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Journal of Bacteriology, March 2000, p. 1616-1623, Vol. 182, No. 6
Department of Biology, Imperial College of
Science, Technology and Medicine, London SW7 2AZ, United Kingdom
Received 29 October 1999/Accepted 14 December 1999
The genome sequence of the extremely thermophilic bacterium
Aquifex aeolicus encodes alternative sigma factor
The special form of bacterial RNA
polymerase (RNAP) containing alternative sigma factor Since this novel form of RNAP was first recognized in enteric bacteria
(25, 29, 47), The A tRNA-binding protein has previously been purified from A. aeolicus (41), and Klenk et al. (33) have
demonstrated activity and thermostability in the RNAP holoenzyme from
its relative Aquifex pyrophilus. Characteristics of the
hyperthermophile A. aeolicus pose important questions about
the evolutionary origins and relationships of the bacteria. According
to 16S ribosomal DNA (rDNA)-based phylogenies, the order
Aquifecales (of which A. aeolicus is a member)
represents the deepest branch of the bacterial evolutionary tree
(7). Thus, it has been argued that by studying these
organisms we can gain insights about the original bacterial ancestor.
In particular, the hyperthermophilic nature of the deepest-branching
organisms has been cited to support the hypothesis of a
hyperthermophilic origin for the bacteria (1). However,
several protein-based phylogenetic analyses, including that of the RNAP
In this paper we describe the heterologous overexpression and
purification of the Computer-based analysis.
DNA sequences were examined for
potential Strains, plasmids, and DNA.
Plasmids are listed in Table
1. Samples of genomic DNA from A. aeolicus and expression strain E. coli C41(DE3) were
kind gifts from R. Huber and J. Walker, respectively (15,
40).
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Functionality of Purified
N
(54) and a NifA-Like Protein from the Hyperthermophile
Aquifex aeolicus
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
N (54, RpoN) and five potential
N-dependent transcriptional activators. Although
A. aeolicus possesses no recognizable nitrogenase
genes, two of the activators have a high degree of sequence similarity
to NifA proteins from nitrogen-fixing proteobacteria. We identified
five putative N-dependent promoters upstream of operons
implicated in functions including sulfur respiration, nitrogen
assimilation, nitrate reductase, and nitrite reductase activity. We
cloned, overexpressed (in Escherichia coli), and purified
A. aeolicus N and the NifA homologue,
AQ_218. Purified A. aeolicus N bound to
E. coli core RNA polymerase and bound specifically to a DNA
fragment containing E. coli promoter glnHp2 and
to several A. aeolicus DNA fragments containing putative
N-dependent promoters. When combined with E. coli core RNA polymerase, A. aeolicus
N supported A. aeolicus NifA-dependent
transcription from the glnHp2 promoter. The E. coli activator PspF
HTH did not stimulate transcription. The
NifA homologue, AQ_218, bound specifically to a DNA sequence centered
about 100 bp upstream of the A. aeolicus glnBA operon and
so is likely to be involved in the regulation of nitrogen assimilation
in this organism. These results argue that the N
enhancer-dependent transcription system operates in at least one
extreme environment, and that the activator and N have coevolved.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
N
or 54 (N-RNAP), initiates transcription
by a mechanism quite distinct from RNAP containing the major
70 sigma factor (35, 39, 48). Transcription
initiation by N-RNAP requires the hydrolysis of
nucleoside triphosphate, catalyzed by activator proteins bound to
upstream activator sequences (enhancer elements). The mechanisms of
regulation involving this form of RNAP are among the most sophisticated
in bacteria (52).
N has been discovered in many
other bacteria, including several proteobacteria, as well as in the
gram-positive Bacillus subtilis (14) and in
Planctomyces limnophila (36). Furthermore,
N appears to be encoded by the genomes of the
hyperthermophile Aquifex aeolicus (15), the
spirochete Borrelia burgdorferi (18), and the
obligate intracellular pathogens Chlamydia trachomatis (51) and Chlamydia pneumoniae (31).
N proteins from Klebsiella pneumoniae and
Escherichia coli have been the subject of much genetic and
biochemical analysis (e.g., see references 8, 20,
and 23). Although considerable progress has been
made towards understanding the mechanisms by which N
performs its function, to date no high-resolution structural data are
available for this system. Thermophilic homologues of mesophilic
proteins have often been shown to be tractable for structural studies.
For example, the crystal structures of the histidine kinase domain of
CheA from Thermotoga maritima and the core RNAP from
Thermus aquaticus have recently been elucidated (4,
57). The publication of the complete genome sequence (15) of A. aeolicus, a hyperthermophilic
bacterium capable of growth at temperatures as high as 95°C, provides
a potentially valuable resource for the study of thermostable proteins.
and ' subunits, have thrown into question the status of the
Aquifecales as the deepest-branching group (e.g., see
reference 33). Based on sequences of
70 sigma factors, Aquifecales appears to be
an early-branching member of the proteobacteria (22).
N and show its core RNAP-binding
activity and sequence-specific DNA binding. We also purified a
NifA-like protein from A. aeolicus and demonstrate
activator-dependent transcription activity of a holoenzyme containing
A. aeolicus N in vitro. We predict possible
functions of the N-RNAP mode of transcription in
A. aeolicus.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
N-dependent promoters using SEQSCAN (B. T. Nixon: http://www.bmb.psu.edu/seqscan/seqform1.htm). Similarity
searches were performed using the PSI-BLAST server (2).
TABLE 1.
Plasmids used in this study
Oligonucleotides, enzymes, and PCR. PCR was carried out using standard protocols with BioTaq (Bioline) or, where proofreading activity was required, Bio-X-Act thermostable DNA polymerase (Bioline). E. coli core RNAP was purchased from Epicentre Technologies.
Cloning.
The sequences of rpoN, encoding
N, and nifA, encoding AQ_218, were amplified
from A. aeolicus genomic DNA by PCR using primers (Table
2, sets A and B) which introduced
NdeI sites immediately upstream of the start codons and
BamHI sites immediately downstream of the stop codons. These
restriction sites were exploited to clone the PCR-amplified fragments
into pET29b+ (Novagen). The inserts in the resulting plasmids
(pDJS42.12 and pDJS48.8) were verified by DNA sequencing.
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Proteins, overexpression, and purification.
Overexpression
and purification of K. pneumoniae N from
pMTHN has been described previously (19). For
A. aeolicus N and AQ_218, cultures of
C41(DE3)(pDJS42.12) and C41(DE3)(pDJS48.8) were grown at 37°C
until mid-log phase, when they were induced with 1 mM
isopropyl--D-thiogalactopyranoside (IPTG) for 12 to 16 h at 25°C. Cells were harvested by centrifugation and
resuspended in 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 5% glycerol
containing a protease inhibitor cocktail (Boehringer). Cells were
disrupted by two passages through a French press. Following
centrifugation at 20,000 × g for 40 min, the
overexpressed protein was found predominantly in the soluble fraction,
which was then heated to 80°C for 10 min and again centrifuged at
20,000 × g for 40 min. The A. aeolicus
protein remained in the soluble fraction and was purified (to more than
95% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis by affinity chromatography on a heparin-Sepharose
column and eluted with a NaCl gradient, using fast-protein liquid
chromatography (Pharmacia). The purified proteins were dialyzed into 50 mM Tris-HCl (pH 8.0)-50 mM NaCl-50% glycerol-1 mM dithiothreitol
(DTT) for storage at 
20°C.
Core-binding assays. Core-binding assays were carried out as described previously (20) but using a Tris-3-(cyclohexylamino)-1-propanesulfonic acid (Tris-CAPS) buffer system (38) at pH 9.5.
Promoter DNA fragments. The wild-type E. coli glnHp2 and mutant glnHp2-m11 fragments (approximately 250 bp) were amplified from pFC60 and pFC60-m11, respectively, using the primers listed in Table 2 (set C). Three putative A. aeolicus promoter DNA fragments, dhsU, glnB, and nirB, were amplified from genomic DNA by PCR using primers in Table 2 (sets D to F). Each of these fragments contained the ATG start codon plus about 240 bp of upstream sequence. All promoter DNA fragments were radioactively end labeled using T4 polynucleotide kinase as described in reference 8. Two additional fragments were amplified from the A. aeolicus glnB region (Table 2, sets G and H). These two fragments differed from each other in that the shorter fragment lacked a 56-bp sequence from the upstream end (see Fig. 4).
Protein-DNA-binding assays.
Binding of N and
holoenzyme to radioactively labeled promoter DNA fragments was detected
by a gel mobility shift assay (8, 9). Holoenzyme was
prepared by mixing E. coli core RNAP and N
subunit (from K. pneumoniae or A. aeolicus) in a
1:2 molar ratio. Assay reactions included concentrations up to 1 µM
of the N protein or 100 nM holoenzyme (see figure
legends for details), 25 nM labeled DNA, and 680 µg of salmon sperm
DNA ml
1 in buffer (40 mM Tris-HCl [pH 8.0], 0.1 mM
EDTA, 10% [vol/vol] glycerol, 100 nM NaCl, 250 mM KCl, and 1 mM
DTT). For AQ_218 binding to DNA, except where stated otherwise, assays
contained 100 nM AQ_218 protein (with respect to monomer), 25 nM
labeled DNA, and up to 204 µg of salmon sperm DNA ml1
in TAPS buffer (50 mM Tris-acetate [pH 8.0], 100 mM potassium acetate, 8 mM magnesium acetate, 27 mM ammonium acetate, 1 mM DTT,
3.5% [wt/vol] polyethylene glycol 8000).
In vitro N transcription activity assays.
Transcription assays were performed as described previously
(20) except that supercoiled pFC60 and pFC60-m11 plasmid
DNAs, containing the wild-type E. coli glnHp2 and mutant
glnHp2-m11 promoters, were used as templates. The
N-RNAP activator PspFHTH (30) is a useful
test activator, since it is constitutively active without a requirement
for covalent modification and is stable. It lacks DNA-binding activity
and exerts its activity while in solution. The assays were performed at
30, 37, and 48°C and contained 50 nM RNAP holoenzyme assembled from
the E. coli core and a sixfold excess of N.
Template DNA (10 nM) was preincubated with holoenzyme for 15 min in STA
buffer (25 mM Tris-acetate) [pH 8.0], 8 mM Mg acetate, 10 mM KCl, 1 mM DTT, 3.5% [wt/vol] polyethylene glycol 6000) with 4 mM GTP, 100 nM activator protein (A. aeolicus NifA-like or E. coli PspFHTH [30]), and 0.1 mM UTP before
heparin, ATP, CTP, and radioactively labeled UTP (1.5 µCi) were
added. Since the initial three nucleotides of the transcript are TGT,
this preincubation would favor the formation of stable initiated complexes.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
|
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The genome of A. aeolicus contains an open reading
frame (ORF) apparently encoding a N protein and five
ORFs predicted to encode NtrC/NifA-family transcriptional activators
(15), designated AQ_218, AQ_164, AQ_230, AQ_1117, and
AQ_1792. ORFs AQ_164, AQ_230, and AQ_1117 resemble the NtrC/AtoC subfamily of activators, members of which contain the receiver component of a two-component signal transduction system, whereas AQ_218
and AQ_1792 closely resemble the VnfA/NifA subfamily (unpublished data), whose members lack the conserved aspartate residue which undergoes phosphorylation in the two-component signal transduction system (43).
Purification of A. aeolicus N and NifA
proteins.
ORFs AQ_599 and AQ_218, encoding A. aeolicus
N and NifA proteins, respectively, were cloned, and the
proteins were overproduced and purified using the T7 system (see
Materials and Methods). The purified N was recognized by
anti-E. coli N antiserum in Western blots
(data not shown).
A. aeolicus N binding to core RNAP.
The purified N from A. aeolicus bound
E. coli core RNAP, as demonstrated by a gel mobility shift
assay (Fig. 1A). The band comprising free
core RNAP was depleted, and new species formed upon addition of the
A. aeolicus N. The lower degree of mobility
of the A. aeolicus N-E. coli core
complex compared to that of the K. pneumoniae
N-E. coli core complex may be explained by
the relatively high pI of the A. aeolicus protein (predicted
to be 8.15, versus 4.60 for K. pneumoniae N;
also compare Fig. 1, lanes 2 and 6). Also, whereas the K. pneumoniae N-E. coli core complex ran as
a single band on the gel, the A. aeolicus
N-E. coli core complex ran as at least two
discrete bands, suggesting more than one conformation, a phenomenon
that has previously been reported for several mutant K. pneumoniae N-E. coli core complexes
(45, 54).
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Sequence-specific DNA-binding activity of A. aeolicus
N.
The purified A. aeolicus
N bound to the E. coli glnHp2 wild-type
promoter with significantly greater affinity than the mutant glnHp2-m11, which has a single base substitution at position
13 (Fig. 1B). This DNA-binding activity was resistant to high levels of nonspecific competitor DNA (680 ng ml1), further
supporting the sequence specificity of the interaction. In contrast to
the 70 family of sigma factors, the A. aeolicus N, like those of the enteric bacteria
(5), clearly has its DNA-binding determinants available
without requiring core binding.
In vitro transcription activity.
The heterologous RNAP
holoenzyme, containing A. aeolicus N and
E. coli core subunits, supported activator-dependent
transcription from the wild-type E. coli glnHp2 promoter in
vitro, giving a transcript of the same length as that from the
holoenzyme containing K. pneumoniae N (see
Fig. 1C). When the E. coli activator protein PspFHTH was added to the transcription assays in place of A. aeolicus AQ_218, transcription levels were very low (PspFHTH is
a good activator of RNAP containing K. pneumoniae
N). Furthermore, there was only a very low level of
transcription activation by AQ_218 with a holoenzyme containing
K. pneumoniae N. This requirement for both
A. aeolicus N and A. aeolicus
activator for appreciable levels of transcription but tolerance of
heterologous core subunits suggests that the A. aeolicus
N and activators have coevolved and might directly
interact with each other during activation of transcription initiation.
Transcription could not be detected from the glnHp2-m11
promoter, demonstrating promoter-specific interaction (data not shown).
N was lower than that of the holoenzyme
containing K. pneumoniae N. It is possible
that at the relatively low temperatures at which the assays were
performed (30, 37, and 48°C, while the optimal growth temperature for
A. aeolicus is about 85°C) the thermophile proteins are in
a "frozen" state that is kinetically unfavorable to the
conformational changes required for transcription initiation. Indeed,
activity increased with temperature over the range tested. Higher
temperatures were not used, since 48°C is close to the upper
temperature limit for E. coli RNAP activity (55).
Many N-dependent promoters have integration host factor
binding sites (26). The A. aeolicus genome
encodes no obvious homologues of himA or himB,
and so involvement, if any, of a sequence-specific DNA bending protein
in A. aeolicus N-dependent transcription is
presently unclear. Also, the fact that purified A. aeolicus
NifA did not require additional factors for the activation of
transcription suggests its activity might be negatively regulated in vivo.
We were unable to rigorously demonstrate that nucleoside hydrolysis by
A. aeolicus NifA is required for transcription initiation due to the apparent instability of open complexes using the
heterologous RNAP holoenzyme (data not shown). However, based on
similarity of central domain sequences in the A. aeolicus
NtrC/NifA family activators to those of well-studied members of this
family (48), we believe that the mechanism of activation is
likely to be similar.
Identification of potential N-dependent promoters in
the A. aeolicus genome.
RNAP containing
N transcribes from promoters with a consensus sequence
of YTGGCACGRNNNTTGCW with
the highly conserved GG and GC motifs at 24 and 12, respectively,
relative to the +1 transcription start site (3). The
A. aeolicus genome sequence was examined for potential
N-dependent promoters. Six good matches to the consensus
were found in predicted noncoding regions immediately upstream of
predicted coding regions (Fig. 2). Four
of the six predicted coding regions showed significant similarity to
genes of known function and are designated glnB,
fhp, dhsU, and nirB. The remaining two
predicted coding regions encode the hypothetical proteins AQ_087 and
AQ_1119.
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N-RNAP in several proteobacteria (37), where
their transcription is activated by the nitrogen-regulatory protein C
(NtrC). However, in the gram-positive B. subtilis,
transcriptional regulation is not dependent on the N
homologue, L (28). It should also be noted
that glnA, encoding glutamine synthetase, is found in the
genomes of Thermotoga maritima (44) and
Mycobacterium spp. (13), which have been
completely sequenced, and yet no homologues of N have
been identified.
A second operon that appears to have a N-RNAP-dependent
promoter contains genes designated fhp, cynS,
glnBi, nasA, and narB. Each of the
ORFs designated fhp (flavohemoprotein), cynS
(cyanate hydrolase), nasA (nitrate transporter), and
narB (nitrate reductase) shows similarity to genes
associated with nitrate reduction (reviewed in reference
42). The glnBi ORF appears to encode a
protein similar to the PII and GlnK family of nitrogen regulatory proteins.
A potential N-dependent promoter is found in the
A. aeolicus genome upstream of dhsU, predicted to
encode a flavocytochrome C sulfide dehydrogenase (10). It
appears that dhsU is part of an operon with soxF
and fccB', which are predicted to encode a Rieske-I
iron-sulfur protein and a sulfide dehydrogenase flavoprotein, respectively (49, 50). Therefore, this operon is almost
certainly involved in sulfur respiration.
In A. aeolicus we have identified a potential
N-dependent promoter upstream of nirB,
predicted to encode the large subunit of cytoplasmic NADH-dependent
nitrite reductase, whose function is detoxification of nitrite formed
as a result of nitrate respiration (12). It appears that
cobA and trpD2 may also be in the nirB operon, and they are predicted, respectively, to encode products involved in cobalamin and tryptophan biosynthesis (17, 27). Although nitrate respiration has not been demonstrated in A. aeolicus, it has been observed in the close relative A. pyrophilus (7). Previously, N-RNAP had
been implicated in the regulation of nitrite reductase in
Pseudomonas stutzeri (24). ORFs AQ_087 and
AQ_1119 have no significant similarity to sequences of known function.
A PSI-BLAST search using AQ_1119 as a probe revealed significant
similarity to an ORF of unknown function, AF0913, in the genome of the
archaeon Archaeglobus fulgidus.
To date, N-RNAP has been implicated in the
transcriptional regulation of such diverse functions as degradation of
xylene and toluene, transport of dicarboxylic acids, pilin synthesis,
nitrogen fixation, hydrogen uptake, flagellar assembly, arginine
catabolism, alginate production, rhamnolipid production, acetoin
catabolism, mannose uptake, proline iminopeptidase activity,
nitrogen assimilation, nitrate respiration (reviewed in references
3, 35, and 39), pathogenesis
(34), development (32), and RNA modification (21). It is also predicted to be involved in the
transcriptional regulation of other factors (46, 53).
Here, we have extended the range of potential functions further to
include sulfur respiration and have provided evidence that
N-RNAP is probably involved in nitrogen assimilation and
nitrate respiration in a hyperthermophile.
N- and N-RNAP holoenzyme binding to
putative A. aeolicus N-dependent
promoters.
We tested N and N-RNAP
holoenzyme binding to 240-bp fragments containing noncoding sequences
upstream of A. aeolicus dhsU, glnB, and
nirB (Fig. 3) in the presence
of high levels of competitor DNA. A. aeolicus
N bound to all three fragments, reducing the
electrophoretic mobility of the labeled DNA (Fig. 3, lanes 13 to 15).
K. pneumoniae N clearly also bound the
dhsU and glnB upstream fragments (Fig. 3, lanes
10 and 11), albeit less stably than did A. aeolicus
N, and only very weakly bound the nirB
upstream fragment (lane 12). The RNAP holoenzyme containing K. pneumoniae N bound to all three fragments to
produce species with much lower degrees of electrophoretic mobility
(Fig. 3, lanes 4 to 6). However, the binding of the RNAP holoenzyme
containing A. aeolicus N appeared to give
rise to complexes with mobility similar to that of those arising from
N and DNA alone (lanes 4 to 6). The amount of stable
holoenzyme-DNA complex formed was below the limit of detection by this
assay. The very low levels of DNA-holoenzyme complex formation, perhaps partly due to the heterologous nature of the system, might explain the
relatively low levels of the transcription (Fig. 1C) on the glnHp2 promoter by RNAP containing A. aeolicus
N.
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N appeared
to bind the A. aeolicus DNA fragments more tightly than did
K. pneumoniae N (compare lanes 10 to 12 with
lanes 13 to 15 in Fig. 3). This was also the case for binding on the
E. coli glnHp2 promoter DNA fragment (data not shown). We
speculate that a stronger DNA-binding activity may be advantageous to
the thermophile in overcoming the kinetic effects of high temperature.
DNA-binding activity of A. aeolicus NifA-like
protein (AQ_218).
The NtrC/NifA family
N-dependent transcriptional activators bind to
enhancerlike elements, usually located up to 200 bp upstream of the
transcription start site (35, 48). Therefore, we tested whether purified protein AQ_218 bound to 240-bp fragments containing noncoding sequences upstream of A. aeolicus dhsU,
glnB, and nirB. In a gel mobility shift assay,
AQ_218 protein bound to all three DNA fragments to give complexes with
reduced mobility (band B in Fig. 4A). The
stability of these complexes was very sensitive to the concentration of
competitor DNA (salmon sperm DNA). Binding to the glnB
fragment appeared to be significantly more resistant to the presence of
competitor DNA than was binding to the dhsU and
nirB fragments. Furthermore, binding to the glnB
upstream sequence gave rise to an additional species that was not
observed in assays containing dhsU and nirB.
Therefore, we examined the glnB upstream DNA sequence and
its interaction with AQ_218 in more detail.
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N-dependent
promoters and five ORFs potentially encoding N-dependent
activators supports the proposition that the enhancer-dependent N-RNAP mode of transcription is functional in A. aeolicus. Direct binding of A. aeolicus promoter
specificity factor N to these sequences supports this
conclusion. We present indirect evidence that the N-RNAP
holoenzyme is involved in sulfur respiration, nitrogen assimilation, reduction of nitrate, and nitrite reductase activity, as well as having
at least two unknown functions, in A. aeolicus. Furthermore, based on a demonstrated specific DNA-binding activity, we propose that
AQ_218, a NifA-like protein from A. aeolicus, is involved in
the regulation of the glnBA operon. Clearly, the NifA
subfamily of proteins is unlikely to have arisen twice independently,
so there are two possible explanations for these proteins' occurrence both in A. aeolicus and in the proteobacteria: (i)
horizontal gene transfer between these bacteria occurred, or (ii) the
lineage containing A. aeolicus split from the proteobacteria
after the evolution of the NifA subfamily. Since NifA has not been
found in any bacteria other than A. aeolicus and azotrophic
proteobacteria, this would suggest that the split was much more recent
than is implied by the 16S rDNA data and is more consistent with
protein-based models of bacterial evolution (e.g., see references
22 and 33). The RNAP holoenzyme
containing purified A. aeolicus N appears to
be silent for transcription, and the A. aeolicus NifA-like protein (AQ_218) overcomes this inhibition to allow transcription, as
is the case for all homologous mesophile systems so far studied. It
seems that the interactions that silence the polymerase, and others
that allow enhancer dependence and which are known to critically involve N region I sequences (20), are at
least partly intact when A. aeolicus N
combines with E. coli core RNAP. To the best of our
knowledge, this is the first report of transcriptional activation using
components from a hyperthermophilic bacterium.
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ACKNOWLEDGMENTS |
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This work was supported by CEC contract Bio4 CT 97-2143 to M.B. M.-T.G. was supported by a CEC Marie Curie fellowship. S.R.W. was supported by the Local Education Authority of Karlsruhe, Germany.
We are grateful to R. Huber for the A. aeolicus genomic DNA,
to J. Walker for C41(DE3), to B. Magasanik for plasmids, and to A. Ishihama for the anti-N antiserum. We thank B. T. Nixon for making available his SEQSCAN program, R. Wassem for helpful
discussion about NifA, and J. Bartley for automated DNA sequencing service.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biology, Sir Alexander Fleming Building, Imperial College of Science, Technology and Medicine, London SW7 2AZ, United Kingdom. Phone: 44 (0) 171 594 5442. Fax: 44 (0) 171 594 5419. E-mail: m.buck{at}ic.ac.uk.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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| 1. | Achenbach-Richter, L., R. Gupta, K. O. Stetter, and C. R. Woese. 1987. Were the original eubacteria thermophiles? Syst. Appl. Microbiol. 9:34-39[Medline]. |
| 2. |
Altschul, S. F., and E. V. Koonin.
1998.
Iterated profile searches with PSI-BLAST a tool for discovery in protein databases.
Trends Biochem. Sci.
23:444-447[CrossRef][Medline].
|
| 3. |
Barrios, H.,
B. Valderrama, and E. Morett.
1999.
Compilation and analysis of 54-dependent promoter sequences.
Nucleic Acids Res.
27:4305-4313 |
| 4. | Bilwes, A. M., L. A. Alex, B. R. Crane, and M. I. Simon. 1999. Structure of CheA, a signal-transducing histidine kinase. Cell 96:131-141[CrossRef][Medline]. |
| 5. | Buck, M., and W. Cannon. 1992. Specific binding of the transcription factor sigma-54 to promoter DNA. Nature 358:422-424[CrossRef][Medline]. |
| 6. | Buck, M., S. Miller, M. Drummond, and R. Dixon. 1986. Upstream activator sequences are present in the promoters of nitrogen fixation genes. Nature 320:374-378[CrossRef]. |
| 7. | Burggraf, S., G. J. Olsen, and K. O. Stetter. 1992. A phylogenetic analysis of Aquifex pyrophilus. Syst. Appl. Microbiol. 15:352-356[Medline]. |
| 8. |
Cannon, W.,
M. T. Gallegos,
P. Casaz, and M. Buck.
1999.
Amino-terminal sequences of N (54) inhibit RNA polymerase isomerization.
Genes Dev.
13:357-370 |
| 9. |
Casaz, P., and M. Buck.
1997.
Probing the assembly of transcription initiation complexes through changes in N protease sensitivity.
Proc. Natl. Acad. Sci. USA
94:12145-12150 |
| 10. |
Chen, Z. W.,
M. Koh,
G. Van Driessche,
J. J. Van Beeumen,
R. G. Bartsch,
T. E. Meyer,
M. A. Cusanovich, and F. S. Mathews.
1994.
The structure of flavocytochrome c sulfide dehydrogenase from a purple phototrophic bacterium.
Science
266:430-432 |
| 11. | Claverie-Martin, F., and B. Magasanik. 1992. Positive and negative effects of DNA bending on activation of transcription from a distant site. J. Mol. Biol. 227:996-1008[CrossRef][Medline]. |
| 12. | Cole, J. 1996. Nitrate reduction to ammonia by enteric bacteria: redundancy, or a strategy for survival during oxygen starvation? FEMS Microbiol. Lett. 136:1-11[CrossRef][Medline]. |
| 13. | Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry, F. Tekaia, K. Badcock, D. Basham, D. Brown, T. Chillingworth, R. Connor, R. Davies, K. Devlin, T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, B. G. Barrell, et al. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537-544[CrossRef][Medline]. |
| 14. |
Debarbouille, M.,
I. Martin-Verstraete,
F. Kunst, and G. Rapoport.
1991.
The Bacillus subtilis sigL gene encodes an equivalent of sigma 54 from gram-negative bacteria.
Proc. Natl. Acad. Sci. USA
88:9092-9096 |
| 15. | Deckert, G., P. V. Warren, T. Gaasterland, W. G. Young, A. L. Lenox, D. E. Graham, R. Overbeek, M. A. Snead, M. Keller, M. Aujay, R. Huber, R. A. Feldman, J. M. Short, G. J. Olsen, and R. V. Swanson. 1998. The complete genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature 392:353-358[CrossRef][Medline]. |
| 16. |
de Zamaroczy, M.,
A. Paquelin, and C. Elmerich.
1993.
Functional organization of the glnB-glnA cluster of Azospirillum brasilense.
J. Bacteriol.
175:2507-2515 |
| 17. |
Escalante-Semerena, J. C.,
S.-J. Suh, and J. R. Roth.
1990.
cobA function is required for both de novo cobalamin biosynthesis and assimilation of exogenous corrinoids in Salmonella typhimurium.
J. Bacteriol.
172:273-280 |
| 18. | Fraser, C. M., S. Casjens, W. M. Huang, G. G. Sutton, R. Clayton, R. Lathigra, O. White, K. A. Ketchum, R. Dodson, E. K. Hickey, M. Gwinn, B. Dougherty, J. F. Tomb, R. D. Fleischmann, D. Richardson, J. Peterson, A. R. Kerlavage, J. Quackenbush, S. Salzberg, M. Hanson, R. van Vug, N. Palmer, M. D. Adams, J. Gocayne, J. C. Venter, et al. 1997. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390:580-586[CrossRef][Medline]. |
| 19. |
Gallegos, M. T., and M. Buck.
1999.
Sequences in N determining holoenzyme formation and properties.
J. Mol. Biol.
288:539-553[CrossRef][Medline].
|
| 20. |
Gallegos, M. T.,
W. V. Cannon, and M. Buck.
1999.
Functions of the 54 Region I in trans and implications for transcription activation.
J. Biol. Chem.
274:25285-25290 |
| 21. |
Genschik, P.,
K. Drabikowski, and W. Filipowicz.
1998.
Characterization of the Escherichia coli RNA 3'-terminal phosphate cyclase and its 54-regulated operon.
J. Biol. Chem.
273:25516-25526 |
| 22. | Gruber, T. M., and D. A. Bryant. 1998. Characterization of the group 1 and group 2 sigma factors of the green sulfur bacterium Chlorobium tepidum and the green non-sulfur bacterium Chloroflexus aurantiacus. Arch. Microbiol. 170:285-296[CrossRef][Medline]. |
| 23. | Guo, Y., L. Wang, and J. D. Gralla. 1999. A fork junction DNA-protein switch that controls promoter melting by the bacterial enhancer-dependent sigma factor. EMBO J. 18:3736-3745[CrossRef][Medline]. |
| 24. |
Hartig, E., and W. G. Zumft.
1998.
The requirement of RpoN (sigma factor 54) in denitrification by Pseudomonas stutzeri is indirect and restricted to the reduction of nitrite and nitric oxide.
Appl. Environ. Microbiol.
64:3092-3095 |
| 25. |
Hirschman, J.,
P. K. Wong,
K. Sei,
J. Keener, and S. Kustu.
1985.
Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor.
Proc. Natl. Acad. Sci. USA
82:7525-7529 |
| 26. | Hoover, T. R., E. Santero, S. Porter, and S. Kustu. 1990. The integration host factor stimulates interaction of RNA polymerase with NifA, the transcriptional activator of nitrogen fixation operons. Cell 63:11-22[CrossRef][Medline]. |
| 27. | Horowitz, H., G. E. Christie, and T. Platt. 1982. Nucleotide sequence of the trpD gene, encoding anthranilate synthetase component II of Escherichia coli. J. Mol. Biol. 156:245-256[CrossRef][Medline]. |
| 28. |
Hu, P.,
T. Leighton,
G. Ishkhanova, and S. Kustu.
1999.
Sensing of nitrogen limitation by Bacillus subtilis: comparison to enteric bacteria.
J. Bacteriol.
181:5042-5050 |
| 29. |
Hunt, T. P., and B. Magasanik.
1985.
Transcription of glnA by purified Escherichia coli components: core RNA polymerase and the products of glnF, glnG, and glnL.
Proc. Natl. Acad. Sci. USA
82:8453-8457 |
| 30. |
Jovanovic, G.,
J. Rakonjac, and P. Model.
1999.
In vivo and in vitro activities of the Escherichia coli 54 transcription activator, PspF, and its DNA-binding mutant, PspFHTH.
J. Mol. Biol.
285:469-483[CrossRef][Medline].
|
| 31. | Kalman, S., W. Mitchell, R. Marathe, C. Lammel, J. Fan, R. W. Hyman, L. Olinger, J. Grimwood, R. W. Davis, and R. S. Stephens. 1999. Comparative genomes of Chlamydia pneumoniae and C. trachomatis. Nat. Genet. 21:385-389[CrossRef][Medline]. |
| 32. |
Keseler, I. M., and D. Kaiser.
1997.
N, a vital protein for Myxococcus xanthus.
Proc. Natl. Acad. Sci. USA
94:1979-1984 |
| 33. | Klenk, H. P., T. D. Meier, P. Durovic, V. Schwass, F. Lottspeich, P. P. Dennis, and W. Zillig. 1999. RNA polymerase of Aquifex pyrophilus: implications for the evolution of the bacterial rpoBC operon and extremely thermophilic bacteria. J. Mol. Evol. 48:528-541[CrossRef][Medline]. |
| 34. | Klose, K. E., and J. J. Mekalanos. 1998. Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle. Mol. Microbiol. 28:501-520[CrossRef][Medline]. |
| 35. |
Kustu, S.,
E. Santero,
J. Keener,
D. Popham, and D. Weiss.
1989.
Expression of 54 (ntrA)-dependent genes is probably united by a common mechanism.
Microbiol. Rev.
53:367-376 |
| 36. | Leary, B. A., N. Ward-Rainey, and T. R. Hoover. 1998. Cloning and characterization of Planctomyces limnophilus rpoN: complementation of a Salmonella typhimurium rpoN mutant strain. Gene 221:151-157[CrossRef][Medline]. |
| 37. | Magasanik, B. 1989. Regulation of transcription of the glnALG operon of Escherichia coli by protein phosphorylation. Biochimie 71:1005-1012[Medline]. |
| 38. | McLellan, T. 1982. Electrophoresis buffers for polyacrylamide gels at various pH. Anal. Biochem. 126:94-99[CrossRef][Medline]. |
| 39. |
Merrick, M. J.
1993.
In a class of its ownthe RNA polymerase sigma factor N (54).
Mol. Microbiol.
10:903-909[Medline].
|
| 40. | Miroux, B., and J. E. Walker. 1996. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260:289-298[CrossRef][Medline]. |
| 41. | Morales, A. J., M. A. Swairjo, and P. Schimmel. 1999. Structure-specific tRNA-binding protein from the extreme thermophile Aquifex aeolicus. EMBO J. 18:3475-3483[CrossRef][Medline]. |
| 42. |
Moreno-Vivián, C.,
P. Cabello,
M. Martínez-Luque,
R. Blasco, and F. Castillo.
1999.
Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases.
J. Bacteriol.
181:6573-6584 |
| 43. |
Morett, E., and L. Segovia.
1993.
The 54 bacterial enhancer-binding protein family: mechanism of action and phylogenetic relationship of their functional domains.
J. Bacteriol.
175:6067-6074 |
| 44. | Nelson, K. E., R. A. Clayton, S. R. Gill, M. L. Gwinn, R. J. Dodson, D. H. Haft, E. K. Hickey, J. D. Peterson, W. C. Nelson, K. A. Ketchum, L. McDonald, T. R. Utterback, J. A. Malek, K. D. Linher, M. M. Garrett, A. M. Stewart, M. D. Cotton, M. S. Pratt, C. A. Phillips, D. Richardson, J. Heidelberg, G. G. Sutton, R. D. Fleischmann, J. A. Eisen, C. M. Fraser, et al. 1999. Evidence for lateral gene transfer between Archaea and Bacteria from genome sequence of Thermotoga maritima. Nature 399:323-329[CrossRef][Medline]. |
| 45. |
Oguiza, J. A.,
M. T. Gallegos,
M. K. Chaney,
W. V. Cannon, and M. Buck.
1999.
Involvement of the N DNA-binding domain in open complex formation.
Mol. Microbiol.
33:873-885[CrossRef][Medline].
|
| 46. | Pallen, M. 1999. RpoN-dependent transcription of rpoH? Mol. Microbiol. 31:393[CrossRef][Medline]. |
| 47. | Reitzer, L. J., and B. Magasanik. 1986. Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter. Cell 45:785-792[CrossRef][Medline]. |
| 48. | Rombel, I., A. North, I. Hwang, C. Wyman, and S. Kustu. 1998. The bacterial enhancer-binding protein NtrC as a molecular machine. Cold Spring Harbor Symp. Quant. Biol. 63:157-166[CrossRef][Medline]. |
| 49. | Schmidt, C. L., S. Anemuller, and G. Schafer. 1996. Two different respiratory Rieske proteins are expressed in the extreme thermoacidophilic crenarchaeon Sulfolobus acidocaldarius: cloning and sequencing of their genes. FEBS Lett. 388:43-46[CrossRef][Medline]. |
| 50. | Simon, J., R. Gross, O. Klimmek, M. Ringel, and A. Kroger. 1998. A periplasmic flavoprotein in Wolinella succinogenes that resembles the fumarate reductase of Shewanella putrefaciens. Arch. Microbiol. 169:424-433[CrossRef][Medline]. |
| 51. |
Stephens, R. S.,
S. Kalman,
C. Lammel,
J. Fan,
R. Marathe,
L. Aravind,
W. Mitchell,
L. Olinger,
R. L. Tatusov,
Q. Zhao,
E. V. Koonin, and R. W. Davis.
1998.
Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis.
Science
282:754-759 |
| 52. | Stock, J. B., A. M. Stock, and J. M. Mottonen. 1990. Signal transduction in bacteria. Nature 344:395-400[CrossRef][Medline]. |
| 53. |
Studholme, D. J., and M. Buck.
2000.
Novel roles of N in small genomes.
Microbiology
146:4-5 |
| 54. |
Studholme, D. J.,
R. D. Finn,
M. K. Chaney, and M. Buck.
1999.
The C-terminal 13 amino acids of N are required for structure and function.
Arch. Biochem. Biophys.
371:234-240[CrossRef][Medline].
|
| 55. |
Wang, J. T.,
A. Syed, and J. D. Gralla.
1997.
Multiple pathways to bypass the enhancer requirement of sigma 54 RNA polymerase: roles for DNA and protein determinants.
Proc. Natl. Acad. Sci. USA
94:9538-9543 |
| 56. |
Wang, L., and J. D. Gralla.
1998.
Multiple in vivo roles for the 12-region elements of sigma 54 promoters.
J. Bacteriol.
180:5626-5631 |
| 57. | Zhang, G., E. A. Campbell, L. Minakhin, C. Richter, K. Severinov, and S. A. Darst. 1999. Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 A resolution. Cell 98:811-824[CrossRef][Medline]. |
| 58. |
Zinchenko, V.,
Y. Churin,
V. Shestopalov, and S. Shestakov.
1994.
Nucleotide sequence and characterization of the Rhodobacter sphaeroides glnB and glnA genes.
Microbiology
140:2143-2151 |
Journal of Bacteriology, March 2000, p. 1616-1623, Vol. 182, No. 6
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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