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Journal of Bacteriology, December 2000, p. 7075-7077, Vol. 182, No. 24
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
Received 21 August 2000/Accepted 4 October 2000
The N-terminal domain of the RNA polymerase 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 Effect of
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Transcription Activation by a Variety of AraC/XylS Family
Activators Does Not Depend on the Class II-Specific Activation
Determinant in the N-Terminal Domain of the RNA Polymerase
Alpha Subunit
ABSTRACT
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Abstract
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subunit (-NTD)
was tested for a role in transcription activation by a variety of
AraC/XylS family members. Based on substitutions at residues 162 to 165 and an extensive genetic screen we conclude that -NTD is not an
activation target for these activators.
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Abstract
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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12-19; S. M. Egan and C. C. Holcroft, unpublished
results; R. Ruiz, J. L. Ramos, and S. M. Egan, unpublished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).
-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).
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).
TABLE 1.
RhaS and RhaR activation with alanine substitutions
in -NTD
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).
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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.
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-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.
-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.
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ACKNOWLEDGMENTS |
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We are very grateful to Richard H. Ebright for providing the
plasmids encoding -NTD derivatives and Robert Martin and Judah Rosner for providing strains.
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.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045. Phone: (785) 864-4294. Fax: (785) 864-5294. E-mail: sme{at}ukans.edu.
Present address: Villa Julie College, Stevenson, MD 21153.
Present address: Life Technologies, Inc., Rockville, MD 20850.
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Journal of Bacteriology, December 2000, p. 7075-7077, Vol. 182, No. 24
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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