Division of Genomic Medicine, F floor, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK1
Author for correspondence: Mark S. Thomas. Tel: +44 114 2712834. Fax: +44 114 2739926. e-mail: m.s.thomas{at}shef.ac.uk
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ABSTRACT |
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Keywords: Burkholderia, Fur repressor, iron regulation, lipoprotein, allantoin
Abbreviations: CF, cystic fibrosis
The GenBank accession numbers for the sequences reported in this paper are AF317836 and AF153356.
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INTRODUCTION |
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The pathogenic mechanisms of B. cepacia remain to be determined, although it has been shown to secrete a number of extracellular products which may contribute to its pathogenicity. These include haemolysin, protease, lipase, phospholipase C and a cytotoxic factor (Nakazawa et al., 1987 ; Lonon et al., 1988
; McKevitt et al., 1989
; Hutchison et al., 1998
; Melnikov et al., 2000
). B. cepacia also elaborates siderophores as a potential mechanism for acquiring iron from the human host. This bacterium can produce up to five different siderophores of which ornibactin and pyochelin are the most prevalent among clinical isolates (Sokol, 1986
; Darling et al., 1998
). The production of these siderophores has been correlated with morbidity and mortality in CF patients and/or shown to contribute to pathology in animal models of respiratory infection (Sokol, 1986
; Sokol & Woods, 1988
; Sokol et al., 1999
).
Iron acquisition mechanisms in bacteria are induced in response to decreased availability of extracellular iron. Furthermore, for many bacteria, an iron-limiting environment acts as a signal to trigger the expression of a wide variety of additional virulence genes. In Gram-negative bacteria, the most common and best-characterized mechanism of gene regulation in response to iron concentration is mediated by the Fur protein (Litwin & Calderwood, 1993 ; Escolar et al., 1999
). Fur (ferric uptake regulator) is a global regulator of iron-regulated gene expression originally identified in Escherichia coli (Hantke, 1984
; Schaffer et al., 1985
). E. coli Fur is a 17 kDa zinc metalloprotein (Schaffer et al., 1985
; Wee et al., 1988
; Althaus et al., 1999
) which, under conditions of iron sufficiency, forms a complex with ferrous ions (Fe2+) and binds as a dimer to a conserved 19 bp palindromic DNA sequence, the Fur box (or iron box), present in the promoter region of many iron-regulated genes (Bagg & Neilands, 1987a
, b
; Calderwood & Mekalanos, 1988
; de Lorenzo et al., 1987
). The consensus Fur box sequence GATAATGATAATCATTATC was originally interpreted to imply recognition of two symmetrically arranged 9 bp half-sites by a dimeric regulatory protein in the classical fashion (Litwin & Calderwood, 1993
). However, this sequence has recently been re-evaluated as comprising three adjacent NAT(A/T)AT repeats which can be extended by addition of further hexameric units in any orientation (Escolar et al., 1998
, 2000
). Binding of the FurFe2+ complex to the Fur box inhibits transcription of downstream genes (Bagg & Neilands, 1987a
; de Lorenzo et al., 1987
). Under iron-limiting conditions, the ferrous ion dissociates from the Fur protein and repression of Fur-regulated genes is relieved. This ensures that repression only occurs when iron is readily available (Escolar et al., 1999
). Genes which are known to be recruited to the Fur regulon include those involved in iron acquisition, pathogenicity, carbon metabolism and resistance to oxidative and acid stresses (Escolar et al., 1999
). Fur homologues have subsequently been identified in many other Gram-negative species (Hamza et al., 1999
; Escolar et al., 1999
) and more recently in some Gram-positive bacteria (Bsat et al., 1998
; Xiong et al., 2000
). Due to the central role that this protein plays in the control of iron-regulated genes, we have cloned and characterized the fur gene of B. cepacia as a starting point for the subsequent identification of genes which comprise the Fur regulon in this organism.
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METHODS |
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Cloning of the B. cepacia and B. pseudomallei fur homologues.
A segment of the B. pseudomallei fur gene was amplified by PCR from chromosomal DNA (extracted with the Qiagen Genomic-tip kit) with primers BF1 (5'-CGGAATTCGGWCTSAARGTTACCSKSCCGCG-3') and BF2 (5'-CGTCTAGACAGCACSCGRTARAYSGTSGCCASRCC-3'), employing 50 cycles of denaturing (1·5 min at 94 °C), annealing (0·5 min at 50 °C) and extending (2·5 min at 72 °C), using DyNAzyme II (Finnzymes). The single PCR product was digested using the primer-derived restriction sites (EcoRI and XbaI) before being cloned into the high copy number vector pUC18 giving rise to pCAL1. Southern blotting, employing the B. pseudomallei fur PCR fragment as a probe, was used to identify chromosomal DNA fragments containing the B. cepacia and B. pseudomallei fur genes essentially according to Sambrook et al. (1989) . DNA was blotted on to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech), following which hybridization was carried out under normal stringency conditions using a probe labelled by the ECL Direct Nucleic Acid Labelling and Detection System (Amersham Pharmacia Biotech). Chromosomal DNA fragments containing fur identified by Southern blotting were eluted from gel slices using the QIAquick Gel Extraction Kit (Qiagen), ligated to pBBR1MCS and used to transform E. coli JM83. Transformants in which the recombinant plasmid harboured the required fur fragment were identified by dot blotting of pooled plasmid minipreparations (10 clones per pool), again using the B. pseudomallei fur PCR fragment as a probe. Individual plasmid clones from within a positive pool were then similarly probed to identify the required plasmid.
DNA sequence analysis.
DNA sequencing was carried out using an automated ABI system with fluorescent labelling and employed a combination of forward and reverse universal primers together with primers designed to anneal to sequences within the cloned chromosomal DNA. Sequences were determined in full on both strands. Sequence similarity searches were implemented with gapped BLASTN and BLASTX (Altschul et al., 1997 ) from NCBI. Amino acid alignments were performed using the CLUSTAL W program at EMBnet-CH (http://www.ch.embnet.org/software/ClustalW.html) and the ALIGN program accessed through the GeneStream network server at Institut Genetique Humaine (http://xylian.igh.cnrs.fr/bin/align-guess.cgi).
Plasmid construction.
Plasmids used in this work are listed in Table 1. Plasmids pAHA21 and pGS301 were constructed in two steps. First, the B. cepacia and B. pseudomallei fur genes and their respective transcription terminators were PCR-amplified with primers BcfurFor (5'-CCCGGATCCCCTCATGACCAATCCGACGGA-3') and BcfurRev (5'-CTTTCGTAAGCTTTCCGGGCG-3') for B. cepacia fur (pCAL4 as template), and BpfurFor (5'-CCCGAATTCCCTCATGA-CCAATCCGACCGA-3') and BpfurRev (5'-TCGCGCGGTCGACAGGC-3') for B. pseudomallei fur (pCAL2 as template), and the products were digested with BamHI and HindIII for B. cepacia fur, and EcoRI and SalI for B. pseudomallei fur, and ligated into the corresponding sites of pUC18, giving rise to pAHA20 and pGS300, respectively. For both fur genes, this manipulation results in placement of an NcoI-compatible BspHI site overlapping the translation initiation codon for fur and thus retains the natural second codon of fur (ACC). The fur genes were then transferred from pAHA20 and pGS300 as BspHIHindIII fragments into the ATG vector pTrc99A digested with NcoI and HindIII, giving rise to pAHA21 and pGS301, respectively.
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Plasmid pJMH203, used to construct the chromosomal furlac fusion in B. cepacia, was generated as follows. A 680 bp fragment containing the B. cepacia fur promoter and first 76 codons of fur was amplified by PCR with the primers BcfurpFor (5'-GCGCGAATTCGCGCGGCCGCCCCGAGAAGTTGACCACGA-3') and BcfurpRev (5'-GCGCTCTAGACTTGCCGGACTCGAAGTTG-3') using pCAL4 as template, and ligated into pUC18 following cleavage of the PCR product and plasmid vector by EcoRI and XbaI. The resultant plasmid was named pJMH202. The fur promoter fragment was then transferred as a NotIEcoRI fragment from pJMH202 into the same sites located upstream of the lacZ gene on the mobilizable suicide vector pUTmini-Tn5TplacZYAB
K. The resultant plasmid, pJMH203, contains a transcriptional fusion of B. cepacia fur to the lacZ gene.
PCR reactions used for plasmid construction employed DyNAzyme II with a regime of 25 cycles of denaturing (94 °C for 4560 s), annealing (4855 °C, depending on the Tm, for 3060 s) and polymerization (72 °C for 6090 s). For amplification of B. pseudomallei fur, DMSO was included at a final concentration of 5% (v/v). The nucleotide sequence of all cloned amplified products was confirmed by automated DNA sequence analysis.
RNA isolation.
RNA was prepared from cultures (10 ml) grown under aeration at 37 °C in M9 medium containing glucose (0·4%) and Casamino acids (0·5%) according to the method of Storz & Altuvia (1994) . Cells were harvested and resuspended in 387 µl TKM (10 mM Tris/HCl, pH 7·5; 10 mM KCl; 5 mM MgCl2) to which 27 µl lysozyme (9 mg ml-1 in TKM) was added and the cell suspension was frozen at -80 °C. To the thawed cells, 57 µl 10% SDS, 30 µl 3 M sodium acetate and 887 µl acid phenol (pH 4·3) (Sigma) were immediately added and the mixture was vortexed, heated at 65 °C for 4 min and vortexed again. The upper phase was re-extracted with 887 µl phenol (including the heating step) and then chloroform extracted before precipitating the nucleic acids with ethanol in the presence of sodium acetate (0·3 M). After washing with 70% ethanol, the precipitate was resuspended in H2O to a final concentration of 45 mg ml-1 and stored frozen (-20 °C).
Primer extension.
Labelled primer was prepared by 5'-end-labelling primer Bcfurtsp (5'-GAATCTTGAGGCGCGGTAG) (10 pmol) with 50 µCi (185 kBq) [-32P]ATP (3000 Ci mmol-1) (NEN) using T4 polynucleotide kinase (10 units) (Promega) in a volume of 20 µl at 37 °C for 45 min. After heat-inactivation of the enzyme, the labelled primer was purified on a Sephadex G-50 column. The primer extension reaction was carried out following incubation of the RNA (4050 µg) with dNTPs (500 µM each dNTP) and 32P-labelled primer (500 c.p.s.) at 80 °C for 10 min in a volume of 16·5 µl and chilling on ice. Primer extension was initiated by addition of 20 units (1 µl) of enhanced AMV reverse transcriptase (Sigma), 2 µl x10 RT buffer (50 mM Tris/HCl, pH 8·3; 40 mM KCl; 8 mM MgCl2; 1 mM DTT) and 0·5 µl human placenta ribonuclease inhibitor (0·5 units) and allowed to continue for 1 h at 42 °C. The reaction was terminated by extraction with phenol/chloroform and the nucleic acids were precipitated with ethanol and resuspended in 3 µl formamide loading solution and 3 µl 0·1 M NaOH. The sample was electrophoresed on a 6% polyacrylamide-urea sequencing gel following denaturation of DNARNA hybrids at 90 °C for 2 min. DNA sequencing reactions were run in parallel with the same primer, using pJMH202 as template.
Expression of B. cepacia and B. pseudomallei fur in E. coli.
To induce high-level synthesis of B. cepacia and B. pseudomallei fur in E. coli, overnight cultures of H1780 containing pAHA21 or pGS301, grown in LB plus ampicillin, were diluted 100-fold into fresh medium and grown at 37 °C to an OD600 of 0·4, at which time IPTG (0·5 mM final concentration) was added and incubation was continued for a further 2 h. Samples from these cultures were then subjected to SDS-PAGE in the presence of ß-mercaptoethanol and either stained with Coomassie brilliant blue or processed for Western blotting. To test for functional complementation of an E. coli fur mutant, cultures of H1780 containing pAHA21, pGS301 and pUNCH600 were grown as described above to an OD600 of 0·4 (but without IPTG induction and with the inclusion of 50 µM FeCl3 or 200 µM 2,2-dipyridyl), at which time ß-galactosidase assays were carried out.
Western blot analysis of Fur protein.
Western blotting (Sambrook et al., 1989 ) was employed for the detection of cross-reactive proteins using a polyclonal rabbit antiserum to Pseudomonas aeruginosa Fur. Whole-cell extracts of cultures grown overnight in LB broth were separated on a 12% SDS-polyacrylamide gel in the presence of ß-mercaptoethanol and electroblotted onto a nitrocellulose membrane (Hybond C; Amersham). The blots were blocked and treated with rabbit anti-P. aeruginosa Fur serum (1:1000 dilution). After washing, the blot was treated with biotinylated goat anti-rabbit IgG, streptavidin-horseradish peroxidase and developed with 4-chloro-1-naphthol substrate.
Fur titration assay (FURTA).
E. coli H1717 containing pHG165, pJMH200 and pGS302 was plated on MacConkey agar (Difco) containing lactose (1%), ampicillin (100 µg ml-1) and 30 µM Fe(NH4)2(SO4)2 and the plates were viewed after incubation at 37 °C for 24 h. Titration of Fur by multicopy Fur boxes was indicated by the formation of red colonies.
Construction of a chromosomal furlac fusion in B. cepacia.
pJMH203 was introduced into B. cepacia 715j by conjugal transfer using the E. coli strain BW19851 as the donor (Herrero et al., 1990 ; de Lorenzo & Timmis, 1994
) and recombinants were selected on M9 minimal agar containing glucose (0·4%), X-Gal (40 µg ml-1) plus antibiotics (25 µg kanamycin ml-1 and 20 µg trimethoprim ml-1).
ß-Galactosidase enzyme assay.
ß-Galactosidase measurements were carried out in triplicate on 3 consecutive days according to the protocol described by Miller and results are presented in Miller units (Miller, 1972 ). In all cases, cultures were grown with aeration at 37 °C following 100-fold dilution of overnight cultures. For measuring the response of the fur promoter to environmental conditions, the B. cepacia furlac fusion strain, AA100, was grown in M9 medium containing glucose (0·4%) and Casamino acids (0·5%), except when the effect of carbon source was assessed, in which case only the carbon source under investigation was included in the medium at 0·4%. To measure the effect of iron limitation on fur transcription, high and low iron conditions were achieved by adding 50 µM FeCl3 and 400 µM Ferrozine. Concentrations of hydrogen peroxide and methyl viologen used were the highest permissible without causing a growth rate defect.
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RESULTS AND DISCUSSION |
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The amplified B. pseudomallei fur gene fragment was used as a probe to locate the entire fur gene within the genomes of B. cepacia and B. pseudomallei following Southern blotting of restriction-enzyme-digested genomic DNA from both organisms. The probe identified a 2·0 kb SalI fragment from B. cepacia and a 1·1 kb SacI fragment from B. pseudomallei containing the respective fur homologues (results not shown) which were selected for cloning into pBBR1MCS. For each of the two cloning experiments, fur-positive recombinant plasmids were identified by dot blotting pooled plasmid extracts and subsequently subjecting individual clones within a positive pool to the same analysis. pBBR1MCS containing the 1·1 kb fur fragment of B. pseudomallei was referred to as pCAL2 and the plasmid carrying the 2·0 kb fur fragment of B. cepacia was named pCAL4.
DNA sequence analysis of B. cepacia and B. pseudomallei fur
The nucleotide sequences of the two cloned fur fragments were determined. The DNA sequence of the 160 bp PCR product was identified within a 426 bp ORF present within the cloned B. pseudomallei fragment (Fig. 1). This ORF has an identical nucleotide sequence to the recently reported sequence of the fur gene of B. pseudomallei strain P844 (Loprasert et al., 2000
). An ORF of 426 bp, exhibiting 90% identity at the nucleotide sequence level with the B. pseudomallei fur sequence, was identified within the cloned B. cepacia DNA (Fig. 1
). The 142-amino-acid polypeptide encoded by the B. cepacia fur homologue has a predicted molecular mass of 16·2 kDa, and exhibits high amino acid sequence identity with Fur proteins from a variety of Gram-negative bacteria. As expected, the B. cepacia Fur homologue exhibits the greatest degree of amino acid sequence identity to the B. pseudomallei Fur protein (95% identity, 99% similarity), with amino acid differences occurring at only seven positions. The highest degree of sequence identity outside the genus was found to the Fur protein of the closely related bacterium R. eutropha (80% identity) but was less marked for the Fur homologues from other ß subclass Proteobacteria such as Bordetella pertussis (61% identity) and Neisseria meningitidis (55% identity). The predicted B. cepacia Fur polypeptide also exhibited a high degree of amino acid sequence identity with the Fur proteins of members of the
subclass, including Legionella pneumophila (56% identity), P. aeruginosa (53% identity) and enterobacteria such as Yersinia pestis and E. coli (both 54% identity).
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Determination of the transcription start point for B. cepacia fur
The transcription start site for the B. cepacia fur gene was determined by primer extension. Fig. 5 shows the result of the primer extension experiment performed with B. cepacia RNA and a B. cepacia fur-specific primer. The extended primer gave rise to two products of 92 and 93 bases in length, indicating that transcription initiates 2829 bp upstream of the initiation codon for fur translation. The location of the transcription start point remained the same irrespective of whether the cells were grown under iron-limiting or iron-replete conditions (result not shown). The predicted -10 sequence (TAACAT) exhibits a good match to the consensus promoter -10 sequence for the major species of eubacterial RNA polymerase holoenzyme (4/6 match), whereas the predicted -35 region (ATATCT) displays a poor match to the consensus (2/6 match) and is separated from the -10 hexamer by a non-standard spacer of 18 bp (Fig. 1
). An analogous primer extension experiment carried out using B. pseudomallei RNA and an appropriate fur-specific primer identified a single extended product and placed the transcription start point for B. pseudomallei fur 29 bp upstream of the start codon for fur translation (result not shown), in agreement with Loprasert et al. (2000)
.
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Regulation of B. cepacia fur
The fur genes of a number of Gram-negative bacteria, including E. coli and Xanthomonas campestris, are negatively autoregulated in the presence of iron, and in at least one case this has been shown to be due to binding of Fur to a Fur-box-like sequence overlapping the fur promoter (de Lorenzo et al., 1988 ; Loprasert et al., 1999
). To ascertain whether or not there are sequences in the promoter region of B. cepacia fur which function as Fur boxes, we carried out a Fur titration assay (FURTA) (Stojiljkovic et al., 1994
). This assay detects Fur-box-like sequences by their ability to compete with a weak Fur box for binding by the E. coli Fur protein. In this system, the competing Fur box is located at the fhuF promoter, which is transcriptionally fused to the lacZ and lacY genes on the chromosome of E. coli strain H1717. Titration of Fur by multicopy Fur boxes is manifested by an increased ß-galactosidase activity and may be monitored at a phenotypic level as a change from a Lac- to a Lac+ phenotype. For this assay, we constructed two plasmids derived from pHG165: one contained
330 bp of the fur promoter region from B. cepacia (pJMH200) and the other, serving as a positive control, contained a consensus E. coli Fur box (pGS302). As expected, E. coli H1717 containing pHG165 produced white (Lac-) colonies on MacConkey-lactose agar containing 30 µM Fe(II) whereas H1717 harbouring pGS302 gave rise to red (Lac+) colonies. However, H1717 containing pJMH200 also gave rise to white colonies.
As Fur indirectly regulates expression of some transcriptional units in response to iron, for example, through the action of a Fur-regulated transcription activator (Heinrichs & Poole, 1996 ), we decided to examine in a more direct way whether fur was regulated by iron in B. cepacia. A mobilizable suicide plasmid was constructed in which the B. cepacia fur promoter region was positioned upstream of the lacZ gene (pJMH203). This plasmid was conjugated into B. cepacia 715j and exconjugants harbouring plasmid co-integrates were selected on M9 agar containing X-Gal. Lac+ recombinants, which appeared at a frequency of
1·0x10-6 per recipient, arose through a single crossover event between cloned B. cepacia sequences present on the plasmid and the fur locus of the bacterium. Southern blotting was used to confirm the integrity and chromosomal location of the integrated plasmid and to distinguish single co-integrates from multiple co-integrates (results not shown). Western blotting of whole-cell extracts with anti-P. aeruginosa Fur antiserum and examination of siderophore production using CAS plates (Schwyn & Neilands, 1987
) was used to confirm that a functional copy of fur remained on the chromosome in strains harbouring single plasmid co-integrates (results not shown).
A representative strain harbouring a single plasmid co-integrate (AA100) was grown at 37 °C under iron-limiting and iron-replete conditions and the ß-galactosidase activity in exponential phase cultures was measured. The assays revealed that transcription of fur in B. cepacia is not significantly influenced by iron availability (Fig. 6). The absence of iron regulation of fur in B. cepacia has also been observed for fur genes in other Gram-negative species such as P. aeruginosa, Bradyrhizobium japonicum, Rhizobium leguminosarum and Campylobacter jejuni (de Luca et al., 1998
; Vasil & Ochsner, 1999
; Hamza et al., 1999
; van Vliet et al., 2000
). The effect of other metal ions (Mn2+, Zn2+, Ni2+ and Cu2+) on the B. cepacia fur promoter was also tested and found to exert negligible effects on fur transcription (data not shown). The furlacZ fusion strain was also used to ascertain whether fur promoter activity responds to other environmental cues which have been shown to influence fur gene expression in some other Gram-negative bacteria. In contrast to the E. coli fur gene, transcription of which is activated by oxidative stress (Zheng et al., 1999
), no effect of hydrogen peroxide or the superoxide generator methyl viologen was observed on fur transcription in B. cepacia (Fig. 6
). The absence of any observable effect of iron or oxidative stress on the transcription of B. cepacia fur parallels the results obtained with the B. pseudomallei fur gene (Loprasert et al., 2000
). As a binding site for CRP (the cAMP receptor protein) is also present at the E. coli fur promoter, suggesting an influence of carbon source availability on iron homeostasis in this bacterium (de Lorenzo et al., 1988
), B. cepacia fur promoter activity was also examined in the presence of different carbon sources. However, the nature of the carbon source (glucose, Casamino acids or succinate) was not found to exert any significant effect on the activity of this promoter (results not shown).
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Downstream of the B. cepacia fur gene, two ORFs can be identified on the 2·0 kb SalI fragment with an orientation opposite to that of fur (data not shown; Fig. 1). The predicted translated product of the gene immediately downstream of fur exhibits a high degree of sequence similarity to a putative ureidoglycolate hydrolase from P. aeruginosa (44% identity) (GenBank no. AE004580). Other homologues exhibiting
40% identity include the ureidoglycolate hydrolases encoded by the allA genes of E. coli and Salmonella enteritidis (also known as glxA2 or ybbT) (Pattery et al., 1999
; Cusa et al., 1999
). The ORF upstream of the B. cepacia allA homologue, for which only partial sequence was obtained, encodes a polypeptide possessing 61% identity with the corresponding region (85 amino acids) of a putative allantoicase (Alc) of P. aeruginosa (GenBank no. AE004580). Both genes are involved in the metabolism of allantoin, an intermediate in the degradation of purines, and are similarly organized in P. aeruginosa.
The nature and organization of the genes flanking fur in B. cepacia is interesting from a phylogenetic point of view. Database searches revealed that the divergent arrangement of the fur and omlA genes is retained in other members of the ß subclass of the Proteobacteria (Fig. 7). This organization has been previously noted in P. aeruginosa and P. fluorescens (Ochsner et al., 1999
), and is also conserved in Xylella fastidiosa and Xanthomonas campestris, all of which are members of the
subclass of the Proteobacteria. However, in other species within the
subclass, such as members of the Enterobacteriaceae (E. coli), Vibrionaceae (Vibrio cholerae) and Pasteurellaceae (Haemophilus influenzae), this relationship does not exist (Fig. 7
). The organization of genes downstream of B. cepacia fur contrasts with the situation in other members of the ß subclass for which DNA sequence information is available. Among the Proteobacteria, recN is most frequently located downstream of fur, and in the case of E. coli and V. cholerae the positional relationship between the omlA/smpA and recN genes is also retained, although these genes are no longer separated by fur (Fig. 7
).
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ACKNOWLEDGEMENTS |
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Received 20 November 2000;
revised 15 January 2001;
accepted 26 January 2001.