![]()
|
|
||||||||
J. Biol. Chem., Vol. 275, Issue 32, 24709-24714, August 11, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,From the Department of Microbial Biotechnology, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco, 28049 Madrid, Spain
Received for publication, April 3, 2000, and in revised form, May 25, 2000
| |
ABSTRACT |
|---|
|
|
|---|
Production of the siderophore aerobactin in
Escherichia coli is transcriptionally metalloregulated
through the iron-dependent binding of the Fur
(ferric uptake regulator) to a
large region (>100 base pairs) within the cognate promoter in
the pColV-K30 plasmid. We show in this article that such an unusually
long operator results from the specific addition of degenerate repeats
5'-NAT(A/T)AT-3' and not from a fortuitous occupation of the DNA
adjacent to the primary binding sites by an excess of the repressor.
Furthermore, the protection pattern revealed by DNase I and hydroxyl
radical footprinting reflected a side-by-side oligomerization of the
protein along an extended DNA stretch. This type of DNA-protein
interactions is more like those observed in some eukaryotic factors and
nucleoid-associated proteins than typical of specific prokaryotic regulators.
Prokaryotic transcriptional regulators bind DNA to repress or
activate expression of specific genes or groups of genes (1). Although
the sequences recognized can be extremely diverse, most regulatory
proteins naturally bind discrete target sites within the bacterial
genome. However, some regulators (typically the nucleoid-associated
proteins; Ref. 2) are also known to bind somewhat degenerated sequences
or structural motifs, thus spreading DNA-protein interactions along
extended nucleotide sequences. This feature is shared with a variety of
eukaryotic regulators, typically those containing zinc fingers such as
the transcription factor TFIIIA (3). In this respect, the Fur
protein of Escherichia coli displays both properties found
in specific transcriptional factors and those in more global
regulators. Fur is the product of the fur
(ferric uptake regulation) gene
(4-7), which controls transcription of iron-dependent
promoters in many prokaryotes. This regulator is a zinc-containing,
Fe2+-binding protein (8) that inhibits transcription of
distinct genes implicated in the response to iron starvation when the
metal is in excess in the medium (9-12). But, in addition, Fur appears to play an important role also in a variety of cell functions unrelated
to iron acquisition, such as the production of several virulence
determinants (13), the defense against oxygen radicals (14, 15), the
acid shock response (16), chemotaxis (17), metabolic pathways, and
others (18-23).
The interaction of the Fur protein-Fe2+ complex with its
operators has been characterized with diverse techniques in several promoters of E. coli and other genera (14, 24-30). These
studies have revealed that every iron-dependent promoter
contains a target DNA sequence with different degrees of similarity to
a palindromic 5'-GATAATGATAATCATTATC-3',
19-bp1 consensus
box (26, 31, 32). More recently, we have reinterpreted such a
consensus as the combination of three repeats of the simpler motif
5'-NAT(A/T)AT-3' (33), in which the thymines would be the bases
determining the type of contact of the Fur protein with such a minimal
unit of interaction. The corollary of this interpretation is that
extended sites for Fur binding could be naturally or artificially assembled by simply adding multiple adjacent 5'-NAT(A/T)AT-3' hexamers
to a minimum of three repeats. This is a very attractive possibility,
because it would permit the generation of repertoires of binding sites
of varying extensions and affinities, which would allow Fur to act in
some promoters as a very specific regulator and in others as a more
general co-regulator (12). Although this notion has been substantiated
using synthetic DNA sequences consisting of synthesized
5'-NAT(A/T)AT-3' hexamers (33), it is unclear whether long
sequence-dependent Fur operators are operative in natural
iron-regulated promoters. The promoter of the operon responsible for
the biosynthesis of the aerobactin siderophore (referred hereafter as
Paer) is particularly interesting in this respect (26, 32,
33, 34). Unlike other promoters controlled by Fur in which the operator
involves a clear-cut target sequence (13, 24, 27-29), Paer
is bound by the repressor to three distinct extents depending on the
concentration of the protein (Fig. 1 and
see below). Although Fur binding to the adjacent sites named I and II
can be justified by their similarity to the consensus, the massive
protection of the further upstream sequences (the so called
polymerization region; Fig. 1) is intriguing, because it does not
contain clear Fur consensus boxes. Such an extensive occupation of the
promoter by the repressor spreading over 100 bp has been revealed not
only by DNase I and hydroxyl radical footprinting (32, see below) but
also visualized directly through electron microcoscopy (35). Other
iron-regulated promoters appear to undergo such an ample occupation as
well (25, 36), so it might be a genuine phenomenon and not an
unspecific protection caused by an excess of the protein.
![]()
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
![]()
View larger version (9K):
[in a new window]
Fig. 1.
Organization of the aerobactin promoter
region. The overall arrangement of functional elements within the
DNA segment placed at 5' in respect to the aerobactin gene cluster is
shown. The promoter region includes two
10/
35 hexamers that define
promoters P1 (proximal) and P2 (distal). The primary target DNA
sequences for the Fur protein (sites I and II) and the upstream
extension are pointed as defined by DNase I footprinting (Ref. 32 and
Fig. 2), with an indication of the two segments with a maximal
coincidence with the 19-bp consensus Fur binding sequences
(5'-GATAATGATAATCATTATC-3', Fur boxes). The transcription start sites
of each of the promoters is indicated as well.
In this work, we show that the as yet unaccounted binding of the Fur
protein to the 5' upstream region of the aerobactin promoter is due to
the functionality of a long operator composed of nine adjacent
5'-NAT(A/T)AT-3' hexamers. This operator, which is entirely sequence-dependent, becomes effective only following the
occupation of the other two sites. These results support the notion
that the binding of Fur to DNA is mediated by the recognition of
hexameric repeats and that an increasing number of adjacent repeats
allow a co-operative binding of the repressor mediated by lateral
protein-protein interactions. Furthermore, we argue that this type of
interaction, which has features reminiscent of some transcription
factors (37), endows the protein with the ability to behave both as a
very specific repressor and as a more general regulator.
| |
EXPERIMENTAL PROCEDURES |
|---|
|
|
|---|
General Procedures-- The Fur protein used in all the assays was purified to homogenity following the metallo-affinity purification protocol of Wee et al. (7). According to Ref. 8, such purification protocol yields a Fur protein containing 1 atom of zinc/repressor monomer, whose DNA binding ability is responsive to Mn2+ in our assays system (see below). Protein Fur concentrations indicated through this work refer to the protein monomer. DNA techniques were run according to published protocols (38).
DNA Templates for Footprinting Assays--
The organization of
the DNA fragments used in footprinting assays is shown in Fig.
2. The fragment wt1 is a 368-bp
EcoRI-PvuII segment from plasmid pUC-LE15 that
contains the region spanning positions
128 to +32 of the aerobactin
promoter region (using as a reference the transcription start site of
the main promoter P1) as an EcoRI-BamHI plus a
vector-born unrelated BamHI-PvuII extension of
208 bp. The strategy for creating the promoter termed
50 is sketched
in Fig. 2A as well. Primers were devised for amplification of the sequence
50 to +32 (thus excluding the P2 promoter) and the
upstream extension region. This fragment was recloned in pUC19 using
the EcoRI and BamHI sites present in the
amplified fragments (BamHI already present and
EcoRI entered with the rightwards primer). To get a template
of identical size to wt1 for the footprint assay, a primer was
engineered that contained a terminal NcoI site located at
exactly the same distance that the EcoRI site of the wt1
promoter (Fig. 2B). Such a segment was then entered at the
single HincII site of the previous pUC19 derivative. This
new plasmid contained an insert in the vector that spans the new
promoter construct
50. The fragment generated after restriction with
NcoI-PvuII allowed a base-wise comparison of its
footprint with the wild type fragment because the end-sites,
EcoRI or NcoI, were located exactly at the same
point.
|
For the second series of templates shown in Fig. 2B,
modified variants of the aerobactin promoter with increasing distance between the Fur box I (Fig. 1) and the downstream protected region were
constructed as follows. In the EcoRI-BamHI insert
of plasmid pUC-LE15 a novel ClaI restriction site was
introduced by site-directed mutagenesis at the boundary between Fur
boxes I and II (Fig. 2B) with the method of Kunkel et
al. (39). Digestion of the resulting construct with
ClaI, filling-in of the cohesive ends, and religation originated a novel NruI as well as +2 bp insertion between
the boxes. The same ClaI-digested plasmid was ligated to the
linker 5'-CGACCATGGT-3', which entered a novel NcoI site as
well as a +10 insertion. Finally, NcoI digestion of the
resulting construct, filling-in the cohesive ends and religation
generated the +14 bp along with a new NsiI site. The mutated
segments were cloned back to pUC19 and used as the source of the
end-labeled restriction fragments employed in the footprinting assays.
To this end, they were excised from these pUC19 derivatives as
EcoRI-PvuII or NcoI-PvuII segments (for labeling of the bottom strand) and purified by
electrophoresis on nondenaturing 5% polyacrylamide gels. The
overhanging ends of the restriction fragments were then filled-in with
[
-32P]dATP and Klenow polymerase, after which they
were further purified from nonincorporated nucleotides on small
Sephadex G-25 columns.
Footprinting with DNase I and Hydroxyl Radicals--
DNA-protein
interactions were probed with DNase I as described in Refs. 32 and 33.
Samples were preincubated for 5 min at 37 °C with the amounts of the
Fur protein indicated in each case. Each tube was then added with 2.5 ng of DNase I and further incubated for 2 min. Reactions were stopped,
nucleic acids precipitated, dried, and directly resuspended in 7 M urea (with tracking dyes). Samples were loaded on 7-10%
polyacrylamyde sequencing gels with 7 M urea. A+G reactions
(40) with the same labeled DNA fragments or sequencing size markers
were loaded onto the gels together with the treated samples.
Footprinting of DNA with hydroxyl radicals generated in situ
with Fe/EDTA/ascorbate were carried out (32) on Fur-DNA mixtures
prepared and preincubated in the same conditions as before.
| |
RESULTS AND DISCUSSION |
|---|
|
|
|---|
Visualization of a Continuous Pattern of Fur-DNA Interactions
through the Aerobactin Promoter Region--
To match faithfully the
extensions of each binding site for the Fur protein along the
Paer promoter with the specific bases involved in
protein-DNA contacts, we conducted the experiment shown in Fig.
3. In it, we compared directly the
protections caused by increasing concentration of Fur-Mn2+
to either DNase I nicking or hydroxyl radical cleavage of
sugar-phosphate bonds (Mn2+ was used instead of
Fe2+ because of its superior stability under the aerobic
conditions of the experiment; Refs. 11, 12, and 26). The reference DNase I footprint to the left of the gel shown in Fig. 3 revealed the
position of each of the known three regions sequentially protected by
growing repressor concentrations. This includes first a 31-bp sequence
(site I) spanning the
35 hexamer of the P1 promoter, an additional
18-19 bp downstream protection (site II) overlapping the
10 box, and
the less defined further upstream region protected toward 5' (26).
Although weaker than those of sites I and II, this last protection
involves exactly 60 bp, so that the addition of all sites covered by
the protein at the higher protein concentration comes to 110 bp. When
the same protein-DNA contacts are inspected in strict parallel with
hydroxyl radical footprinting, some salient features become apparent.
First, that the distinction between the three binding regions revealed
by DNase becomes less clear-cut. At the higher Fur concentration, the
whole of the 110-bp DNA sequence displays a continuous and repetitive
pattern of two protected residues/four nonprotected bases. The frame of
such a regular pattern is shifted only once and by one base at the very
boundary between the DNase I site I and the upstream protected region, to then resume the previous two protected/four nonprotected mold through the further upstream region. Furthermore, the data of Fig. 3
suggest that the occupation of such an upstream region does not
commence until the site II is fully bound by the repressor. Because we
could not distinguish discrete binding sites within the 110-bp region
but rather a continuum of repetitive interactions, we wondered whether
the entire DNA stretch actually functions as a natural, extended
operator of the type predicted by the reinterpretation of the Fur
consensus sequences presented before (33).
|
The Primary Fur Binding Site in the Paer Promoter Nucleates the
Occupation of the Adjacent Downstream Sequence--
Because the 50-bp
sequence of the Paer promoter spanning DNase sites I and II
interacts invariably with the Fur protein with the 6-bp periodicity
discussed above, we first addressed whether such sites are independent
(as they should be by the 19-bp consensus criterium) or site II is a
sequence-dependent extension of site I. Such secondary
sites are protected in most E. coli Fur-regulated promoters,
although the sequences can be very variable (24, 28, 29, 14). To
address this issue, we engineered a ClaI site next to the
Fur box of site I (primary binding site of the protein). This site was
employed to insert extra bases that changed the relative orientation of
the downstream site II by 2, 10, or 14 bp (Fig. 2B). The
expected result of such insertions was to either offset moderately the
two target sequences (+2) or to separate them but keep the phase of the
DNA helix (+10) or to entirely offset and separate sites I and II
(+14). The resulting promoter variants were then footprinted with DNase
I in the presence of growing concentrations of Fur-Mn2+,
with the results shown in Fig. 4.
|
The 2-bp insertion (Fig. 4) between the sites entered a change in the extension and strength of the occupation of site II. The first 31 bp (site I) were protected to the same extension and the same protein concentration as the wild type promoter. However, occupation of site II (which was displaced further downstream: upwards in Fig. 4) required a significantly higher Fur-Mn2+ level. Interestingly, the protection of the 5' extension region occurred at the very same protein concentration as in the wild type promoter, thus suggesting that such an extension is entirely independent of the presence of protein bound to site II. Although these results indicated that occupation of site II is co-operative with that of site I, they do not rule by themselves that both sites are indeed independent. This issue, however, was unequivocally ascertained by the results of the +10 and +14 promoters. Regardless of the maintenance of the DNA helix phase (+10) or its full disruption (+14), the increased distance between sites I and II resulted in the inability of the downstream site to bind any protein. In both cases, the protection of site I was in all comparable with the wild type Paer promoter. These data favor the notion that site II is an extension of site I rather than a separate target sequence. As in the case of the +2 template, the separation of the sites by longer insertions did not affect at all the upstream 5' extensions, which were detected to the same extent and apparent intensity as in the wild type promoter.
Extensive Binding of Fur to the DNA Adjacent to the Primary Binding Site in the Aerobactin Promoter Is Sequence-specific-- The results above gave a preliminary hint on whether the lateral enlargements of the protection caused by Fur on most iron-regulated promoters of E. coli (24, 28, 29, 14) is sequence-specific or whether they just reflect an artifactual occupation caused by a high protein concentration in vitro. This is a reasonable doubt, because such prolongation not always matches the 19-bp consensus Fur box (24, 28, 29, 14). The data of Fig. 4 show that not any sequence adjacent to site I within the aerobactin promoter is suitable to become protected by a high concentration of the repressor. Furthermore, extensions require a certain frame and distance in respect to the primary site. However, the secondary site does include a sequence stretch similar to the 19-bp consensus, thereby suggesting that frame, distance, and specific sequence are all necessary for the enlargement toward the site II.
This is, however, less clear at the third protected region, where the
extension phenomenon is far more dramatic that in site II (Fig. 3). In
this case, the upstream protection observed spans 60 additional base
pairs. Although such a protection has a clear directionality and
defined boundaries, the sequence involved does not show any significant
homology with the reference 19-bp Fur consensus box (26). It is
thus conceivable that such a massive protection is not specific and
therefore irrelevant to understand the metalloregulation of the
promoter. To ascertain this question, we simply prepared a new DNA
template (Fig. 2A) in which we faithfully replaced the
upstream DNA by an unrelated sequence. The substitution was such that a
DNA fragment of a size identical to that bearing the wild type
Paer promoter could be examined in parallel in DNase I
footprinting assays. The results shown in Fig.
5 indicated that the unrelated sequence
failed to bear any visible extension of the footprint, even at the
higher protein concentrations. Furthermore, the 5' boundary of the
protection was located exactly at the point where the heterologous
sequence started (marked with an arrow in Fig. 5). We thus
conclude that the binding of Fur to the third region is indeed
sequence-dependent. We argue below that this cannot be
explained with the generally accepted 19-bp consensus model, but it is
perfectly compatible with the notion that a shorter 5'-NAT(A/T)AT-3'
motif is the basic unit of Fur binding.
|
Reinterpretation of the Fur Operator within the Aerobactin
Promoter--
The data presented in this work support the hypothesis
(33) that Fur binding sites do not follow the standard palindromic organization of target sequences for regulators in prokaryotic promoters (1). Instead, Fur operators of different extensions can be
formed by addition, in any orientation, of a minimum of three
NAT(A/T)AT hexamers. Although this was shown to be true for
artificially assembled NAT(A/T)AT multimers (33), this report shows
that this is the case also for a natural promoter such as that of the
aerobactin operon, whose extended binding sites for the repressor
cannot be easily explained with the generally accepted 19-bp consensus
model. Fig. 6 shows a reinterpretation of
the pattern of Fur-DNA interactions in the promoter as the result of a
single, enlarged operator that is formed by additions of up to 18 boxes
whose frame give a maximum match to the reference AT(A/T)AT pentamer.
Such boxes are separated in all cases by one intervening extra base.
The one exception is at the boundary between the site I and the
protected upstream region, which lacks such an additional base, a fact
that is faithfully reflected in the hydroxyl radical footprint of the
region (Fig. 3). It seems that either the deletion of 1 base or the
addition of two bases between boxes (as in the artificial promoter +2,
see Fig. 4) flaws the co-operative occupation of adjacent hexamers but
does not inhibit it. In fact, it is revealing that such a naturally
existing deletion between site I and the upstream extension is required
to frame maximally the further upstream sequence to the reference
NAT(A/T)AT motif. But how does this hypothesis equate the actual
data?
|
The sequence that is protected by the lowest concentrations of Fur-Mn2+ includes 31 bp and, according to the hydroxyl radical footprinting of Fig. 3, consists of a whole of five adjacent hexamers, three of them with a nearly perfect match to NAT(A/T)AT. The side repeats contain less conserved T residues, and thus their occupation requires a higher repressor concentration, which establishes the pause in the protection that is clearly revealed by DNase I footprint (Fig. 3) and which defines site II. Such second site would include three additional repeats. This extension certainly requires protein-protein interactions with the repressor already bound to site I, to compensate the divergence in the sequence. In fact, some hexamers have only a limited match with the consensus. Thus, the downstream sequence may not bind by itself to the Fur-Mn2+ complex, but it does in the context of the whole promoter. Finally, the long upstream extension can also be sorted out as an array of adjacent Fur-binding hexamers frameshifted by one base in respect to the sequence of boxes included in sites I and II. Although such a shift may explain the lower affinity, the hydroxyl radical data of Fig. 3 shows that the shift resettles the pattern of protein-DNA interactions to the maximum match with the NAT(A/T)AT array. It thus appears that the suboptimal alignment with the primary sites and the considerable sequence divergence of the upstream region are balanced by a higher number of boxes that, as a result, produce an unusually long operator.
Conclusion--
Although not to the same dramatic extent as the
aerobactin system, many if not all iron-regulated promoters of E. coli (14, 22, 24, 28, 29, 36) contain Fur target sequences that spread beyond the core iron box. No natural Fur binding
sites have been found to give less than a 31-bp footprint with DNase I,
although the minimal operator is only 19 bp. It thus looks likely that
such adjacent sequences are not casual but are indeed arrayed in a
configuration of various 6-bp repeats with a potential to interact
specifically with the Fur protein as a whole. Extended sites might
tolerate a degree of divergence in the sequences involved, which could
be compensated by the higher overall affinity. These additional
contacts might strengthen the overall binding of the DNA segment to the
regulator and do explain why the protection is not limited to the
consensus Fur box. The 6-bp box criteria accounts for the
variability and extension of the sequences protected by Fur in most
iron-regulated promoters and is also compatible with the relatively
high amount of Fur molecules (approximately 5000) found inside the cell
(16, 41). The published DNase I footprinting assays on several
promoters (14, 24, 28, 29) can be consistently reinterpreted as arrays
of hexameric sequences akin to those of the aerobactin promoter, in
which the key T residues are conserved to various degrees. This mode of Fur-DNA interaction, in which new Fur molecules must necessarily bind
adjacent hexamers through side-by-side oligomerization, explains the
gradual physiological response observed in Fe2+-responsive
systems, because it would make possible an entire range of repression
levels of iron-controlled promoters (12). The affinity for specific
promoters would vary depending on the number of repeats present on each
operator and the conservation of their sequences, thus generating a
hierarchy of transcriptional responses depending on small changes in
the iron status of the cell. Such an ability of Fur to control
promoters through extensive DNA-protein interactions makes this protein
to be mechanistically closer to general regulators than to specific
transcriptional factors. In fact, because Fur is a Zn-containing
protein (8), it is curious that the type of DNA-protein interactions
reported here have certain reminiscence to the occupation of adjacent
DNA sites by individual zinc fingers within eukaryotic transcription factors such as transcription factor TFIIIA (3, 37).
| |
FOOTNOTES |
|---|
* This work was supported by Contracts BIO4-CT97-2040 and QLRT-1999-00041 of the European Union and by Grant BIO98-0808 of the Comisiòn Interministerial de Ciencia y Tecnología.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Fellowship of Fundación Ramón Areces.
Present address: Inst. für Genetik. Biozentrum, Weinbergweg, 22 06120 Halle (Saale) Germany.
§ To whom correspondence should be addressed: Dept. of Microbial Biotechnology, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, 28049 Madrid, Spain. Tel.: 34-91-585-4536; Fax: 34-91-585-4506; E-mail: vdlorenzo@cnb.uam.es.
Published, JBC Papers in Press, May 31, 2000, DOI 10.1074/jbc.M002839200
| |
ABBREVIATIONS |
|---|
The abbreviation used is: bp, base pair(s).
| |
REFERENCES |
|---|
|
|
|---|
| 1. | Ptashne, M. (1992) A Genetic Switch , Cell Press and Blackwell Scientific Publications, Cambridge, MA |
| 2. | Azam, T. A., and Ishihama, A. (1999) Proc. Natl. Acad. Sci. U. S. A. 274, 33105-33113 |
| 3. | Wuttke, D. S., Foster, M. P., Case, D. A., Gottesfeld, J. M., and Wright, P. E. (1997) J. Mol. Biol. 273, 183-206 |
| 4. | Bagg, A., and Neilands, J. B. (1985) J. Bacteriol. 161, 450-453 |
| 5. | Hantke, K. (1984) Mol. Gen. Genet. 197, 337-341 |
| 6. | Saito, I., Wormald, M. R., and Williams, R. J. P. (1991) Eur. J. Biochem. 197, 29-38 |
| 7. | Wee, S., Neilands, J. B., Bittner, M. L., Hemming, B. C., Haymore, B. L., and Seetharam, R. (1988) Biol. Metals 1, 62-68 |
| 8. | Althaus, E. W., Outten, C. E., Olson, K. E., Cao, H., and O'Halloran, T. V. (1999) Biochemistry 38, 6559-6569 |
| 9. | Bagg, A., and Neilands, J. B. (1987) Microbiol. Rev. 51, 509-518 |
| 10. | Earhart, C. F. (1996) Escherichia coli and Salmonella: Cellular and Molecular Biology , 2nd Ed. , pp. 1075-1090, ASM Press, Washington, D.C. |
| 11. | Escolar, L., de Lorenzo, V., and Pérez-Martín, J. (1997) Mol. Microbiol. 26, 799-808 |
| 12. | Escolar, L., Pérez-Martín, J., and de Lorenzo, V. (1999) J. Bacteriol. 181, 6223-6229 |
| 13. | Litwin, M., and Calderwood, S. (1993) Clin. Microbiol. Rev. 6, 137-149 |
| 14. | Tardat, B., and Touati, D. (1993) Mol. Microbiol. 9, 53-63 |
| 15. | Zheng, M., Doan, B., Schneider, T. D., and Storz, G. (1999) J. Bacteriol. 181, 4639-4643 |
| 16. | Hall, H. K., and Foster, J. W. (1996) J. Bacteriol. 178, 5683-5691 |
| 17. | Karjalainen, T. K., Evans, D. G., Evans, D. J., Graham, D. Y., Jr., and Lee, C. H. (1991) Microb. Pathog. 11, 317-323 |
| 18. | Hantke, K. (1987) Mol. Gen. Genet. 210, 135-139 |
| 19. | Makemson, J. C., and Hastings, J. (1982) Curr. Microbiol. 7, 181-186 |
| 20. | McCarter, L., and Silverman, M. (1989) J. Bacteriol. 171, 731-736 |
| 21. | Ochsner, U. A., and Vasil, M. L. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4409-4414 |
| 22. | Stojiljkovic, I., Bäumler, A. J., and Hantke, K. (1994) J. Mol. Biol. 236, 531-545 |
| 23. | Tsolis, R. M., Bäumler, A. J., Stojiljkovic, I., and Heffron, F. (1995) J. Bacteriol. 177, 4628-4637 |
| 24. | Brickman, T. J., Ozenberger, B. A., and McIntosh, M. A. (1990) J. Mol. Biol. 212, 669-682 |
| 25. | Chai, S., Welch, T. J., and Crosa, J. H. (1998) J. Biol. Chem. 273, 33841-33847 |
| 26. | de Lorenzo, V., Wee, S., Herrero, M., and Neilands, J. B. (1987) J. Bacteriol. 169, 2624-2630 |
| 27. | Desai, P. J., Angerer, A., and Genco, C. A. (1996) J. Bacteriol. 178, 5020-5023 |
| 28. | Griggs, D. W., and Konisky, J. (1989) J. Bacteriol. 171, 1048-1054 |
| 29. | Hunt, M. D., Pettis, G. S., and McIntosh, M. A. (1994) J. Bacteriol. 176, 3944-3955 |
| 30. | Watnick, P. I., Butterton, J. R., and Calderwood, S. B. (1998) Gene 209, 65-70 |
| 31. | Calderwood, S., and Mekalanos, J. J. (1988) J. Bacteriol. 170, 1015-1017 |
| 32. | de Lorenzo, V., Giovannini, F., Herrero, M., and Neilands, J. B. (1988) J. Mol. Biol. 203, 875-884 |
| 33. | Escolar, L., Pérez-Martín, J., and de Lorenzo, V. (1998) J. Mol. Biol. 283, 537-547 |
| 34. | Bindereif, A., and Neilands, J. B. (1985) J. Bacteriol. 162, 1039-1046 |
| 35. | Le Cam, E., Fréchon, D., Barray, M., Fourcade, A., and Delain, E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11816-11820 |
| 36. | Fréchon, D., and Le Cam, E. (1994) Biochem. Biophys. Res. Commun. 201, 346-355 |
| 37. | Pizzi, S., Dieci, G., Frigeri, P., Piccoli, G., Stocchi, V., and Ottonello, S. (1999) J. Biol. Chem. 274, 2539-4838 |
| 38. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
| 39. | Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382 |
| 40. | Maxam, A. M., and Gilbert, W. (1980) Methods Enzymol. 65, 499-560 |
| 41. | Watnick, P. I., Eto, T., Takahashi, H., and Calderwood, S. B. (1997) J. Bacteriol. 179, 243-247 |
This article has been cited by other articles:
![]() |
Z. Chen, K. A. Lewis, R. K. Shultzaberger, I. G. Lyakhov, M. Zheng, B. Doan, G. Storz, and T. D. Schneider Discovery of Fur binding site clusters in Escherichia coli by information theory models Nucleic Acids Res., November 7, 2007; 35(20): 6762 - 6777. [Abstract] [Full Text] [PDF] |
||||
![]() |
R. Quatrini, C. Lefimil, D. S. Holmes, and E. Jedlicki The ferric iron uptake regulator (Fur) from the extreme acidophile Acidithiobacillus ferrooxidans Microbiology, June 1, 2005; 151(6): 2005 - 2015. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Rau, S. Wyllie, J. Whittimore, and J. E. Raulston Identification of Chlamydia trachomatis Genomic Sequences Recognized by Chlamydial Divalent Cation-Dependent Regulator A (DcrA) J. Bacteriol., January 15, 2005; 187(2): 443 - 448. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. L. Lavrrar and M. A. McIntosh Architecture of a Fur Binding Site: a Comparative Analysis J. Bacteriol., April 1, 2003; 185(7): 2194 - 2202. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. Baichoo and J. D. Helmann Recognition of DNA by Fur: a Reinterpretation of the Fur Box Consensus Sequence J. Bacteriol., November 1, 2002; 184(21): 5826 - 5832. [Abstract] [Full Text] [PDF] |
||||
![]() |
T. Hoffmann, A. Schutz, M. Brosius, A. Volker, U. Volker, and E. Bremer High-Salinity-Induced Iron Limitation in Bacillus subtilis J. Bacteriol., February 1, 2002; 184(3): 718 - 727. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. R. Echenique, C. W. Dorsey, L. C. Patrito, A. Petroni, M. E. Tolmasky, and L. A. Actis Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron Microbiology, October 1, 2001; 147(10): 2805 - 2815. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. A. Lowe, A. H. Asghar, G. Shalom, J. G. Shaw, and M. S. Thomas The Burkholderia cepacia fur gene: co-localization with omlA and absence of regulation by iron Microbiology, May 1, 2001; 147(5): 1303 - 1314. [Abstract] [Full Text] |
||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |