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
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ABSTRACT |
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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.
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INTRODUCTION |
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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
-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.
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METHODS |
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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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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 KpnISacII 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, EcoRIBamHI 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).
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RESULTS AND DISCUSSION |
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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
28, which, consequently, would give rise to a lower expression of Class 3 promoters.
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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.
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ACKNOWLEDGEMENTS |
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Received 12 December 2001;
revised 14 December 2001;
accepted 17 December 2001.
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