Expression of Hemin Receptor Molecule ChuA Is Influenced by RfaH in Uropathogenic Escherichia coliStrain 536

Bacterial strains and culture conditions.The uropathogenicE. coli strain 536 was isolated from a patient with acute pyelonephritis (3). In the mutant strain 536rfaH::cat, the rfaH gene was inactivated by insertion of a chloramphenicol acetyltransferase (cat) cassette. The insertion was performed by allelic exchange as previously described (23).trans-complementation of rfaH was achieved by supplying the mutant strain with the plasmid pSMK1, which carriedrfaH together with its promoter region cloned into the vector pGEM-T Easy (Promega). The strains used in Southern hybridization experiments are listed in Table1. The enterohemorrhagic E. coli (EHEC) strain 95004730 and the enteroaggregativeE. coli (EAggEC) strain DPA065 were provided by Robert Pringle (Victorian Infectious Diseases Reference Laboratory, North Melbourne, Australia) and Anna Giammanco (Dipartimento di Igiene e Microbiologia, University of Palermo, Palermo, Italy), respectively. The origins of all otherE. coli wild-type strains are referenced in Table 1. Bacteria were grown routinely in Luria-Bertani broth or Luria-Bertani broth solidified with 1.5% agar (Difco, Detroit, Mich.). In iron-restricted studies, a 0.4 mM concentration of the iron chelator 2,2′-dipyridyl (Sigma, Deisenhofen, Germany) was added to the media. When appropriate, the medium was supplemented with the following antibiotics at the indicated concentrations: ampicillin, 100 μg/ml; chloramphenicol, 30 μg/ml.

Expression of ChuA is decreased in the rfaH mutant of strain 536.The ChuA protein levels expressed in E. coli 536 and its derivatives were determined by Western blotting (Fig. 1A). Whole-cell extracts obtained from bacteria grown in normal and iron-restricted media were separated on a 10% polyacrylamide gel and were blotted onto a nitrocellulose membrane. The blocked membranes were treated with an anti-HemR antiserum (kindly provided by J. Heesemann) and were developed as described elsewhere (28). HemR is the hemin receptor protein of Yersinia enterocolitica. The HemR antiserum was proven to be cross-reactive with ChuA of E. coli 536 (data not shown). The quantity of ChuA protein was strongly reduced in the rfaH-negative strain compared to the wild type.trans-complementation of the mutant strain withrfaH (on pSMK1) restored higher levels of ChuA. No ChuA protein was detectable in the absence of 2,2′-dipyridyl, indicating that expression of ChuA is dependent on the availability of free iron.

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Fig. 1.

Influence of RfaH on chuA expression ofE. coli strain 536. (A) Detection of ChuA levels by Western blot analysis of whole-cell extracts of E. colistrain 536 and its derivatives using a HemR-specific antiserum. The strains were grown in the presence (+) or absence (−) of 0.4 mM 2,2′-dipyridyl. (B) Analysis of chuA transcript levels of E. coli strain 536 and its derivatives. An enhanced chemiluminescence-labeled chuA-specific probe was hybridized to total RNA isolated from strain 536 (lane 1), 536rfaH::cat (lane 2), 536rfaH::cat (pSMK1) (lane 3). The 23S and 16S rRNA were stained with 0.3% methylene blue after transfer of separated total RNA to a nylon membrane (internal control).

To investigate whether the altered expression of ChuA protein was a consequence of decreased chuA transcription in therfaH mutant, we performed Northern blot analysis (Fig. 1B). Total RNA was isolated from bacteria harvested from iron-restricted medium using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Northern blot analysis was performed as described previously (1). Ten micrograms of isolated RNA per lane was separated on a 1.2% agarose-formaldehyde gel and was transferred overnight to a nylon membrane (Biodyne B; Pall Ltd., Portsmouth, England) by capillary blotting. The DNA probe specific for the 3′ end of chuA was generated by PCR using the primers 5′-GTCGCTTCTATACCAACTATTGGGTG-3′ and 5′-CCGTTACGACCATCCTGTG-3′ and was labeled with the ECL direct labeling system (Amersham-Pharmacia, Freiburg, Germany). Hybridization was carried out overnight at 42°C as described by Amersham-Pharmacia. Before luminography, the membrane was washed twice for 15 min in 0.5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.4% sodium dodecyl sulfate (SDS) (50°C) and then twice for 5 min in 2× SSC (20°C). The chuA-specific DNA probe hybridized to a 2.2 to 2.3-kb mRNA, which corresponds to thechuA transcript (Fig. 1B). The absence of an intactrfaH gene resulted in reduced levels of chuAmRNA; however, the length of the transcript was not altered. Overexpression of RfaH (536rfaH::cat carrying pSMK1) manifested in an increased chuA transcription compared to the level found in the wild-type strain. RfaH has been known as a regulator which influences the transcription of long operons encoding cell surface and extracellular components that are important for bacterial fertility and virulence. These include the hly, rfa, rfb, tra, cps, and kps operons that direct the synthesis of α-hemolysin, lipopolysaccharide core, O antigen, F factor, and group I and group II capsule, respectively (2). The hemin receptor ChuA is also anchored in the outer membrane of pathogenic E. coli strains and is considered to be a potential virulence factor. As the encoding gene (chuA) is transcribed as a monocistronic mRNA, the way RfaH is involved in transcriptional regulation of chuAtranscription seems to be inconsistent with the present view that RfaH acts as a transcriptional antiterminator without affecting transcription initiation (18).

Coregulation of different determinants involved in pathogenicity is energetically advantageous for pathogenic bacteria. This is especially true for components of a complex system which are functionally related. α-Hemolysin expression and hemin uptake are both regulated by iron (16, 21), suggesting that the utilization of heme compounds liberated from eukaryotic cells is an important iron acquisition strategy during infection. Coupled regulation by RfaH gives further evidence that the function of theE. coli hemin uptake system (chu) is dependent on secreted hemolysin.

Sequence analysis of the chuA gene of E. coli strain 536.Sequencing of the chuA gene was performed from a cosmid clone of E. coli 536 using an ABI Prism 310 automatic sequencer. It was previously shown that RfaH-regulated operons carry a conserved region known as the JUMPStart sequence (2). Within the chuA gene ofE. coli strain 536, a similar motif was identified. A comparison of this motif to JUMPStart sequences of other E. coli operons known to be regulated by RfaH is shown in Fig.2. The 39-bp region found in thechuA gene is located 1,158 bp downstream of the start codon. It contains an ops-like motif with an additional conserved C base located downstream of the ops element. In the 5′ region of the JUMPStart sequence, a relatively well-conserved direct repeat could be identified with relevant spacing similar to those of other JUMPStart sequences.

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Fig. 2.

Comparison of JUMPStart sequences from differentE. coli operons. Boldface letters denote theops element; underlined bases represent the imperfect repeats within the JUMPStart sequences. Accession numbers or references for the following sequences are as follows: cps,AF104912 ; kps, X53819 ; rfb, U09876 ;tra, U01159 ; rfa, M86935 ; p152hly, M14107 and X07565 ; 2001 hly, reference24; J96 hly, M10133 ; 536 hlyI andhlyII, G. Nagy and G. Blum-Oehler, unpublished data; 536chu, AF280396 ; EDL933 chu, U67920 .

The 1,983-bp coding region of chuA shows high homology to the corresponding sequences derived from E. coliO157:H7 (38) and S. dysenteriae(22). The potential promoter region is located about 300 bp upstream of the start codon, and is overlapped by a putative Fur box. The presence of this motif neighboring the promoter explains the observed effect of iron availability on ChuA protein levels. In contrast to the high homology between the coding regions of differentchuA and shuA determinants, the E. coli 536-specific chuA upstream region showed less similarity to the corresponding regions of E. coli O157:H7 and S. dysenteriae. A 74-bp region located between the putative promoter and the start codon of the E. coli 536-specific determinant is replaced by a totally different 73-bp motif in S. dysenteriae and E. coli O157:H7 (Fig.3). In the uropathogenic strain, this region is flanked by 6-bp direct repeats that might have served as a site for recombination. In E. coli O157:H7, this region is bordered by similar, nevertheless imperfect, repeats.

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Fig. 3.

Genetic map of the uropathogenic E. colistrain 536-specific chuA gene (A) and that of EHEC strain EDL933 (B). The chuA coding regions are indicated by boxes; the 5′ flanking regions are indicated by single lines. The arrows labeled with P denote the promoters. The sequences of the upstream element specific for strain 536 or strain EDL933 are given. Bases in boldface type represent the direct repeats flanking dissimilar regions. The numbers and thick lines denote the probes used for Southern hybridization (see text). The sequences of oligonucleotides used as probes are underlined.

To investigate the distribution of the two different identified 5′-flanking sequences of chuA, several E. coli strains representing different pathogroups were tested by Southern hybridization. Chromosomal DNA was isolated as described before (9). The DNA was digested with BglI prior to separation on a 0.8% agarose gel and subsequent transfer to a nylon membrane (Biodyne B; Pall Ltd.) The presence of chuAin the genomes was proven by hybridization of a 600-bp probe derived from the well-conserved 3′ part of chuA (probe-1). Generation, labeling, and detection of the chuA-specific probe as well as the hybridization procedure were performed as described above for Northern blot analysis. Oligonucleotides derived from the dissimilar upstream regions were used to analyze the distribution of the different 5′ flanking regions. Probe-2 (derived from strain 536 [Fig. 3]) (5′-TGA ATT ATC AGA AAT ATT CGG CAA TTT TAC GGG ATA TAT ACG CTA ATA GCT TCC CGT GGT GAT ATC TAA TCA-3′) and probe-3 (derived from the strain EDL933 [Fig. 3]) (5′-CGA GTT ATC AGG CAA TTT CAT GGG ATA TAA ACG C-3′) were purchased from ARK Scientific GmbH (Darmstadt, Germany). The probes were labeled with digoxigenin using the DIG Oligonucleotide 3′-End Labeling kit (Roche, Mannheim, Germany). Prehybridization and hybridization were carried out in high-SDS hybridization buffer at 30°C for 4 h and overnight, respectively. The filters were washed twice for 10 min at room temperature in 2× SSC–0.1% SDS. Hybridized oligonucleotides were detected using the DIG Luminescent Detection kit (Roche) following the standard protocol provided by the manufacturer. The results of the hybridization experiments are summarized in Table 1.

The probe specific for the 3′ end of the chuA gene (probe-1) hybridized with numerous intestinal and all extraintestinal pathogenic E. coli strains. However, thechuA-specific probe hybridized to two distinct bands: to a larger DNA fragment (∼12 kb) in case of the extraintestinal and some of the enteropathogenic E. coli (EPEC) strains, whereas in EHEC O157, enteroinvasive E. coli(EIEC) and some other EPEC strains the chuA probe hybridized to a smaller fragment (∼11 kb). In correspondence with former investigations, none of the tested non-O157-EHEC, EAggEC, and enterotoxigenic E. coli representatives carriedchuA (41). The oligonucleotide specific for thechuA upstream region of strain 536 (probe-2) hybridized to all strains that carried chuA on the 12-kb fragment, while that which originated from the O157:H7 strain EDL933 (probe-3) hybridized to the 11-kb fragment, suggesting that two distinct variants of the chuA determinant, which show differences in their flanking sequences, exist. The existence of these two variants and their patterned distribution among different pathogroups provides further evidence for the clonality of E. colipathogens. Whether the differences in the chuA upstream regions have any influence on the regulation of chuAexpression still needs to be clarified.

Nucleotide sequence accession number.The nucleotide sequence of the E. coli strain 536-specific chuA gene has been deposited in the GenBank database (accession numberAF280396 ).