AEM
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
AEM.00276-07v1
73/17/5389    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bender, K. S.
Right arrow Articles by Wall, J. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bender, K. S.
Right arrow Articles by Wall, J. D.
Agricola
Right arrow Articles by Bender, K. S.
Right arrow Articles by Wall, J. D.
Applied and Environmental Microbiology, September 2007, p. 5389-5400, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.00276-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Analysis of a Ferric Uptake Regulator (Fur) Mutant of Desulfovibrio vulgaris Hildenborough{triangledown}

Kelly S. Bender,1,10,{dagger} Huei-Che Bill Yen,1,10 Christopher L. Hemme,2,3,10 Zamin Yang,2,10 Zhili He,2,3,10 Qiang He,4,10 Jizhong Zhou,2,3,10 Katherine H. Huang,5,10 Eric J. Alm,5,6,10 Terry C. Hazen,7,10 Adam P. Arkin,5,8,9,10 and Judy D. Wall1,10*

Department of Biochemistry, University of Missouri—Columbia, Columbia, Missouri 65211,1 Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831,2 Institute for Environmental Genomics, Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019,3 Department of Civil and Environmental Engineering, Temple University, Philadelphia, Pennsylvania 19122,4 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720,5 Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,6 Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720,7 Department of Bioengineering, University of California, Berkeley, California 94720,8 Howard Hughes Medical Institute, Chevy Chase, Maryland 20815,9 Virtual Institute for Microbial Stress and Survival ,{ddagger},{ddagger}

Received 2 February 2007/ Accepted 12 June 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous experiments examining the transcriptional profile of the anaerobe Desulfovibrio vulgaris demonstrated up-regulation of the Fur regulon in response to various environmental stressors. To test the involvement of Fur in the growth response and transcriptional regulation of D. vulgaris, a targeted mutagenesis procedure was used for deleting the fur gene. Growth of the resulting {Delta}fur mutant (JW707) was not affected by iron availability, but the mutant did exhibit increased sensitivity to nitrite and osmotic stresses compared to the wild type. Transcriptional profiling of JW707 indicated that iron-bound Fur acts as a traditional repressor for ferrous iron uptake genes (feoAB) and other genes containing a predicted Fur binding site within their promoter. Despite the apparent lack of siderophore biosynthesis genes within the D. vulgaris genome, a large 12-gene operon encoding orthologs to TonB and TolQR also appeared to be repressed by iron-bound Fur. While other genes predicted to be involved in iron homeostasis were unaffected by the presence or absence of Fur, alternative expression patterns that could be interpreted as repression or activation by iron-free Fur were observed. Both the physiological and transcriptional data implicate a global regulatory role for Fur in the sulfate-reducing bacterium D. vulgaris.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Iron is an essential nutrient for most bacteria because of its role as an enzymatic cofactor and electron transport protein component. In addition to the metabolic importance of iron, pathogenic bacteria use its availability as an environmental signal for regulation of virulence genes. Despite the metabolic dependence on iron, cellular concentrations must be intricately regulated in aerobic environments to prevent Fe(II)-mediated formation of reactive oxygen species via Fenton chemistry (72). In most bacteria, this complex regulation is carried out by the ferric uptake regulator protein (Fur) (23, 29). The traditional mode of Fur regulation has been described as follows: under iron-replete conditions, the Fur protein and its corepressor [Fe(II)] block the transcription of iron uptake and storage genes. When iron becomes limiting, the Fur repressor is no longer saturated with Fe(II) and cannot bind the operator, leading to transcription of genes involved in iron uptake and storage. Since iron is found predominantly as insoluble ferric hydroxides in aerobic environments, bacteria have evolved a mechanism for uptake dependent on the synthesis and transport of specialized chelators called siderophores. Thus, Fur also regulates the synthesis of siderophores in bacteria studied to date (29, 65, 68). In addition to its role as the primary regulator responding to available iron, Fur has also been shown to play a global regulatory role in oxidative stress response, acid tolerance response, virulence factor synthesis, and motility (2, 23, 29).

Because of the insolubility and sequestration of iron in aerobic and host environments, studies involving iron regulation have focused primarily on aerobic/facultative and pathogenic microbes, respectively. The role of Fur or iron metabolism in strict anaerobes has received little attention. In anaerobic environments, enough iron is expected to be in the Fe(II) form that its accessibility should not be limiting. Also, the likelihood of Fe(II)-mediated formation of reactive oxygen species via Fenton chemistry is decreased by limited oxygen exposure. Thus, elaborate iron regulation would seem to be less critical in anaerobes. However, recent genomic studies of metal-reducing {delta}-proteobacteria have indicated the presence of not one but three fur paralogs, fur (DVU0942), perR (DVU3095), and zur (DVU1340), in these anaerobes (61). While the roles of these regulators remain unclear, the sulfate reducers Desulfovibrio vulgaris Hildenborough and Desulfovibrio desulfuricans G20 appear to have extended Fur regulons compared to the predictions from genomes of other {delta}-proteobacteria (61). Regulon members include ferrous iron transporter genes (feoAB, DVU2572/71), a flavodoxin gene (fld, DVU2680), genes for P-type and ABC ATPases, and genes possessing GGDEF and HD domains. The proteins encoded by the latter genes are predicted to have cyclic di-GMP synthesis and hydrolysis activity, respectively, that could allow second-messenger concentrations to be responsive to Fur signals (66). A large cluster of genes predicted to be involved in biopolymer transport, such as tonB (DVU2390), were also suggested to be part of the Fur regulon (61). However, siderophore production by Desulfovibrio has not been documented, nor have genes for siderophore synthesis been identified. The annotation of a putative transporter for the siderophore enterobactin (fepC, DVU0648) suggests a possible mechanism for acquiring insoluble iron via chelators produced by other bacteria.

Desulfovibrio species are anaerobic sulfate-reducing bacteria (SRB) known for their ability to corrode ferrous metals as well as to reduce heavy metals such as uranium(VI), chromium(VI), and technetium(VII). D. vulgaris Hildenborough is also believed to possess a robust iron requirement based on its abundance of iron-containing cytochromes, hydrogenases, and electron transport proteins, as determined from genome analysis (36). Despite the solubility of iron in anaerobic environments, the degree to which Fe(II) is accessible to sulfate reducers is unknown, especially since sulfide, a by-product of sulfate reduction, complexes with Fe(II) to form insoluble pyrite (FeS). Another notable aspect of iron metabolism in Desulfovibrio is the production of a ferritin protein containing a unique heme group (58, 62). Ferritin and bacterioferritin proteins are produced by aerobic bacteria to sequester free iron in a nonreactive insoluble Fe(III) form (1, 2, 72). The role of these proteins in anaerobic bacteria has been assumed to be in oxygen defense (60, 62), but their intrinsic regulatory mechanisms are currently unknown.

Even though Desulfovibrio species are considered strict anaerobes, they do possess numerous oxygen detoxification proteins, such as rubrerythrin, rubredoxin oxidoreductase, and superoxide dismutase, all of which contain iron (19, 24, 45). The role of iron in response to oxygen stress was underscored by recent research with Bacillus subtilis indicating that iron oxidation was likely the initiating event for detection of peroxides via the metal-dependent peroxide sensor/regulator PerR (41). The annotation of a perR gene in SRB suggests that the relationship between these anaerobes and iron may be more complex than previously considered. Here we describe a marker exchange method for gene deletion in D. vulgaris Hildenborough and the physiology and transcriptional profile of the resulting {Delta}fur strain. The transcriptional data are strengthened by comparison with previous studies documenting the differentially expressed genes in stressed D. vulgaris cells (12, 35, 46, 47). While Fur in D. vulgaris appears to regulate genes traditionally involved in Fe(II) uptake, our data suggest a diverse regulatory pattern for Fur in SRB. To our knowledge, this is the first report of a {Delta}fur deletion in a sulfate-reducing bacterium.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains, plasmids, and media.
Bacterial strains and vectors used in this study are listed in Table 1. D. vulgaris cultures were grown anaerobically as previously described (57) in two defined media: LS4D, routinely used in transcriptomic and proteomic studies of this bacterium so data can be compared, and Yen45, used to reduce the formation of precipitates when ion concentrations were altered. LS4D (60 µM total iron) consisted of 60 mM sodium lactate, 50 mM Na2SO4, 8.0 mM MgCl2, 20 mM NH4Cl, 2.2 mM K2HPO4, 0.6 mM CaCl2, 30 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)], 12.5 ml trace mineral solution per liter (7), NaOH to a pH of 7.2, and 1.0 ml 10x vitamin solution per liter (7) (added after autoclaving). Yen45 (30 µM total iron) consisted of 60 mM sodium lactate, 30 mM Na2SO4, 8.0 mM MgCl2, 20 mM NH4Cl, 2 mM phosphate buffer (K2HPO4/NaH2PO4, pH 7.4), 0.6 mM CaCl2, 30 mM Tris-HCl (pH 7.4), and, per liter, 2 ml modified trace mineral solution (7) (modified by omission of nitrilotriacetic acid and FeCl2), 0.24 ml iron solution (125 mM FeCl2/250 mM EDTA, pH 7.3), 20 ml sterile 1 M NaHCO3 (added after autoclaving), and 1.0 ml sterile 10x vitamin solution (7) (added after autoclaving). As the reductant for both media, 5 ml per liter of an anaerobic titanium citrate solution was used. This solution contained 20% (wt/vol) titanium(III) chloride, 0.2 M sodium citrate, and 8.0% (wt/vol) sodium carbonate. For plating, LS4D medium was supplemented with 0.2% (wt/vol) yeast extract, 1.5% (wt/vol) agar, and 1.2 mM thioglycolate. Cells were distributed in 4 ml of molten top agar (30 mM PIPES, 1.5% agar).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Bacteria and plasmids used in this study

 
Deletion cassette construction.
Deletion cassettes were constructed by a method similar to the molecular bar-coding methods described for Saccharomyces cerevisiae (27, 69). Briefly, PCR primer sets were designed to amplify approximately 800 bp up- and downstream of the fur open reading frame (ORF) (DVU0942). Primer nucleotide sequences are listed in Table 2 along with genome locations relative to the fur ORF. For future tracking of the mutant in a mixed population, unique barcode sequences were added between the common sequences and Kmr sequences of primers P5 and P6. The following PCR mixture was used to amplify the upstream (primers P1 and P2) and downstream (primers P3 and P4) regions flanking the fur ORF from D. vulgaris as well as the Kmr cassette from pSC27 (63) (primers P5 and P6): 1x Herculase buffer (Stratagene, La Jolla, CA), 0.25 mM each deoxynucleoside triphosphate, 2.5 pmol each primer, 2.5 units Herculase polymerase (Stratagene), and 1 µl template DNA in a total volume of 40 µl. Reaction mixtures were cycled according to the following program: 94°C denaturation for 60 s followed by 5 cycles consisting of 94°C for 30 s, 45°C for 30 s, and 70°C for 70 s and then 24 cycles consisting of 94°C for 30 s, 54°C for 30 s, and 70°C for 70 s, ending with a 6-min extension at 70°C. Products were gel purified using Quantum Prep Freeze'N Squeeze DNA gel extraction spin columns (Bio-Rad, Hercules, CA) and immediately used for fusion PCR.


View this table:
[in this window]
[in a new window]

  		TABLE 2. PCR primers for deletion cassette construction

 
For the fusion PCR, the following reaction mixture was used: 1x Herculase buffer, 0.25 mM each deoxynucleoside triphosphate, 4 units Herculase polymerase, and 50 to 100 ng of each of the three products generated in the PCRs described above (approximately 3 µl each). Before addition of the polymerase, the reaction mixture was heated to 94°C for 30 s. Reaction mixtures were subjected to four cycles consisting of 94°C for 30 s, 55°C for 60 s, 72°C for 3 min 30 s, and then 4 U more of polymerase and 25 pmol each of primers P1 and P4 were added. This mixture was then cycled 25 times at 94°C for 30 s, 55°C for 60 s, and 72°C for 3 min 30 s, ending with an 8-min extension at 72°C for 8 min. The resulting fusion PCR product was gel purified as described above and ligated into the EcoRV site of pBluescript SK(+) (Stratagene, La Jolla, CA) to generate pMO707. Prior to electroporation of pMO707 into D. vulgaris, the construct was sequenced to check for proper arrangement and lack of sequence errors.

Transformation and mutant selection.
Approximately 109 to 1010 D. vulgaris cells, harvested at early stationary phase (optical density at 600 nm [OD600] of ca. 1.0) from LS4D modified to contain 0.2% yeast extract, were electroporated with 5 µg of the knockout vector (pMO707) and 1 µg lambda DNA, as an additional substrate for nucleases in the recipient. Prior to electroporation, the cells were washed twice with ice-cold 1 mM MgCl2-10% (vol/vol) glycerol. Electroporations were carried out in a total volume of 75 µl with a BTX electroporation pulse generator, model ECM630 (Genetronix, San Jose, CA). The parameters obtained for the electroporations were 1.75 kV, 25 µF, and 250 {Omega}. Immediately following transformation, the cells were recovered in 1 ml LS4D medium supplemented with 0.2% (wt/vol) yeast extract. Following 4 h of incubation at 37°C, the cultures were diluted to 5 ml in the same medium containing 400 µg G418/ml and allowed to grow overnight. The next day the transformation cultures were plated (1 ml/plate) on solidified LS4D plus 0.2% (wt/vol) yeast extract medium containing 400 µg G418/ml. Resulting transformants were analyzed for deletion of the fur gene via PCR that targeted genome regions outside of the fur knockout cassette (primers P7 and P8 [Table 2]) and Southern analyses of genomic DNAs. One confirmed fur deletion mutant was selected and designated JW707.

Nucleic acid procedures.
Genomic DNA was extracted with a Wizard genomic purification kit (Promega, Madison, WI). Southern analyses employed Zeta-Probe (Bio-Rad, Hercules, CA) membranes and were performed according to the manufacturer's instructions. For Northern analysis, RNA was isolated from exponential-phase (OD600 ~ 0.4) cultures using RNAwiz (Ambion, Austin, TX) according to the manufacturer's protocol. Prior to electrophoresis, contaminating DNA was removed from the RNA preparation using DNA-free DNase (Ambion). A total of 10 µg RNA per lane was separated in a gel of 1.2% (wt/vol) agarose with 1x formaldehyde-MOPS-EDTA sodium acetate buffer (Sigma, St. Louis, MO). Following electrophoresis, the RNA was transferred to Zeta-Probe membranes using the downward transfer method described in Ambion technical bulletin 169. The RNA was then permanently affixed to the membrane via UV cross-linking at 120 mJ/cm2.

Southern and Northern hybridizations were performed overnight at 42°C using 5 µl 32P-labeled PCR products in ULTRAhyb solution (Ambion). Blots were washed twice in 2x SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7)-0.1% (wt/vol) SDS for 5 min and twice in 0.1x SSC-0.1% (wt/vol) SDS for 15 min before being placed on film. Probes for both Southern and Northern analyses were generated via PCR (see Table 3 for primer sequences and product sizes) and labeled with [{alpha}-32P]dCTP using a Prime-It II random primer labeling kit (Stratagene).


View this table:
[in this window]
[in a new window]

  		TABLE 3. PCR primers for gene probe construction

 
Phenotypic analysis.
Unless otherwise noted, 2% (vol/vol) subcultures from active cultures in standard Yen45 medium were used as the inoculum for phenotypic analyses. Response to iron limitation was monitored via optical density at 600 nm in Yen45 medium modified by omission of all known sources of iron and addition of 6, 3, or 1 µM FeCl2. The inocula for testing the growth responses to limiting iron were from medium containing 1 µM added FeCl2. Modified Yen45 lacking added iron was analyzed by inductively coupled plasma spectrometry by the University of Missouri Agriculture Experiment Station Lab and found to contain 0.3 µM iron. To determine resistance to MnCl2, CFU were counted following a 112-h exposure to 20, 30, or 40 mM MnCl2. Growth responses to high concentrations of nitrate and nitrite were monitored via optical density of LS4D cultures amended to contain 50 or 100 mM nitrate or 2 or 5 mM nitrite at time zero. Responses to increased osmolarity were determined via optical density and microscopic observations following the addition of 300 mM NaCl or KCl (46) at the time of subculture. Osmoprotection was assayed by the addition of 2 mM glycine betaine at time zero to subcultures containing 300 mM NaCl or KCl.

Microarray analysis.
Volumes of 600 ml of D. vulgaris Hildenborough wild type or JW707({Delta}fur) were grown in LS4D as 100-ml batches in six 125-ml bottles to a cell density of ca. 3 x 108 cells/ml (OD600 ~ 0.4). The 100-ml aliquots were used as inocula for six replicate bottles, each containing 900 ml LS4D, and the cultures were grown to log phase at 30°C in anaerobic chambers. At log phase (OD600 = 0.38 for WT and 0.33 for JW707), 250 ml of each culture was harvested for sampling. For iron-limited experiments, the same protocol was followed with modified LS4D containing 5 µM FeCl2 instead of 60 µM. Cultures were sampled at log phase, OD600 = 0.13 for WT and 0.17 for JW707. Cell harvesting, RNA extraction, and microarray analyses were carried out as described previously (46).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mutagenesis of D. vulgaris fur gene.
Plasmid pMO707 (Fig. 1) was transferred to wild type D. vulgaris via electroporation. Positive (pSC27) and negative (lambda DNA) control DNAs were also electroporated using 109 to 1010 recipient cells for each transformation. To select for transformants, cells were plated on LS4D medium modified to contain 0.2% (wt/vol) yeast extract and 400 µg G418/ml and colonies appeared after four days at 30°C. The transformation efficiency for pMO707 was ca.1.2 x 10–7 per recipient cell, while the efficiency for the stable plasmid pSC27 was 1.4 x 10–6 per recipient cell. No G418 resistant colonies resulted from plating the negative control transformation. Colonies from the pMO707 transformation were subcultured into liquid medium of the same composition as that used for plating and a second single-colony isolation was made. Deletion of the fur gene via marker exchange was verified by a change in the size of a PCR product generated from primers complementary to regions up- and downstream of the fur gene (Fig. 2A). While the resulting PCR product from the wild-type fur region was 2168 bp, the product from two selected transformants (JW706 and JW707) was 671 bp larger (2,839 bp total). This difference correlates to the larger size of the Kmr determinant replacing the fur gene. Southern analysis with probes internal to fur or the Kmr determinant also corroborated replacement of the fur gene with the Kmr determinant (data not shown) and JW707 was selected for further analysis.


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 1. Map of pMO707 containing the fur deletion construct. A 2,706-bp PCR product containing the pSC27 Kmr determinant flanked by DNA sequences upstream (768 bp) and downstream (831 bp) of the D. vulgaris fur gene was inserted into the EcoRV site of the pBluescript SK(+) multicloning site. Both pBluescript and pMO707 are unstable in D. vulgaris. Numbers indicate positions of sequences upstream and downstream of the fur gene; BC indicates molecular barcodes allowing mutant tracking in a mixed population.

 

Figure 2
View larger version (49K):
[in this window]
[in a new window]

  		FIG. 2. (A) PCR analysis of Kmr D. vulgaris transformants using primers targeting regions outside of the fur knockout cassette (P7/P8). Lane 1, wild type; lane 2, transformant A; lane 3, transformant B; lane 4, pMO707; lane 5, no DNA; lane M, 1-kb marker. An increase in product size from 2,168 bp to 2,839 bp indicates exchange of the fur gene for the Kmr determinant, creating JW707. (B) Northern analyses of total RNA (10 µg) from JW707. Lane 1, wild type; lane 2, JW707; lane M, RNA marker. Probes used for hybridization are indicated above the blot. nt, nucleotides.

 
Northern analysis.
RNAs from exponential-phase (OD600 ~ 0.4) wild-type and JW707 cultures grown in standard LS4D medium were analyzed for the expression of the fur gene as well as other genes predicted to be regulated by iron (61): fld (DVU2680; flavodoxin), sodB (DVU2410; superoxide dismutase), and feoA (DVU2572; ferrous iron transporter) (Fig. 2B). Hybridization analysis with the 334-bp fur probe indicated the presence of a single ~500-bp transcript for the wild type, but as expected, no signal was present in the JW707 lane (Fig. 2B, lane 2). This transcript size correlates with the predicted fur ORF, including a possible promoter sequence. For the fld gene, a weak band corresponding to a ~650-nucleotide transcript was observed for the wild type, while an intense band was present for JW707. While the fld ORF was annotated to be 447 bp, the Fur binding site was predicted to be 182 bp upstream (61). This hybridization signal is indicative of a sharp increase in expression. No transcript was evident for the sodB gene in either the wild-type or the JW707 samples, indicating expression at a level below detection (Fig. 2B). Results from the feoA probe indicated a large signal smear for JW707, with limited hybridization for the wild type. This lack of hybridization specificity may be due to the operon of which feoA is a part (DVU2571 to DVU2572), as well as the two genome copies of the feoA gene (DVU2572 and DVU2574). Predicted operon arrangements for the targeted genes are indicated in Table 4.


View this table:
[in this window]
[in a new window]

  		TABLE 4. Effect of {Delta}fur on expression of selected genes in D. vulgaris Hildenborougha

 
Response to iron.
Growth curves for both the wild type and JW707 in Yen45 (30 µM FeCl2) were identical, indicating that the fur gene is not essential for anaerobic dissimilatory sulfate reduction by D. vulgaris under iron-replete conditions (Fig. 3A). To test the effect of iron limitation on the fur mutant, growth was analyzed in Yen45 medium containing 1 and 6 µM added FeCl2. Inocula used in this experiment were 2% (vol/vol) of active cultures in medium containing 1 µM added FeCl2 to limit carryover of excess iron. Resulting growth curves for both iron levels were similar between the wild type and JW707 (data not shown). The Fur mutant did not exhibit a growth advantage over the wild type when iron was limiting.


Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 3. Growth curves of the D. vulgaris wild type (open symbols) and mutant strain JW707 (filled symbols) under various conditions. (A) Growth under iron-replete conditions (30 µM FeCl2). (B) Response to osmolarity stress, with sodium as 300 mM NaCl (circles) or 300 mM NaCl plus 2 mM glycine betaine (squares). (C) Response to osmolarity stress, with potassium as 300 mM KCl (circles) or 300 mM KCl plus 2 mM glycine betaine (squares). (D) Response to nitrite at 2 mM NaNO2 (circles) and 5 mM NaNO2 (squares). Curves are representative of three trials at 37°C.

 
Metal sensitivity.
Since a common trait of bacterial {Delta}fur mutants is increased resistance to Mn(II) (8, 30, 32, 40, 55), the effect of increasing concentrations of MnCl2 on the growth of both wild-type and JW707 strains was tested. Excessive precipitation caused by the addition of MnCl2 to the growth medium (data not shown) mandated that CFU be monitored as an indicator of Mn(II) effects on growth. Following 112 h of exposure to 20, 30, or 40 mM MnCl2, cultures were plated on Yen45 medium. Wild-type untreated cultures had more CFU than untreated JW707; however, with 40 mM MnCl2, the number of wild-type CFU declined from 1.3 x 108 to 1.4 x 105 (Fig. 4). By comparison, the numbers of CFU for JW707 achieved in cultures exposed to MnCl2 were similar, with 3.7 x 107 CFU in the untreated control and 1.8 x 107 CFU after exposure to 40 mM MnCl2 (Fig. 4).


Figure 4
View larger version (9K):
[in this window]
[in a new window]

  		FIG. 4. Growth response of the D. vulgaris wild type (open symbols) and mutant strain JW707 (filled symbols) to MnCl2 treatment. Curves are representative of three trials at 37°C.

 
Response to osmolarity shock.
Previous studies with wild-type D. vulgaris indicated that salt stress reduced growth rate and increased cell length and that these effects could be countered by addition of the osmoprotectant glycine betaine (46). Interestingly, the putative Fur regulon members were among the most highly differentially transcribed genes during stress with NaCl or KCl (46) Therefore, the response of the {Delta}fur mutant to salt stress was tested. Growth curves of cultures in medium supplemented with 300 mM additional NaCl or KCl (Fig. 3B and C, respectively) were similar and growth rates decreased for both the wild type and JW707. Microscopic observations of the salt-stressed cultures showed that 91.4% ± 2.8% (standard deviation) of wild-type cells were elongated from 1.5 µm to 5 µm or more. Although JW707 cells were not counted, the fraction elongated appeared to be similar to that of the wild type. Addition of 2 mM glycine betaine with the salt protected the wild type from the decrease in growth rate (Fig. 3B and C) and restored the cell morphology to normal. However, the growth rate of JW707 remained 75% of the rate of uninhibited cells regardless of the presence of 2 mM glycine betaine. Interestingly, microscopic observations indicated that the osmolyte apparently restored the mutant cell lengths to ~1.5 µM (data not shown).

Response to nitrate/nitrite.
Tests of JW707 growth responses to nitrate or nitrite stress were prompted by data from proteomic and microarray analyses of wild-type D. vulgaris (35, 59). The transcript analyses indicated upregulation of both the fur regulon and genes predicted to be involved in iron binding, particularly under nitrite stress. Significant differences in growth between the wild type and JW707 were not observed in Yen45 medium containing 50 mM NaNO3 (data not shown). Increasing this NaNO3 concentration to 100 mM did not have an effect on the final growth extent, but a 60-h lag phase for the wild type and an 80-h lag phase for JW707 did occur (data not shown). Growth of the wild type in Yen45 medium supplemented with 2 mM NaNO2 resulted in a lag phase of 60 h, whereas no growth was detected in medium supplemented with 5 mM NaNO2 after 117 h (Fig. 3D). In contrast, both 2 and 5 mM NaNO2 completely inhibited the growth of JW707. Since comparable cell numbers were used as inocula for the parent and mutant, the results indicate an increased sensitivity to nitrite by the {Delta}fur mutant.

Expression profile of JW707.
Transcriptional arrays covering 3,482 of the 3,531 (98.6%) protein coding sequences in the D. vulgaris genome were used to identify genes affected by deletion of the fur gene. In addition, the effect of iron concentration on the global transcription of the fur mutant was also determined by performing two separate experiments with growth medium containing 60 µM and 5 µM added FeCl2. Samples were taken at similar optical densities for both JW707 and the parental wild-type strain, with differential gene expression calculated as log2 ratios using the following formula: log2(transcripts of JW707) – log2(transcripts of wild type). Following normalizations for signal intensities (13) and sector-based artifacts, the significance of the ratios was calculated as a Z score. Generally, ratios of ≥ 1.6 (≥3-fold change in expression) were selected for further analysis.

Evidence that Fur is a global regulator in D. vulgaris derives from the observation that changes in gene expression with fur deleted were identified in 12 functional categories based on the annotation by The Institute for Genomic Research. Transcript analysis revealed 34 and 50 genes differentially expressed at least threefold in response to the fur deletion (compared to the wild type) under iron-replete (60 µM) and iron-limited (5 µM) conditions, respectively (Fig. 5A). Comparison of the two data sets indicated that expression levels for 13 genes were affected under both iron conditions (Table 4). Under iron-replete conditions, 30 genes were up-regulated and 4 genes were down-regulated, with 44% of the total population predicted to encode hypothetical or conserved hypothetical proteins. Under iron-limiting conditions, 32 genes were up-regulated and 18 genes were down-regulated, with 30% of the total population predicted to encode hypothetical or conserved hypothetical proteins.


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 5. (A) Genes differentially expressed threefold in the {Delta}fur mutant, JW707, compared to wild-type cells, under iron-replete (+Fe; 60 µM) and iron-limited (–Fe; 5 µM) conditions. (B) Overlap of genes differentially expressed threefold in JW707 iron-limited (–Fe; 5 µM) conditions compared with JW707 iron-replete conditions versus those differentially expressed in iron-limited wild-type cells compared to iron-replete wild-type cells.

 
When JW707 responses to limiting iron were compared to its responses in iron-replete conditions, 22 genes were increased in expression, compared to 22 that were decreased. In contrast, when the wild type was subjected to limited iron, 50 genes were increased in transcription and only 18 were decreased relative to iron-replete cells (Fig. 5B). Thus, the genes differentially expressed in JW707 may be candidates for iron regulation. Genes expressed differently in the wild type should include those regulated by Fur and those regulated by iron concentration. Curiously, only three genes meeting the stringent criterion of a threefold increase in expression were common to iron-restricted JW707 and iron-restricted wild type: these were annotated as rubrerythrin (rbr2; DVU2318), chemotaxis protein (cheY2; DVU2073) (Table 5, category IV), and an ABC transporter permease protein (DVU2385) (Table 4).


View this table:
[in this window]
[in a new window]

  		TABLE 5. D. vulgaris Hildenborough transcriptional responses to {Delta}fur and FeCl2 concentrations suggesting different regulatory systems

 
Transcriptional profile of the predicted Fur regulon.
Computational analysis of the upstream regions of feoAB operons from multiple {delta}-proteobacterial genomes revealed a conserved 17-bp palindromic motif corresponding to a putative Fur binding site (61). A genomic scan of the D. vulgaris genome with this motif revealed 17 sites upstream of eight genes or operons, constituting the putative Fur regulon (61). The prediction that these sites are operator sites for Fe(II)-bound Fur appears to have been supported by some of the transcriptional responses of the wild type compared to those of the Fur mutant (Table 4). Comparison of the transcripts from the wild type under iron-limited versus iron-replete conditions also supported the prediction that Fur is not autogenously regulated in D. vulgaris (61) (data not shown). The lack of correlation between expression changes in the Fur regulon and the fur gene in microarray data from stressed D. vulgaris cells (35, 46) also suggests that fur is not autogenously controlled.

The DVU2681 gene, encoding a hypothetical 60-amino-acid protein, exhibited the most up-regulation in the transcriptional profiling of JW707: log2 ratios of 5.31 and 2.30 under iron-replete and -restricted conditions, respectively. It should be noted that DVU2681 is located directly downstream of the flavodoxin gene (DVU2680) that is annotated as "iron repressed" and, by Northern analysis, shown to be greatly increased in transcription in the {Delta}fur mutant (Fig. 2B). However, DVU2681 is transcribed in the opposite orientation. Other putative Fur-regulated operons, such as those encoding the annotated GGDEF domain protein (DVU0763) and the HD domain protein (DVU3123) (Table 4), did not appear to respond to decreased iron concentration. In the putative genYZ operon, only genZ (DVU0303) expression was consistent with iron-bound Fur repression. The latter two genes were predicted to be members of a Fur regulon unique to metal-reducing {delta}-proteobacteria, though possible functions are unknown (61). In contrast, the hypothetical iron-regulated P-type ATPase gene (DVU3330) that was predicted to be monocistronic responds as if it were part of an operon of two to four genes (Table 4). A similar expansion of Fur or iron influence on expression can be seen downstream of the foxR regulatory gene (DVU2378), which appears to include 12 genes (Table 4). This cluster is composed of ABC transporters (DVU2380, DVU2384 to DVU2387), the biopolymer transporters (TolRQ; DVU2388 to DVU2389), and an energy-generating mechanism for transporting polymers across the membrane (TonB [DVU2390] and a TonB receptor [DVU2383]). While these genes have been shown to be involved in iron acquisition via siderophore production and transport in many bacteria (39, 44, 56), D. vulgaris has not been shown to produce siderophores (unpublished data), nor have genes for siderophore biosynthesis been recognized in the genome. Whether the transcriptional changes are a direct response to Fur deletion or are mediated by influences on activities of other regulators, such as FoxR, will need to be addressed with further experimentation.

Other iron-related genes.
Transcriptional responses of genes that might be predicted to be involved with iron metabolism from the reported roles of their orthologs in other bacteria were examined (data not shown). Expression profiles of a putative siderophore uptake system encoded by DVU0650 to DVU0646 and of two iron storage proteins, bacterioferritin (DVU1397) and ferritin (DVU1568), did not support a role for Fur regulation or a clear response to iron concentrations. Two proteins requiring iron for function, Fe hydrogenase (DVU1771) and ferredoxin II (DVU0305), were also not significantly altered in expression in the absence of Fur.

Selected genes believed to be involved in the oxidative stress response of D. vulgaris were also examined for transcriptional responses in the deletion mutant (data not shown). Only the relative transcription for the gene annotated to encode alkyl hydroperoxide reductase C (DVU2247) was consistent with iron-bound Fur-dependent repression, although the changes did not meet our cutoff for significance. This gene product is reported to reduce hydrogen peroxide and to protect the cell from reactive oxygen species formed while iron acquisition systems are induced (inactive Fur repressor). Changes in the expression of genes for the putative cytochrome d ubiquinol oxidase (DVU3270 to DVU3271), superoxide dismutase (DVU2410), and catalase (DVUA0091) were not consistent with classical Fur-mediated regulation. As has been shown in other systems (4, 11, 25), the catalase gene transcription was increased by limiting iron, an example of regulation of a gene located on the megaplasmid in D. vulgaris.

Other potential modes of maintaining iron homeostasis.
Iron-responsive regulation through the Fe(II)-bound Fur repressor is the most commonly identified microbial mechanism for maintaining iron homeostasis (31). However, a second Fe(II)-responsive repressor, DtxR, has been identified, primarily in gram-positive bacteria (31), and a role for Fe(III)-specific two-component regulatory systems has been recognized (56). From sequence analysis of D. vulgaris, homology to DtxR proteins has not been recognized, nor have Fe(III)-specific histidine kinases and response regulators been annotated. However, the expression of a number of genes would be consistent with alternative modes of regulation (Table 5), as have been identified recently for Helicobacter pylori (14, 21). Category I illustrates the predicted pattern for Fe(II)-bound Fur regulation: increased expression in the absence of Fur in plentiful iron and when Fe(II) is limiting in wild-type cells. Other candidates are shown in Table 4. The two hypothetical genes shown in category I (Table 5) are highly regulated, but possible functions remain obscure. Category II is exemplified by amino acid biosynthesis genes for tryptophan and for methionine. These genes are up-regulated in the absence of Fur and low iron and decreased in expression in the presence of Fur and low iron, a pattern consistent with repression by iron-free Fur. Category III lists a few examples of genes that exhibit expression patterns that could be compatible with iron-free Fur induction. In limiting iron, low levels of expression are observed in the absence of Fur, but high levels of expression are measured when Fur is present. In wild-type cells with 5 µM FeCl2, Fur would induce but the addition of 60 µM FeCl2 would prevent induction. The apparent positive regulation by iron-free Fur in D. vulgaris requires confirmation before mechanisms are sought.

Regulation in response to iron concentrations that is not mediated by Fur may also be apparent in D. vulgaris. Categories IV and V show transcription patterns that seem consistent with iron repression and iron induction, respectively. For iron repression, candidate regulated genes were increased in expression in limiting iron regardless of the presence of Fur. For iron induction, the opposite pattern of transcript levels was seen. The two genes whose expression was found to be increased over threefold in both iron-limited and iron-replete conditions, rbr2 and DVU2541, are among those in category IV (Table 5). Curiously, category V includes several genes present on the 200-kb megaplasmid in wild-type D. vulgaris.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Genetic system.
Here we describe a variation on the genetic system for D. vulgaris (26). We constructed the knockout cassette by fusion PCR to delete target genes via double recombination following electroporation. The development of this gene deletion method for Desulfovibrio was necessitated by a gene reversion event observed following plasmid interruption of the cycA gene in D. desulfuricans G20 (57). Due to the possibility of verifying deletion mutants within 2 weeks following cassette construction, this method can be used for rapid production of D. vulgaris deletion mutants. In 1997, Fu and Voordouw described the first case of targeted gene deletion in D. vulgaris (26). This method is dependent on Cmr selection and SacB counterselection following two separate recombination events and has been successful in generating at least 11 D. vulgaris deletion mutants (reference 5 and references therein; 75). Because the SacB counterselection procedure involves DNA transfer by conjugation as well as lengthy transformant screening, its use in high-throughput mutant construction is not practical.

A clean selection method is also key for the success of a genetic system, and developing such a method for Desulfovibrio has been problematic due to broad-range antibiotic resistance. While the method described by Fu and Voordouw utilizes the Cmr determinant (26), chloramphenicol resistance has been problematic in our hands. We have found that 400 µg of G418 (gentamicin) per ml of LS4D medium provides clean selection when the Kmr determinant is used. The utility of this method is illustrated by the 1.25 x 10–7 transformation efficiency obtained and the successful deletion of the fur gene in all three of the transformants screened via phenotypic (MnCl2 resistance) and molecular (PCR and Southern analysis) methods.

The method described here is also dependent on DNA delivery by electroporation, which had not been successful in D. vulgaris until now. We added a high concentration of deletion vector as well as lambda DNA to an electroporation procedure first described for Desulfovibrio fructosivorans (64). The intrinsic nucleases of D. vulgaris likely linearize the plasmid and promote double recombination events and thus marker exchange between the vector and genome (5). Other deletion mutants screened in our laboratory using this deletion procedure follow the same pattern (data not shown).

Physiology of JW707.
The similar decreases in growth rate for both the wild type and JW707 with 5 µM added FeCl2 indicated that both cultures were limited for iron, but not differentially (data not shown). Thus, regardless of iron concentration, growth of the {Delta}fur mutant was neither inhibited nor promoted by the derepression of putative ferrous iron uptake genes (feoAB) (Fig. 2B and Table 4). Similarly, neither Dichelobacter nodosus nor Shewanella oneidensis Fur mutants exhibited a detectable phenotype when grown anaerobically (52, 71). Without oxygen present, toxic radical formation resulting from increased ferrous iron and the Fenton reaction would be less likely. However, further tests are needed to determine if the cellular iron content of the D. vulgaris Fur mutant differs from that of the wild type.

Osmotic stress.
Salt stress in Bacillus subtilis has been shown to induce iron acquisition systems; therefore, salinity has been proposed to cause iron limitation (37, 70). Analysis of the D. vulgaris transcriptome and proteome under both NaCl and KCl stress indicated the same phenomenon, Fur regulon induction in response to salt stress (46). In contrast, physiological studies indicated growth inhibition of JW707 equivalent to that of the wild type upon exposure to 300 mM NaCl or KCl (Fig. 3). Therefore, increased expression of the Fur regulon is not sufficient for overcoming this osmotic stress in D. vulgaris.

NO3–/NO2– stress.
The effect of nitrate on D. vulgaris growth is of specific concern for environmental bioremediation applications. High levels of nitrate in uranium-contaminated have been documented sites (http://www.esd.ornl.gov/nabirfrc/) (20), and nitrate is believed to have an inhibitory affect on sulfate reduction through the intermediate nitrite (42, 49, 50). A previous proteomic analysis of wild-type D. vulgaris showed that the presence of 105 mM NaNO3 induced proteins involved in the ionic stress response (59), although transcriptional profiling has indicated a response unique to nitrate that is not a composite of salt and nitrite (unpublished data).

The increased sensitivity to nitrite stress for JW707 was unexpected based on previous transcript analyses of wild-type D. vulgaris (35). An increased expression of the Fur regulon in the wild type was observed under nitrite stress (35). Therefore, derepression of the regulon through deletion of the repressor was expected to provide an advantage for JW707 exposed to nitrite. However, the Fur mutant was completely inhibited by 5 mM NaNO2 (Fig. 3D). Like JW707, an Escherichia coli {Delta}fur mutant was also more sensitive to NaNO2 regardless of the upregulation of genes involved in iron uptake (48). This increased expression of the Fur regulon following 1 mM NaNO2 treatment was hypothesized to be a consequence of NO nitrosylation of the Fe(II) in Fur that inactivated the repressor and likely other iron-containing genes (15). Direct evidence for this mechanism of iron modification in nitrite-treated D. vulgaris remains to be obtained.

Transcriptional profile of JW707.
While the overall physiology of D. vulgaris was not dramatically affected by deleting the fur gene, a diverse transcriptional response occurred for JW707 compared to the wild type. In many bacteria, Fur negatively regulates siderophore production and transport (reference 67 and references therein). Despite the up-regulation of a 12-gene cluster containing genes predicted to be involved in siderophore uptake (e.g., TolRQ) (Table 4), no genes for siderophore production have been annotated in the D. vulgaris genome. This may allow D. vulgaris to save energy by simply stealing iron-complexed siderophores produced by other bacteria, as suggested for the spirochete Leptospira biflexa (43). In other bacteria, similar TonB/ABC and biopolymer uptake systems have also been shown to transport vitamin B12, phages, colicins, and maltodextrins into the cell (51, 54). Further studies are needed to determine what role the large (12-gene) cluster plays in D. vulgaris.

The high level of differential expression for the feoAB operon, apparently regulated by Fur and its corepressor Fe(II) (Fig. 2 and Table 4), is interpreted as evidence that the FeoAB system is the primary iron uptake mechanism in D. vulgaris. This follows from the prediction that Fe(II) predominates in anaerobic environments. However, genes predicted to be involved in iron storage, such as bfr and ftn, did not exhibit a strong transcriptional response to either iron concentration or the deletion of the fur gene.

Oxidative stress.
The Fur regulator has been shown to be intricately involved in the oxidative stress response of some bacteria (2, 33, 34). Thus, it can be argued that regulation by Fur is linked to an increased uptake of iron that can generate reactive oxygen species upon oxygen exposure. Despite the anaerobic lifestyle of D. vulgaris, it is known to survive exposure to oxygen and contains several oxidative stress response genes within its genome. As such, it was not surprising to find that ahpC, rdl, cydA, and katA were differentially expressed at low levels in this study. However, only ahpC appeared to be classically regulated by iron-bound Fur (data not shown). The diverse regulatory pattern of the other oxidative stress response genes may be explained by coregulation by PerR, a homolog to Fur that also responds to iron concentration (9, 11). In fact, ahpC and rdl are predicted to possess a PerR binding site within their promoter regions (61). Coregulation of katA and ahpC by Fur and PerR has also been proposed in the microaerophilic Campylobacter jejuni (3, 38, 73). Further studies are needed to determine if Fur and PerR regulation overlap in D. vulgaris.

Other mechanisms of Fur regulation.
In addition to the traditional repressor role of iron-bound Fur, alternative activation and repression by iron-free Fur were inferred from the transcription patterns seen in D. vulgaris. These two forms of regulation have recently been described in Helicobacter pylori (14, 18, 22). While iron-bound Fur has been shown in the literature to be a positive regulator, it is possible that some of the affected genes are actually repressed by iron-free Fur. This form of Fur regulation was described for the pfr (which encodes a ferritin) and sodB genes of H. pylori (18, 22). Some of the genes for which iron-free Fur repression is consistent in D. vulgaris are the seven genes of the tryptophan operon and metK (Table 5).

Alternatively, the transcription of flagellar genes (DVU1443 to DVU1445) appeared to be consistent with induction by iron-free Fur in D. vulgaris, and DVU1444 expression changes met the criteria used as an example in this category (Table 5). This form of positive regulation by iron-free Fur has been reported only for flaB, a major flagellin gene, in H. pylori (14). It is tempting to infer that motility may be decreased in the presence of plentiful iron for both D. vulgaris and H. pylori. Another example of positive regulation by Fur is activation by iron-bound Fur, suggested for a collection of Neisseria meningitidis genes involved in both aerobic and anaerobic respiration (17). However, expression patterns suggesting this form of regulation were not observed in this study.

Iron regulation independent of Fur.
Differential expression of genes that is independent of Fur implies that other regulators may respond to iron concentrations within the cell. Possible examples of these regulators include PerR, DtxR, NikR, Irr, and RirA (6, 9, 10, 14, 16, 28). However, only perR has been annotated in the D. vulgaris genome (61), with DVU2318 (encoding a rubrerythrin) being the only gene in the iron-responsive category predicted to be PerR regulated (Table 5) (61). Thus, it appears that other iron-responsive regulators may be present in D. vulgaris but have not yet been identified. Based on the iron-dependent regulation of megaplasmid-borne genes (Table 5), these iron-responsive regulators may not be limited to the D. vulgaris chromosome but may also be present on the megaplasmid. However, preliminary searches for regulatory motifs upstream of genes showing similar differential expression patterns have been unsuccessful. This is not too surprising because of the small number of candidate genes involved in each category, although both the genes from D. vulgaris and the orthologs from D. desulfuricans G20 were included in the search (data not shown).

In summary, both the physiological and microarray data indicate a global regulatory role for Fur in D. vulgaris, including involvement in the osmotic and nitrite stress responses. While a traditional repressor role for iron-bound Fur was observed, expression data also suggest activation by iron-free Fur. The patterns of apparent transcription regulation presented here require further confirmation but indicate that alternate models of iron homeostasis may be functioning in D. vulgaris and possibly anaerobes in general. Specifically, studies to determine the roles of iron-free Fur and the TonB/TolQR system in addition to identification of other iron-dependent regulators are needed. These issues are especially pertinent because of the differential expression of the D. vulgaris Fur regulon upon exposure of the cells to various environmental stressors (35, 46, 74). Information from these studies may prove integral in the design of future bioremediation strategies for the removal of heavy metals.


   ACKNOWLEDGMENTS
 
This work was part of the Virtual Institute for Microbial Stress and Survival (http://vimss.lbl.gov), supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomics Program: GTL through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.

We thank Barbara Giles for sharing her technical expertise regarding Desulfovibrio mutant construction.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry, 117 Schweitzer Hall, Columbia, MO 65211. Phone: (573) 882-8726. Fax: (573) 882-5635. E-mail: wallj{at}missouri.edu Back

{triangledown} Published ahead of print on 13 July 2007. Back

{dagger} Present address: Department of Microbiology, Southern Illinois University, Carbondale, IL 62901. Back

http://vimss.lbl.gov. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Andrews, S. C. 1998. Iron storage in bacteria. Adv. Microb. Physiol. 40:281-351.[Medline]
  2. Andrews, S. C., A. K. Robinson, and F. Rodriguez-Quinones. 2003. Bacterial iron homeostasis. FEMS Microbiol. Lett. 27:215-237.[CrossRef]
  3. Baillon, M.-L. A., A. H. M. van Vliet, J. M. Ketley, C. Constantinidou, and C. W. Penn. 1999. An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni. J. Bacteriol. 181:4798-4804.[Abstract/Free Full Text]
  4. Barriere, C., R. Bruckner, D. Centeno, and R. Talon. 2002. Characterisation of the katA gene encoding a catalase and evidence for at least a second catalase activity in Staphylococcus xylosus, bacteria used in food fermentation. FEMS Microbiol. Lett. 216:277-283.[Medline]
  5. Bender, K. S., H.-C. Yen, and J. D. Wall. 2006. Analysing the metabolic capabilities of Desulfovibrio species through genetic manipulation. Biotechnol. Genet. Eng. Rev. 23:157-174.
  6. Boyd, J., M. N. Oza, and J. R. Murphy. 1990. Molecular cloning and DNA-sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae. Proc. Natl. Acad. Sci. USA 87:5968-5972.[Abstract/Free Full Text]
  7. Brandis, A., and R. K. Thauer. 1981. Growth of Desulfovibrio species on hydrogen and sulfate as sole energy source. J. Gen. Microbiol. 12:249-252.
  8. Brickman, T. J., and S. K. Armstrong. 1995. Bordetella pertussis fur gene restores iron repressibility of siderophore and protein expression to deregulated Bordetella bronchiseptica mutants. J. Bacteriol. 177:268-270.[Abstract/Free Full Text]
  9. Bsat, N., A. Herbig, L. Casillas-Martínez, P. Setlow, and J. D. Helmann. 1998. Bacillus subtilis contains multiple Fur homologues; identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors. Mol. Microbiol. 29:189-198.[CrossRef][Medline]
  10. Chao, T.-C., J. Buhrmester, N. Hansmeier, A. Puhler, and S. Weidner. 2005. Role of the regulatory gene irrA in the transcriptional response of Sinorhizobium meliloti to iron limitation. Appl. Environ. Microbiol. 71:5969-5982.[Abstract/Free Full Text]
  11. Chen, L., L. Keramati, and J. D. Helmann. 1995. Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions. Proc. Natl. Acad. Sci. USA 92:8190-8194.[Abstract/Free Full Text]
  12. Chhabra, S. R., Q. He, K. H. Huang, S. P. Gaucher, E. J. Alm, Z. He, M. Z. Hadi, T. C. Hazen, J. D. Wall, A. P. Arkin, and A. K. Singh. 2006. Global analysis of heat shock response in Desulfovibrio vulgaris Hildenborough. J. Bacteriol. 188:1817-1828.[Abstract/Free Full Text]
  13. Colantuoni, C., G. Henry, S. Zeger, and J. Pevsner. 2002. Local mean normalization of microarray element signal intensities across an array surface: quality control and correction of spatially systematic artifacts. BioTechniques 32:1316-1320.[Medline]
  14. Danielli, A., D. Roncarati, I. Delany, V. Chiarini, R. Rappuoli, and V. Scarlato. 2006. In vivo dissection of the Helicobacter pylori Fur regulatory circuit by genome-wide location analysis. J. Bacteriol. 188:4654-4662.[Abstract/Free Full Text]
  15. D'Autreaux, B., D. Touati, B. Bersch, J. M. Latour, and I. Michaud-Soret. 2002. Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron. Proc. Natl. Acad. Sci. USA 99:16619-16624.[Abstract/Free Full Text]
  16. Delany, I., R. Ieva, A. Soragni, M. Hilleringmann, R. Rappuoli, and V. Scarlato. 2005. In vitro analysis of protein-operator interactions of the NikR and Fur metal-responsive regulators of coregulated genes in Helicobacter pylori. J. Bacteriol. 187:7703-7715.[Abstract/Free Full Text]
  17. Delany, I., R. Rappuoli, and V. Scarlato. 2004. Fur functions as an activator and as a repressor of putative virulence genes in Neisseria meningitidis. Mol. Microbiol. 52:1081-1090.[CrossRef][Medline]
  18. Delany, I., G. Spohnn, R. Rappuoli, and V. Scarlato. 2001. The Fur repressor controls transcription of iron-activated and -repressed genes in Helicobacter pylori. Mol. Microbiol. 42:1297-1309.[CrossRef][Medline]
  19. Dos Santos, W. G., I. Pacheco, M. Y. Liu, M. Teixeira, A. V. Xavier, and J. Le Gall. 2000. Purification and characterization of an iron superoxide dismutase and catalase from the sulfate-reducing bacterium Desulfovibrio gigas. J. Bacteriol. 182:796-804.[Abstract/Free Full Text]
  20. Elias, D. A., L. R. Krumholz, D. Wong, P. E. Long, and J. M. Suflita. 2003. Characterization of microbial activities and U reduction in a shallow aquifer contaminated by uranium mill tailings. Microb. Ecol. 46:83-91.[CrossRef][Medline]
  21. Ernst, F. D., S. Bereswill, B. Waidner, J. Stoof, U. Mäder, J. G. Kusters, E. J. Kuipers, M. Kist, A. H. M. van Vliet, and G. Homuth. 2005. Transcriptional profiling of Helicobacter pylori Fur- and iron-regulated gene expression. Microbiology 151:533-546.[Abstract/Free Full Text]
  22. Ernst, F. D., G. Homuth, J. Stoof, U. Mäder, B. Waidner, E. J. Kuipers, M. Kist, J. Kusters, S. Bereswill, and A. H. M. van Vliet. 2005. Iron-responsive regulation of the Helicobacter pylori iron-cofactored superoxide dismutase SodB is mediated by Fur. J. Bacteriol. 187:3687-3692.[Abstract/Free Full Text]
  23. Escolar, L., J. Perez-Martin, and V. de Lorenzo. 1999. Opening the iron box: transcriptional metalloregulation by the Fur protein. J. Bacteriol. 181:6223-6229.[Free Full Text]
  24. Frazão, C., G. Silva, C. M. Gomes, P. Matias, R. Coelho, L. Sieker, S. Macedo, M. Y. Liu, S. Oliveira, M. Teixeira, A. V. Xavier, C. Rodrigues-Pousada, M. A. Carrondo, and J. L. Gall. 2000. Structure of a dioxygen reduction enzyme from Desulfovibrio gigas. Nat. Struct. Biol. 7:1041-1045.[CrossRef][Medline]
  25. Frederick, J. R., J. G. Elkins, N. Bollinger, D. J. Hassett, and T. R. McDermott. 2001. Factors affecting catalase expression in Pseudomonas aeruginosa biofilms and planktonic cells. Appl. Environ. Microbiol. 67:1375-1379.[Abstract/Free Full Text]
  26. Fu, R., and G. Voordouw. 1997. Targeted gene-replacement mutagenesis of dcrA, encoding an oxygen sensor of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. Microbiology 143:1815-1826.[Abstract]
  27. Giaever, G., A. M. Chu, L. Ni, C. Connelly, L. Riles, S. Veronneau, S. Dow, A. Lucau-Danila, K. Anderson, B. Andre, A. P. Arkin, A. Astromoff, M. El-Bakkoury, R. Bangham, R. Benito, S. Brachat, S. Campanaro, M. Curtiss, K. Davis, A. Deutschbauer, K. D. Entian, P. Flaherty, F. Foury, D. J. Garfinkel, M. Gerstein, D. Gotte, U. Guldener, J. H. Hegemann, S. Hempel, Z. Herman, D. F. Jaramillo, D. E. Kelly, S. L. Kelly, P. Kotter, D. LaBonte, D. C. Lamb, N. Lan, H. Liang, H. Liao, L. Liu, C. Luo, M. Lussier, R. Mao, P. Menard, S. L. Ooi, J. L. Revuelta, C. J. Roberts, M. Rose, P. Ross-Macdonald, B. Scherens, G. Schimmack, B. Shafer, D. D. Shoemaker, S. Sookhai-Mahadeo, R. K. Storms, J. N. Strathern, G. Valle, M. Voet, G. Volckaert, C. Y. Wang, T. R. Ward, J. Wilhelmy, E. A. Winzeler, Y. Yang, G. Yen, E. Youngman, K. Yu, H. Bussey, J. D. Boeke, M. Snyder, P. Philippsen, R. W. Davis, and M. Johnston. 2002. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387-391.[CrossRef][Medline]
  28. Hamza, I., Z. Qi, N. D. King, and M. R. O'Brian. 2000. Fur-independent regulation of iron metabolism by Irr in Bradyrhizobium japonicum. Microbiology 146:669-676.[Abstract/Free Full Text]
  29. Hantke, K. 2001. Iron and metal regulation in bacteria. Curr. Opin. Microbiol. 4:172-177.[CrossRef][Medline]
  30. Hantke, K. 1987. Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K12: fur not only affects iron metabolism. Mol. Gen. Genet. 210:135-139.[CrossRef][Medline]
  31. Hantke, K., and V. Braun. 2000. The art of keeping low and high iron concentrations in balance, p. 275-288. In G. Storz and R. Hengge-Aronis (ed.), Bacterial stress responses. ASM Press, Washington, D.C.
  32. Haraszthy, V. I., S. F. Jordan, and J. J. Zambon. 2006. Identification of Fur-regulated genes in Actinobacillus actinomycetemcomitans. Microbiology 152:787-796.[Abstract/Free Full Text]
  33. Harris, A. G., F. E. Hinds, A. G. Beckhouse, T. Kolesnikow, and S. L. Hazell. 2002. Resistance to hydrogen peroxide in Helicobacter pylori: role of catalase (KatA) and Fur, and functional analysis of a novel gene product designated ‘KatA-associated protein’, KapA (HP0874). Microbiology 148:3813-3825.[Abstract/Free Full Text]
  34. Hassett, D. J., P. A. Sokol, M. L. Howell, J. F. Ma, H. T. Schweizer, U. Ochsner, and M. L. Vasil. 1996. Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities. J. Bacteriol. 178:3996-4003.[Abstract/Free Full Text]
  35. He, Q., K. H. Huang, Z. He, E. J. Alm, M. W. Fields, T. C. Hazen, A. P. Arkin, J. D. Wall, and J. Zhou. 2006. Energetic consequences of nitrite stress in Desulfovibrio vulgaris Hildenborough, inferred from global transcriptional analysis. Appl. Environ. Microbiol. 72:4370-4381.[Abstract/Free Full Text]
  36. Heidelberg, J. F., R. Seshadri, S. A. Haveman, C. L. Hemme, I. T. Paulsen, J. F. Kolonay, J. A. Eisen, N. Ward, B. Methe, L. M. Brinkac, S. C. Daugherty, R. T. Deboy, R. J. Dodson, A. S. Durkin, R. Madupu, W. C. Nelson, S. A. Sullivan, D. Fouts, D. H. Haft, J. Selengut, J. D. Peterson, T. M. Davidsen, N. Zafar, L. Zhou, D. Radune, G. Dimitrov, M. Hance, K. Tran, H. Khouri, J. Gill, T. R. Utterback, T. V. Feldblyum, J. D. Wall, G. Voordouw, and C. M. Fraser. 2004. The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. Nat. Biotechnol. 22:1-6.[CrossRef][Medline]
  37. Hoffmann, T., A. Schütz, M. Brosius, A. Völker, U. Völker, and E. Bremer. 2002. High-salinity-induced iron limitation in Bacillus subtilis. J. Bacteriol. 184:718-727.[Abstract/Free Full Text]
  38. Holmes, K., F. Mulholland, B. M. Pearson, C. Pin, J. McNicholl-Kennedy, J. M. Ketley, and J. M. Wells. 2005. Campylobacter jejuni gene expression in response to iron limitation and the role of Fur. Microbiology 151:243-257.[Abstract/Free Full Text]
  39. Köster, W. 2001. ABC transporter-mediated uptake of iron, siderophores, heme, and vitamin B12. Res. Microbiol. 152:291-301.[Medline]
  40. Lam, M. S., C. M. Litwin, P. A. Carroll, and B. Calderwood. 1994. Vibrio cholerae fur mutations associated with loss of repressor activity: implications for the structural-functional relationships of fur. J. Bacteriol. 176:5108-5115.[Abstract/Free Full Text]
  41. Lee, J. W., and J. D. Helmann. 2006. The PerR transcription factor senses H2O2 by metal-catalysed histidine oxidation. Nature 440:363-367.[CrossRef][Medline]
  42. Londry, K. L., and J. M. Suflita. 1999. Use of nitrate to control sulfide generation by sulfate-reducing bacteria associated with oily waste. J. Ind. Microbiol. Biotechnol. 22:582-589.[CrossRef][Medline]
  43. Louvel, H., I. Saint Girons, and M. Picardeau. 2005. Isolation and characterization of FecA- and FeoB-mediated iron acquisition systems of the spirochete Leptospira biflexa by random insertional mutagenesis. J. Bacteriol. 187:3249-3254.[Abstract/Free Full Text]
  44. Moeck, G. S., and J. W. Coulton. 1998. TonB-dependent iron acquisition: mechanisms of siderophore-mediated active transport. Mol. Microbiol. 28:675-681.[CrossRef][Medline]
  45. Moura, I., P. Tavares, and N. Ravi. 1994. Characterization of 3 proteins containing multiple iron sites—rubrerythrin, desulfoferrodoxin, and a protein containing a six-iron cluster. Methods Enzymol. 243:216-240.[Medline]
  46. Mukhopadhyay, A., Z. He, E. J. Alm, A. P. Arkin, E. E. Baidoo, S. C. Borglin, W. Chen, T. C. Hazen, Q. He, H. Y. Holman, K. Huang, R. Huang, D. C. Joyner, N. Katz, M. Keller, P. Oeller, A. Redding, J. Sun, J. D. Wall, J. Wei, Z. Yang, H.-C. Yen, J. Zhou, and J. D. Keasling. 2006. Salt stress in Desulfovibrio vulgaris Hildenborough: an integrated genomics approach. J. Bacteriol. 188:4068-4078.[Abstract/Free Full Text]
  47. Mukhopadhyay, A., A. M. Redding, M. P. Joachimiak, A. P. Arkin, S. E. Borglin, P. S. Dehal, R. Chakraborty, J. T. Geller, T. C. Hazen, Q. He, D. C. Joyner, V. J. J. Martin, J. D. Wall, Z. K. Yang, J. Zhou, and J. D. Keasling. 2007. Cell wide responses to low oxygen exposure in Desulfovibrio vulgaris Hildenborough. J. Bacteriol. 189:5996-6010.[Abstract/Free Full Text]
  48. Mukhopadhyay, P., M. Zheng, L. A. Bedzyk, R. A. LaRossa, and G. Storz. 2004. Prominent roles of the NorR and Fur regulators in the Escherichia coli transcriptional response to reactive nitrogen species. Proc. Natl. Acad. Sci. USA 101:745-750.[Abstract/Free Full Text]
  49. Myhr, S., B. L. P. Lillebo, E. Sunde, J. Beeder, and T. Torsvik. 2002. Inhibition of microbial H2S production in an oil reservoir model column by nitrate injection. Appl. Microbiol. Biotechnol. 58:400-408.[CrossRef][Medline]
  50. Nemati, M., T. J. Mazutinec, G. E. Jenneman, and G. Voordouw. 2001. Control of biogenic H2S production with nitrite and molybdate. J. Ind. Microbiol. Biotechnol. 26: