SoxS consists of a single domain and can activate
class I and class II promoters (8). Activation of class II
promoters was not significantly decreased upon deletion of -CTD
(15), and residues at the C-terminal end of
70 are not essential for transcription activation by
SoxS (Jair and Wolf, unpublished). To test for a role of -NTD in
SoxS activation, we assayed strains bearing translational fusions of
four class II SoxS-dependent promoters (fumC,
micF, nfo, and sodA) (Table 4) and two class I SoxS-dependent
promoters (zwf and fpr) (data not shown).
Cultures were grown in LB medium-ampicillin (125 µg/ml), induced for
1 h with 0.5 mM paraquat, and then assayed as previously described
(23). The -NTD derivatives conferred no significant effects on transcription activation of the class I or class II SoxS-dependent promoters.
Genetic screen for mutations in rpoA resulting in SoxS
activation defects.
Given that the mutations at residues 162 to
165 had no effect, a screen for other rpoA mutations
affecting activation of class II SoxS-dependent promoters was designed.
To construct the screening strain, we moved a soxR
constitutive mutation (31), which provided an
intermediate level of SoxS, into a strain that contained a fumC-lacZ fusion (21). This strain was
transformed with derivatives of plasmid pREII (5) in
which the entire rpoA gene had been subjected to PCR
mutagenesis with Taq polymerase (32) using primers with the same sequence as those used by Niu et al.
(24). The transformants were screened on lactose-tetrazolium
plates (23) containing ampicillin (100 µg/ml) and
kanamycin (20 µg/ml). The strain carrying the
soxRC1 allele produced white colonies with light
pink centers whereas a similarly uninduced isogenic strain with the
wild-type allele of soxR produced red colonies. In the
presence of paraquat, both strains produced white colonies. We
demonstrated that less than a twofold reduction in lacZ
expression in this strain resulted in reddish colonies that were
clearly distinguishable from the pink-centered wild-type colonies.
Approximately 24,000 transformants were screened from 26 independent
mutagenesis reactions. No mutations in -NTD were isolated that
conferred a defective phenotype; however, the screen readily yielded
mutations in -CTD.
We next confined a genetic screen to -NTD (by PCR mutagenesis of
only the XbaI-to-HindIII fragment of
pREII) to determine whether any -NTD substitutions conferred
activation defects. Twenty independent PCR mutagenesis mixtures
were ligated into pREII, and 18,000 transformants were
screened. Only one transformant with an activation-deficient phenotype
was identified, and this plasmid turned out to have a rearrangement
that produced an -CTD deletion. As a control, we also screened for
mutations in -CTD and found 16 apparent activation-deficient mutants
among just two independent PCRs and 2,000 transformants. Therefore,
while we readily obtained mutations in the -CTD by using
this mutagenesis strategy, we were again unable to isolate any
mutations in the -NTD that influenced SoxS activation.
Remarks.
From the results of this work, we conclude that
transcription activation by RhaS, RhaR, XylS, MarA, and SoxS does not
require contact with the 162-to-165 determinant of -NTD, nor, most
likely, any other portion of -NTD. The activators tested in
this study represent a diverse set of AraC/XylS family proteins, which,
with the exception of particularly related pairs (MarA/SoxS and
RhaS/RhaR), share only 24 to 28% amino acid sequence identity. It is
likely, therefore, that our conclusions apply to many other AraC/XylS family members.
Work in the laboratory of S.M.E. was supported by Public Health Service
grant GM55099 from the National Institute of General Medical Sciences
and the Franklin Murphy Molecular Biology Endowment. Work in the
laboratory of R.E.W. was supported by Public Health Service grant
GM27113 from the National Institutes of General Medical Sciences. R.R.
received a travel fund from the Spanish Ministry of Education to visit
the laboratory of S.M.E. at the University of Kansas. Work in the
laboratory of J.L.R. was funded by grant BIO-97-0641.
| 1.
|
Abril, M. A.,
C. Michan,
K. N. Timmis, and J. L. Ramos.
1989.
Regulator and enzyme specificity of TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway.
J. Bacteriol.
171:6782-6790[Abstract/Free Full Text].
|
| 2.
|
Barbosa, T. M., and S. B. Levy.
2000.
Differential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA.
J. Bacteriol.
182:3467-3474[Abstract/Free Full Text].
|
| 3.
|
Bhende, P. M., and S. M. Egan.
1999.
Amino acid-DNA contacts by RhaS: an AraC family transcription activator.
J. Bacteriol.
181:5185-5192[Abstract/Free Full Text].
|
| 4.
|
Bhende, P. M., and S. M. Egan.
2000.
Genetic evidence that transcription activation by RhaS involves specific amino acid contacts with sigma 70.
J. Bacteriol.
182:4959-4969[Abstract/Free Full Text].
|
| 5.
|
Blatter, E. E.,
W. Ross,
H. Tang,
R. L. Gourse, and R. H. Ebright.
1994.
Domain organization of RNA polymerase subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding.
Cell
78:889-896[CrossRef][Medline].
|
| 6.
|
Egan, S. M., and R. F. Schleif.
1994.
DNA-dependent renaturation of an insoluble DNA binding protein. Identification of the RhaS binding site at rhaBAD.
J. Mol. Biol.
243:821-829[CrossRef][Medline].
|
| 7.
|
Egan, S. M., and R. F. Schleif.
1993.
A regulatory cascade in the induction of rhaBAD.
J. Mol. Biol.
234:87-98[CrossRef][Medline].
|
| 8.
|
Fawcett, W. P., and R. E. Wolf, Jr.
1994.
Purification of a MalE-SoxS fusion protein and identification of the control sites of Escherichia coli superoxide-inducible genes.
Mol. Microbiol.
14:669-679[CrossRef][Medline].
|
| 9.
|
Gallegos, M.-T.,
R. Schleif,
A. Bairoch,
K. Hofmann, and J. L. Ramos.
1997.
AraC/XylS family of transcriptional regulators.
Microbiol. Mol. Biol. Rev.
61:393-410[Abstract].
|
| 10.
|
Gonzalez-Perez, M. M.,
J. L. Ramos,
M. T. Gallegos, and S. Marques.
1999.
Critical nucleotides in the upstream region of the XylS-dependent TOL meta-cleavage pathway operon promoter as deduced from analysis of mutants.
J. Biol. Chem.
274:2286-2290[Abstract/Free Full Text].
|
| 11.
|
Hansen, L. H.,
S. J. Sorensen, and L. B. Jensen.
1997.
Chromosomal insertion of the entire Escherichia coli lactose operon, into two strains of Pseudomonas, using a modified mini-Tn5 delivery system.
Gene
186:167-173[CrossRef][Medline].
|
| 12.
|
Holcroft, C. C., and S. M. Egan.
2000.
Roles of cyclic AMP receptor protein and the carboxyl-terminal domain of the subunit in transcription activation of the Escherichia coli rhaBAD operon.
J. Bacteriol.
182:3529-3535[Abstract/Free Full Text].
|
| 13.
|
Jair, K.,
R. G. Martin,
J. L. Rosner,
N. Fujita,
A. Ishihama, and R. E. Wolf, Jr.
1995.
Purification and regulatory properties of MarA protein, a transcriptional activator of Escherichia coli multiple antibiotic and superoxide resistance promoters.
J. Bacteriol.
177:7100-7104[Abstract/Free Full Text].
|
| 14.
|
Jair, K.-M.,
X. Yu,
K. Skarstad,
B. Thony,
N. Fujita,
A. Ishihama, and R. E. Wolf, Jr.
1996.
Transcriptional activation of promoters of the superoxide and multiple antibiotic resistance regulons by Rob, a binding protein of the Escherichia coli origin of chromosomal replication.
J. Bacteriol.
178:2507-2513[Abstract/Free Full Text].
|
| 15.
|
Jair, K.-W.,
W. P. Fawcett,
N. Fujita,
A. Ishihama, and R. E. Wolf, Jr.
1996.
Ambidextrous transcriptional activation by SoxS: requirement for the C-terminal domain of the RNA polymerase alpha subunit in a subset of Escherichia coli superoxide-inducible genes.
Mol. Microbiol.
19:307-317[CrossRef][Medline].
|
| 16.
|
Landini, P.,
J. A. Bown,
M. R. Volkert, and S. J. W. Busby.
1998.
Ada protein-RNA polymerase subunit interaction and subunit-promoter DNA interactions are necessary at different steps in transcription activation at the Escherichia coli ada and aidB promoters.
J. Biol. Chem.
273:13307-13312[Abstract/Free Full Text].
|
| 17.
|
Landini, P., and S. J. Busby.
1999.
The Escherichia coli Ada protein can interact with two distinct determinants in the 70 subunit of RNA polymerase according to promoter architecture: identification of the target of Ada activation at the alkA promoter.
J. Bacteriol.
181:1524-1529[Abstract/Free Full Text].
|
| 18.
|
Landini, P.,
T. Gaal,
W. Ross, and M. R. Volkert.
1997.
The RNA polymerase subunit carboxyl-terminal domain is required for both basal and activated transcription from the alkA promoter.
J. Biol. Chem.
272:15914-15919[Abstract/Free Full Text].
|
| 19.
|
Lonetto, M. A.,
V. Rhodius,
K. Lamberg,
P. Kiley,
S. Busby, and C. Gross.
1998.
Identification of a contact site for different transcription activators in region 4 of the Escherichia coli RNA polymerase 70 subunit.
J. Mol. Biol.
284:1353-1365[CrossRef][Medline].
|
| 20.
|
Martin, R. G.,
W. K. Gillette,
S. Rhee, and J. L. Rosner.
1999.
Structural requirements for marbox function in transcriptional activation of mar/sox/rob regulon promoters in Escherichia coli: sequence, orientation and spacial relationship to the core promoter.
Mol. Microbiol.
34:431-441[CrossRef][Medline].
|
| 21.
|
Martin, R. G.,
W. K. Gillette, and J. L. Rosner.
2000.
Promoter discrimination by the related transcriptional activators MarA and SoxS: differential regulation by differential binding.
Mol. Microbiol.
35:623-634[CrossRef][Medline].
|
| 22.
|
Martin, R. G., and J. L. Rosner.
1997.
Fis, an accessorial factor for transcriptional activation of the mar (multiple antibiotic resistance) promoter of Escherichia coli in the presence of the activator MarA, SoxS, or Rob.
J. Bacteriol.
179:7410-7419[Abstract/Free Full Text].
|
| 23.
|
Miller, J. H.
1972.
Experiments in molecular genetics.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 24.
|
Niu, W.,
Y. Kim,
G. Tau,
T. Heyduk, and R. H. Ebright.
1996.
Transcription activation at class II CAP-dependent promoters: two interactions between CAP and RNA polymerase.
Cell
87:1123-1134[CrossRef][Medline].
|
| 25.
|
Osborne, D. J.,
R. W. Pickup, and P. A. Williams.
1988.
The presence of two homologous meta pathway operons on TOL plasmid pWW53.
J. Gen. Microbiol.
134:2965-2975[Medline].
|
| 26.
|
Ramos, J. L.,
A. Stolz,
W. Reineke, and K. N. Timmis.
1986.
Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria.
Proc. Natl. Acad. Sci. USA
83:8467-8471[Abstract/Free Full Text].
|
| 27.
|
Rhee, S.,
R. G. Martin,
J. L. Rosner, and D. R. Davies.
1998.
A novel DNA-binding motif in MarA: the first structure for an AraC family transcriptional activator.
Proc. Natl. Acad. Sci. USA
95:10413-10418[Abstract/Free Full Text].
|
| 28.
|
Silhavy, T. J.,
M. L. Berman, and L. W. Enquist.
1984.
Experiments with gene fusions.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 29.
|
Tobin, J. F., and R. F. Schleif.
1990.
Purification and properties of RhaR, the positive regulator of the L-rhamnose operons of Escherichia coli.
J. Mol. Biol.
211:75-89[CrossRef][Medline].
|
| 30.
|
Tobin, J. F., and R. F. Schleif.
1990.
Transcription from the rha operon psr promoter.
J. Mol. Biol.
211:1-4[CrossRef][Medline].
|
| 31.
|
Tsaneva, I. R., and B. Weiss.
1990.
soxR, a locus governing a superoxide response regulon in Escherichia coli K-12.
J. Bacteriol.
172:4197-4205[Abstract/Free Full Text].
|
| 32.
|
Zhou, Y. H.,
X. P. Zhang, and R. H. Ebright.
1991.
Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase.
Nucleic Acids Res.
19:6052[Free Full Text].
|
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Abstract
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Present address: Villa Julie College, Stevenson, MD 21153.
Present address: Life Technologies, Inc., Rockville, MD 20850.
