Intracellular cyclic AMP concentration is decreased in Salmonella typhimurium fur mutants

Susana Campoy1, Mónica Jara1, Núria Busquets1, Ana M. Pérez de Rozas2, Ignacio Badiola2 and Jordi Barbé1,2

Department of Genetics and Microbiology, Universitat Autònoma de Barcelona1 and Centre de Recerca en Sanitat Animal (CReSA), Universitat Autònoma de Barcelona Institut de Recerca i Tecnologia Agroalimentària (UAB-IRTA)2, Bellaterra, 08193 Barcelona, Spain

Author for correspondence: Jordi Barbé. Tel: +34 93 581 1837. Fax: +34 93 581 2387. e-mail: jordi.barbe{at}uab.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
It is known that the Fur protein negatively regulates iron-uptake systems in different bacterial species, including Salmonella typhimurium. In this study it has been shown that the intracellular concentration of cyclic AMP (cAMP) is lower in a knockout S. typhimurium fur mutant than in the wild-type strain. According to this, the expression of two cAMP-regulated genes, such as pepE (encoding an {alpha}-aspartyl dipeptidase) and the Escherichia coli lac operon, is decreased in S. typhimurium fur cells in comparison with wild-type cells. Introduction of an additional mutation in cpdA, encoding a cyclic 3',5'-cAMP phosphodiesterase, recovers wild-type intracellular cAMP concentration in the S. typhimurium fur mutant. Likewise, expression of pepE and the E. coli lac operon was the same in the S. typhimurium fur cpdA double mutant and the wild-type strain. Moreover, these results also demonstrate that the S. typhimurium Fur protein positively regulates the expression of the flhD master operon governing the flagellar regulon. This positive control must be mediated by binding of the S. typhimurium Fur protein to the flhD promoter as indicated by the fact that this promoter tests positive in a Fur titration assay.

Keywords: gene regulation, iron-uptake system, cpdA

Abbreviations: DPD, 2,2-dipyridyl; FURTA, Fur titration assay

The GenBank accession number for the sequence reported in this paper is AF268282.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
In Escherichia coli the iron-uptake system is under the control of the fur gene product (Hantke, 1984 ), a 17 kDa protein presenting Fe2+-dependent DNA-binding activity (Bagg & Neilands, 1987 ). Genes under fur control require the presence in their promoters of at least three contiguous NAT(A/T)AT-like hexamers, in either direct or inverse orientation, to which the Fur protein binds (Escolar et al., 1999 ). This sequence, known as the Fur box, seems to be widespread in bacteria since its presence and functionality have been described in the promoter of iron-regulated genes of several bacterial species belonging to groups as different as Enterobacteriaceae, Pseudomonadaceae, Neisseriaceae and Gram-positive bacteria (Escolar et al., 1999 ; Ratledge & Dover, 2000 ). It has also been reported that the Escherichia coli Fur protein is a positive regulator of sodB gene expression, although the precise mechanism of this stimulatory effect has not been established since a putative Fur box seems not to be present in the promoter of this gene (Dubrac & Touati, 2000 ). However, the Helicobacter pylori Fur protein can activate frpB gene transcription by directly binding its promoter (Delany et al., 2001 ). The Salmonella typhimurium fur gene and several genes which are under its control have been identified (Ernst et al., 1978 ; Foster & Hall, 1992 ; Tsolis et al., 1995 ). The Fur protein is also involved in the acid tolerance response of S. typhimurium (Wilmes-Riesenberg et al., 1996 ), although its role in iron uptake and acid resistance is physiologically and genetically separable (Hall & Foster, 1996 ).

The product of the crp gene is another global regulator which, by binding to cyclic AMP (cAMP), controls cellular catabolism (including aerobic and anaerobic respiration), at least in the Enterobacteriaceae (Kolb et al., 1993 ). Intracellular cAMP concentration is negatively modulated by the presence of glucose. As the glucose level decreases, the intracellular level of cAMP rises and an active cAMP-CRP complex is formed which transcriptionally regulates the expression of numerous genes (Ishizuka et al., 1993 ).

It has been suggested that the Fur protein could also act as an internal iron chelator, avoiding a dangerously high increase in reactive ferrous iron concentrations within bacterial cells (Abdul-Tehrani et al., 1999 ). In this respect, it is known that double recA fur mutants of E. coli are not viable when growing in the presence of oxygen (Touati et al., 1995 ). This fact is attributed to the interaction of reactive oxygen species (such as the superoxide radical generated during aerobic respiration) with a higher availability of free Fe(II) in the cytoplasm of such double mutants (Touati et al., 1995 ; Henle & Linn, 1997 ; Abdul-Tehrani et al., 1999 ).

The presence of a putative sequence to which the cAMP-CRP complex binds in the E. coli fur promoter has been suggested on the basis of computational analysis (Zheng et al., 1999 ; Gelfand et al., 2000 ). In agreement with this possibility, it has been recently demonstrated that the fur gene of Pasteurella multocida, which belongs to the {gamma}-Proteobacteria, as does E. coli, is positively regulated by the cAMP-CRP complex (Bosch et al., 2001 ). On the basis of these data, a close relationship between the metabolism of both cAMP and iron in bacterial cells could be hypothesized. To test this putative relationship, the intracellular levels of cAMP and the expression of several genes regulated by this nucleotide have been studied in an S. typhimurium fur knockout mutant.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. E. coli and S. typhimurium strains were grown in LB broth (Miller, 1991 ). CAS plates (Schwyn & Neilands, 1987 ) were used to confirm the constitutive synthesis of siderophores characteristic of fur mutants. Antibiotics were added to the culture medium at the concentrations reported by Jordan et al. (1996) . When necessary, chelating agent 2,2-dipyridyl (DPD) was used at 50 µg ml-1. Induction of the lac operon in S. typhimurium cells carrying the F'128 (Pro+ Lac+ zzf::Tn10 dtet) plasmid was analysed by the addition of IPTG to the desired culture at a final concentration of 10 mM. To measure expression of lacZ fusions, samples for the ß-galactosidase assays were taken, in all cases, from cultures in mid-exponential-growth phase (OD550 about 0·4) and enzymic activity was determined as reported by Miller (1991) . In the qualitative Fur titration assay (FURTA), 1 mM FeSO4-supplemented Lac EMBO agar plates (Stojiljkovic et al., 1994 ) were used. For quantitative analysis of FURTA experiments, cells grown on these plates were collected, resuspended in LB medium and their ß-galactosidase activities measured.


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Table 1. Bacterial strains and plasmids used in this study

 
Genetic techniques and DNA manipulations.
Biparental and triparental matings using pRK2013 as the mobilizing plasmid were performed as described by Jordan et al. (1996) . S. typhimurium chromosome exchange markers, P22 HT-mediated transductions and plasmid electroporation were performed as described by Jordan et al. (1996) . In all cases, the absence of the P22 HT prophage in the transductants obtained was determined by streaking them on green plates (Davis et al., 1980 ).

Standard DNA techniques, including restriction enzyme digests, ligation, transformation and plasmid purification, have been described elsewhere (Jordan et al., 1996 ). cpdA and promoters of pepE, as well as of all flagellar genes used in this work, were isolated from S. typhimurium ATCC 14028 chromosomal DNA by PCR amplification using the appropriate oligonucleotide primers. These primers (Table 2) were designed based on data obtained through early release of the S. typhimurium genome sequence (http://www.genome.wustl.edu/gsc) by the Genome Sequencing Center of Washington University, USA. Oligonucleotide primers were supplied by Roche Diagnostics. To facilitate subcloning of PCR DNA fragments and construction of the lacZ fusions, specific restriction sites were incorporated at their 5' ends (Table 2).


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Table 2. Oligonucleotide primers used in this work

 
Isolation of a S. typhimurium fur knockout mutant.
To isolate the fur gene, a pRK404 plasmid-based genomic library of S. typhimurium was introduced by triparental mating into the H1780 E. coli fur reporter strain, which is a fur-deficient mutant containing a fusion between the fur-controlled promoter of the fiu gene and lacZ in its chromosome (Hantke, 1987 ). After plating in LB medium supplemented with X-Gal, ferric sulfate (100 µM) and kanamycin (50 µg ml-1), five white clones were detected whose plasmids were retransformed into H1780, again giving white colonies. Since restriction analysis indicated that all five clones contained the same 1 kb size fragment, only one of these plasmids (pUA931) was selected for subsequent work.

Further subcloning and sequencing of several internal fragments enabled us to obtain the sequence of the S. typhimurium fur gene present in plasmid pUA931 (GenBank accession no. AF268282).

To obtain an S. typhimurium fur knockout mutant, a 3·5 kb chloramphenicol resistance cassette was inserted into the internal Asp700 site of the cloned fur gene. A KpnI–SacII 4·5 kb fragment containing the fur::Cm construction was then cloned in the pGP704 suicide vector and introduced into a RifR derivative of the S. typhimurium ATCC 14028 wild-type strain by triparental mating. Chloramphenicol-resistanttransconjugants were screened for loss of vector-mediated ampicillin resistance to detect putative mutants which had exchanged their wild-type gene for the inactivated fur gene as a consequence of a double cross-over event. For one of these strains, UA1784, this was unequivocally confirmed by PCR amplification of chromosomal DNA using Furup and Furdw primers, Southern dot blotting and constitutive synthesis of siderophores on CAS plates (data not shown).

It has been suggested that most rifampicin-resistant mutants of S. typhimurium are affected in their gene expression pattern (Björkman et al., 1998 ). To prevent any putative interference of the RifR mutation in the behaviour of our fur mutant, the fur::Cm region from strain UA1784 was transferred by P22-mediated transduction to wild-type (Rifs) cells of S. typhimurium ATCC 14028. The PCR profile of the chromosome of 10 CmR transductants, when amplified with Furup and Furdw primers, and inoculation in CAS plates revealed that all of them contained the desired fur::Cm mutation. One of these transductants, UA1779, was kept for further work.

Construction of lacZ fusions and ß-galactosidase assays.
A PCR-fragment of about 300 bp containing the promoter and a fragment of its coding region was cloned for each gene in the pGEM-T vector (Promega) to construct the desired lacZ fusion. Upper primers used for the construction of lacZ fusions contained an EcoRI restriction site at their 5' ends, whereas lower primers presented a BamHI site at their 5' ends (Table 2). For each fusion, EcoRI–BamHI restriction fragments were recovered from the appropriate pGEM-T derivative and subcloned into pUJ8 upstream of the promoterless trp'-'lacZ region. Afterwards, the NotI fragment harbouring the created fusion was recovered from agarose gels, filled-in with T4 DNA polymerase to obtain blunt ends and inserted into the single SmaI cloning site of the low-copy-number pLV106 plasmid. To prevent any possible effect of the pLV106-tet promoter on the expression of the gene to be studied, only clones containing a pLV106 plasmid carrying the lacZ fusion in the opposite transcriptional direction to this promoter were selected for further work. Finally, plasmids containing the constructed fusions were introduced by biparental mating into desired S. typhimurium strains. The activity of ß-galactosidase was assayed as described by Miller (1991) . The enzyme units reported here were the means of at least three independent assays and all values were reproducible to within an error of ±10%.

cAMP determinations.
The intracellular concentration of cAMP was determined using the cAMP enzyme immunoassay kit (Amersham Pharmacia Biotech), according to the instructions specified by the manufacturer. To do this, culture samples at different points during the exponential growth phase (OD550 of 0·2, 0·4 and 0·8 for cells growing in the absence of the chelating agent DPD, and 0·1, 0·2 and 0·4 for those growing in the presence of DPD) were taken. After boiling for 5 min in lysis buffer and centrifugation at 1500 g for 3 min at 4 °C, the supernatants were immediately frozen for use later in the assay. The intracellular concentration of cAMP obtained was in the range of values reported by Saier et al. (1975) in S. typhimurium cells. All cAMP determinations were carried out independently at least three times and the standard deviation among each one of the triplicates was never higher or lower than 10%.

Protein analysis.
Outer-membrane proteins from S. typhimurium wild-type or fur strains were extracted from cultures grown under the desired conditions as described by Ferreiros et al. (1990) . Briefly, cultures were centrifuged at 48000 g and pellets were resuspended in 0·1 M acetate buffer/0·2 M lithium chloride at pH 5·8, incubated for 2 h at 45 °C in a shaking water bath and passed through a 21-gauge needle. These suspensions were then centrifuged at 10000 g, the pellets being discarded. Membrane fragments were obtained from the supernatant by centrifugation at 30000 g for 2·5 h, and the pellet was resuspended in distilled water. The protein concentration of outer-membrane samples was determined by the Lowry method and their profiles were examined by 12% PAGE in the presence of SDS (Laemmli, 1970 ).

To confirm the identity of the 52 kDa protein, SDS-PAGE gels were electroblotted onto polyvinylidene difluoride membranes (Bio-Rad) and stained with Coomassie blue. This protein was then recovered from the membrane and its N-terminal amino acid sequence was determined by Edman degradation using Protein Sequencer 477A (Applied Biosystems).


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Expression of cAMP-regulated genes in S. typhimurium fur cells
The synthesis of flagella in S. typhimurium requires more than 50 genes, which are distributed in 17 identified operons (Chilcott & Hughes, 2000 ). Expression of these genes follows a hierarchic cascade, known as the flagellar regulon, in which three classes of promoters have been identified. Class 1 consists of the promoter of the transcriptional unit flhDC, also known as the master operon (Chilcott & Hughes, 2000 ). The master operon is under cAMP control (Yokota & Gots, 1970 ; Silverman & Simon, 1974 ). FlhD and FlhC are the activators of Class 2 promoters (Kutsukake et al., 1990 ; Liu & Matsumura 1994 ). They regulate, among other genes involved in flagellum biosynthesis, the expression of fliA, which is an alternative sigma factor ({sigma}28) specifically required for the transcription of Class 3 promoters (Ohnishi et al., 1990 ). Promoters of the fliC and fljB genes encoding, respectively, each of the two different flagellins which S. typhimurium cells can display, belong to Class 3 (Chilcott & Hughes, 2000 ).

To determine the effect of the fur mutation in the flagellar regulon, genes belonging to each one of the three promoter classes were selected. Expression of flhD (Class 1), fliA and flgA (Class 2), and fliC (Class 3) promoters was analysed through lacZ fusions. Results obtained indicated that all three genes display a significantly lower transcription in the fur mutant than in the wild-type strain (Fig. 1). From these data it can be inferred that the inhibition of fliC gene expression should be attributed to the hierarchic organization of the flagellar regulon. Thus, the decrease in flhCD operon transcription would lead to a lower concentration of sigma factor {sigma}28, which, consequently, would give rise to a lower expression of Class 3 promoters.



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Fig. 1. Basal expression of several promoters belonging to the S. typhimurium flagellar regulon. ß-Galactosidase synthesis was measured from a fusion of each promoter with lacZ in both wild-type (wt) and fur (Fur-) strains growing in LB medium in the absence or presence of either glucose or DPD. ß-Galactosidase activities were measured from samples taken from mid-exponential-phase cultures (OD550 about 0·4).

 
It must be noted that the addition of DPD did not modify either fliC expression in wild-type or fur cells, nor the difference existing between these strains (Fig. 1). Furthermore, the presence of glucose produces a decrease in fliC expression in the wild-type strain of a similar magnitude as that reported for E. coli (Bertin et al., 1994 ), but does not produce any effect in the fur mutant (Fig. 1). The same results were obtained when SDS-PAGE profiles of outer-membrane proteins of wild-type and fur cells were analysed. Hence, the fur mutant showed a significant decrease in the amount of a 52 kDa protein (Fig. 2) corresponding to the fliC gene product, as determined by sequencing its N-terminal end (data not shown). Likewise, and in agreement with data obtained from fliC-lacZ fusions, the presence of DPD does not abolish the difference existing in the concentration of the fliC gene product between wild-type and fur strains (Fig. 2). All of these data indicate that the decrease in transcription of flagellar genes is not related to iron starvation, but rather to the lack of the Fur protein.



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Fig. 2. SDS-PAGE profiles of outer-membrane proteins from wild-type and fur cells growing in LB medium in the presence or absence of DPD. White and black arrows indicate the fur-dependent high-molecular-mass proteins and the 52 kDa protein corresponding to the product of fliC, respectively.

 
To ascertain whether transcription of other cAMP-CRP-dependent promoters is affected in the fur mutant, the induction of the E. coli lac operon, as well as the basal expression of the cAMP-regulated S. typhimurium pepE gene (Conlin et al., 1994 ), encoding an {alpha}-aspartyl dipeptidase, was analysed. As seen in Fig. 3, expression of both transcriptional units (E. coli lac operon and S. typhimurium pepE gene) was also reduced in the fur strain, but was not affected by the presence of DPD.



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Fig. 3. Basal expression of the S. typhimurium pepE promoter and IPTG-mediated induction of the E. coli lac operon. Expression of the lac operon was measured 90 min after IPTG addition. The lac operon was present on the F'128 plasmid in both wild-type and fur strains of S. typhimurium growing in LB medium in the presence or absence of DPD. ß-Galactosidase activities were measured from samples taken from mid-exponential-phase cultures (OD550 about 0·4).

 
Intracellular concentration of cAMP in S. typhimurium fur cells
The data shown above confirm that a connection between the behaviour of fur cells and cAMP may exist. For this reason, we decided to analyse the concentration of this nucleotide at different points during the exponential growth phase, in both wild-type and fur strain cultures, in either the presence or absence of glucose, as well as when DPD had been added. Fig. 4(a) shows how the fur mutant has a lower intracellular concentration of cAMP than wild-type cells, regardless of the OD550 of the culture from which the sample was taken. Moreover, wild-type and fur cells growing in the presence of glucose have the same cAMP level (Fig. 4b), although this is lower than that observed in the absence of this carbon source. Likewise, the intracellular concentration of cAMP is practically the same in both wild-type and fur strains in the presence of DPD (Fig. 4c), with the level being slightly higher in fur cells.



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Fig. 4. Intracellular cAMP concentration of wild-type and fur strains of S. typhimurium at different points during exponential growth phase. Growth occurred in LB medium (a) in the presence of either glucose (b) or DPD (c). Growth of wild-type and fur cells is represented by closed and open circles, respectively. Samples were taken at OD550 values of 0·1, 0·2 and 0·4, or at OD550 values of 0·2, 0·4 and 0·8, depending on whether DPD was added or not, respectively. In all cases, values are presented in nmol cAMP (mg total protein of culture samples)-1.

 
Role of cpdA in the behaviour of the S. typhimurium fur mutant
It has been shown that the enzymic activity of the cyclic 3',5'-cAMP phosphodiesterase of E. coli encoded by cpdA is strongly stimulated in vitro by iron (II) (Nielsen et al., 1973 ; Imamura et al., 1996 ). Likewise, it is known that the Fe(II)/Fe(III) ratio is higher in Fur mutants than in wild-type E. coli cells and that part of this Fe(II) is not bound to iron-storage proteins as firmly in this mutant as in the wild-type strain (Abdul-Tehrani et al., 1999 ). Results reported above indicate that the lower concentration of cAMP in S. typhimurium fur mutants is related to intracellular iron availability. So, iron-depleted cells (those being cultured in the presence of DPD) of both wild-type and fur strains showed the same cAMP levels (Fig. 4c). In contrast, the fur mutant, which shows a constitutive expression of iron-uptake mechanisms and lacks the predicted protective effect of the Fur protein in iron-overloading conditions (Touati et al., 1995 ), presents a lower level of cAMP (Fig. 4a) when growing in an iron-rich medium (in the absence of DPD). For all of these reasons, we decided to analyse whether the product of cpdA is responsible for the low cAMP level of S. typhimurium fur cells. To carry this out, the S. typhimurium cdpA gene was isolated by using oligonucleotide primers shown in Table 2 and mutagenized by insertion of an {Omega}Km resistance cassette into its internal HindIII site. This cpdA::{Omega}Km construction was introduced by marker exchange into both S. typhimurium RifR wild-type and Fur- strains and its presence confirmed by both PCR and Southern analysis (data not shown). Fig. 5 indicates that the intracellular concentration of cAMP is restored in double cpdA fur mutants, this value being slightly higher than that shown by wild-type cells.



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Fig. 5. Intracellular cAMP concentration in wild-type, fur, cpdA and fur cpdA cells of S. typhimurium in mid-exponential-phase growth (OD550 about 0·4). In all cases, values are presented in nmol cAMP (mg total protein of culture samples)-1.

 
According to these results, expression of both pepE and the E. coli lac operon in the S. typhimurium cpdA fur double mutant is practically the same as in wild-type cells (Fig. 6). In agreement with previous studies (Nielsen et al., 1973 ; Imamura et al., 1996 ), the higher level of Fe(II) in S. typhimurium Fur cells could stimulate the activity of the cyclic phosphodiesterase encoded by cpdA which would then produce a decrease in the intracellular cAMP level. In accordance with this hypothesis, the addition of exogenous cAMP to S. typhimurium fur cultures did restore the wild-type level expression of either pepE or the E. coli lac operon (data not shown). Furthermore, and giving support to the role of cAMP in the behaviour of the S. typhimurium fur mutant, expression of the pepE and lac operons was the same in fur, crp and fur crp strains of S. typhimurium (data not shown). As anticipated, the level of expression was lower in all these mutants than in wild-type cells (data not shown).



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Fig. 6. Basal expression of the flhD, fliC and pepE promoters and IPTG-mediated induction of the E. coli lac operon in wild-type, fur, cpdA and fur cpdA cells of S. typhimurium growing in LB medium. ß-Galactosidase activities were measured from samples taken from mid-exponential-phase cultures (OD550 about 0·4).

 
In contrast to the findings described above, S. typhimurium cpdA fur cells did not restore wild-type levels of either flhD or fliC transcription (Fig. 6). Consistent with this finding, the amount of the product of fliC in outer-membrane profiles of both fur and fur cpdA mutants was the same when analysed by SDS-PAGE (data not shown). This fact indicates that, regardless of cAMP, the Fur protein must be involved in the control of the flagellar regulon and that its presence is absolutely required to achieve an optimal expression of the flhD promoter, despite there being enough intracellular cAMP concentration. In this respect, it is worth noting that the S. typhimurium flhD promoter tested positive, in both qualitative and quantitative assays, in the FURTA test used to determine if the Fur protein binds to a given promoter in vivo (Stojilkovic et al., 1994 ). Thus, E. coli H1717 (which is the basis of the FURTA test) harbouring a pUA949 plasmid derivative, including the S. typhimurium flhD promoter, expressed 80 Miller units of ß-galactosidase. This same strain containing the pUA949 plasmid alone showed a ß-galactosidase activity of only 2 Miller units. This result demonstrates that the Fur protein must bind to the S. typhimurium flhD promoter. It must be also noted that a putative Fur Box containing a perfect Fur-motif [NAT(A/T)AT] surrounded by copies of this hexamer presenting various degrees of conservation is located 107 bp upstream of the translational start codon of the S. typhimurium flhD gene.

Virulence of S. typhimurium fur cells is strongly reduced when orally inoculated, but only slightly affected when intraperitoneally challenged (Garcia del Portillo et al., 1993 ). Attenuation of orally inoculated fur cells has been demonstrated to be due to the extreme sensitivity of these cells to acid conditions (Wilmes-Riesenberg et al., 1996 ). Moreover, the virulence decrease of intraperitoneally inoculated fur cells had been attributed to its high sensitivity to superoxide (Touati et al., 1995 ). In contradiction to this last hypothesis, it has been established that S. typhimurium fur and wild-type strains present the same viability inside macrophage cells (Garcia del Portillo et al., 1993 ). The lower intracellular concentration of cAMP in S. typhimurium fur cells could explain its virulence decrease in comparison to the wild-type strain when such strains are intraperitoneally inoculated into mice, since appropriate levels of cAMP are required for S. typhimurium cells to be virulent (Curtiss & Kelly, 1987 ).

In summary, S. typhimurium fur mutants present reduced cAMP levels which results in an indirect reduction of expression of cAMP-regulated genes, such as pepE and lac. This decrease can be compensated by the introduction of a knockout mutation in cpdA, encoding a cyclic 3',5'-cAMP phosphodiesterase. Moreover, the S. typhimurium flhD master operon, controlling flagellar gene expression, is positively regulated by Fur through an iron-independent mechanism that requires further characterization.


   ACKNOWLEDGEMENTS
 
This work was funded by Grants BIO99-0779 of the Ministerio de Ciencia y Tecnología de España and 1999SGR-106 of the Comissionat per Universitats i Recerca de la Generalitat de Catalunya. Susana Campoy, Mónica Jara and Nuria Busquets were recipients of a predoctoral fellowship from the Direcció General d’Universitats de la Generalitat de Catalunya. We are deeply indebted to Josep Elias for his contribution to improve our installations and to Joan Ruiz and Susana Escribano for their excellent technical assistance.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 12 December 2001; revised 14 December 2001; accepted 17 December 2001.



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