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Journal of Bacteriology, March 2007, p. 1803-1815, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.00451-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A Novel Protein, TtpC, Is a Required Component of the TonB2 Complex for Specific Iron Transport in the Pathogens Vibrio anguillarum and Vibrio cholerae

Michiel Stork,^1,2 Ben R. Otto,² and Jorge H. Crosa^1*

Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon 97239-3098,¹ Department of Molecular Microbiology, Institute of Molecular Cell Biology, Faculty of Earth and Life Science, Vrije Universiteit, Amsterdam, The Netherlands²

Received 1 April 2006/ Accepted 4 December 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Active transport across the outer membrane in gram-negative bacteria requires the energy that is generated by the proton motive force in the inner membrane. This energy is transduced to the outer membrane by the TonB protein in complex with the proteins ExbB and ExbD. In the pathogen Vibrio anguillarum we have identified two TonB systems, TonB1 and TonB2, the latter is used for ferric-anguibactin transport and is transcribed as part of an operon that consists of orf2, exbB2, exbD2, and tonB2. This cluster was identified by a polar transposon insertion in orf2 that resulted in a strain deficient for ferric-anguibactin transport. Only the entire cluster (orf2, exbB2, exbD2 and tonB2) could complement for ferric-anguibactin transport, while just the exbB2, exbD2, and tonB2 genes were unable to restore transport. This suggests an essential role for this Orf2, designated TtpC, in TonB2-mediated transport in V. anguillarum. A similar gene cluster exists in V. cholerae, i.e., with the homologues of ttpC-exbB2-exbD2-tonB2, and we demonstrate that TtpC from V. cholerae also plays a role in the TonB2-mediated transport of enterobactin in this human pathogen. Furthermore, we also show that in V. anguillarum the TtpC protein is found as part of a complex that might also contain the TonB2, ExbB2, and ExbD2 proteins. This novel component of the TonB2 system found in V. anguillarum and V. cholerae is perhaps a general feature in bacteria harboring the Vibrio-like TonB2 system.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Iron is an essential element for all organisms but is not freely available because in the environment iron is found in the form of insoluble ferric-oxyhydroxides, and in vertebrates this metal is mainly bound to heme, transferrin, lactoferrin, and ferritin (29, 40). Thus, bacteria have evolved mechanisms to acquire the otherwise-unavailable iron. Bacterial iron acquisition systems can be divided in two main groups: those that utilize iron directly from the host proteins, such as transferrin and heme/hemoglobin, and those that produce intermediaries, such as hemophores and siderophores, which scavenge heme or iron, respectively, from any iron-chelating compound (13, 29, 30, 40). In both cases, uptake of the iron source requires a specific outer membrane receptor. The transport across the outer membrane is an active process in which the energy required is generated from the proton motive force in the inner membrane and transduced by the TonB protein in complex with ExbB and ExbD to the outer membrane receptor (28). The latter proteins belong to the MotB/TolR/ExbB and MotA/TolQ/ExbD family of proteins, respectively. Uptake of group B colicins and infection by phages T1 and 80 also requires the energy transduced by the TonB system (35). The TonB system is a complex consisting of the proteins TonB, ExbB, and ExbD, and it was demonstrated in Escherichia coli to be required for the TonB protein to bind the outer membrane receptor at a conserved region called the TonB box (2, 28). This complex is believed to have more than one molecule of each protein, and the ratio between TonB and the outer membrane receptor is 2:1 (18). In E. coli, TonB was found to be part of both the inner and outer membranes, where it is possibly associated with the outer membrane receptor (22).

We have previously identified in the fish pathogen Vibrio anguillarum two TonB systems (36). The first, TonB1, is able to transport heme and ferrichrome, whereas the second TonB system, TonB2, in addition to the transport of heme and ferrichrome, can also facilitate the uptake of catechol siderophores, including the endogenous catechol/hydroxamate siderophore anguibactin (36). The TonB2 system is also essential for virulence in rainbow trout, and its genes are transcribed as an iron-regulated operon (36). We describe here the previously uncharacterized gene, ttpC, that is located upstream of the exbB2, exbD2, and tonB2 genes and demonstrate that this novel protein is essential for TonB2-mediated iron transport in V. anguillarum. Homologues of the ttpC gene are found as part of the tonB2-like system in all other Vibrio species examined, suggesting that it could also play a role in the tonB2-mediated iron transport in these bacteria. In the present study we confirmed this hypothesis in one of these microorganisms, V. cholerae, and we show that TtpC is essential for TonB2-mediated transport of enterobactin in this bacterium.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in the present study are described in Table 1. V. anguillarum and V. cholerae were cultured at 25°C in either Trypticase soy broth or agar supplemented with 1% NaCl (TSBS or TSAS, respectively). For experiments determining iron uptake characteristics, the strains were first grown on TSAS supplemented with the appropriate antibiotics and passed to M9 minimal medium (32) supplemented with 0.2% Casamino Acids, 5% NaCl, and the appropriate antibiotics. To achieve iron-limiting conditions, 2 µM ethylenediamine-di-(o-hydroxyphenyl acetic acid) (EDDA) was added to the medium, while for iron-rich conditions the medium was supplemented with 4 µg of ferric ammonium citrate/ml. The antibiotic concentrations used for V. anguillarum were ampicillin at 250 to 1,000 µg/ml, rifampin at 100 µg/ml, chloramphenicol (Cm) at 10 µg/ml, kanamycin (Km) at 200 µg/ml and for V. cholerae were Km at 200 µg/ml and trimethoprim (Tp) at 100 µg/ml.

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TABLE 1. Bacterial strains and plasmids

E. coli strains were grown in Luria-Bertani (LB) medium in the presence of the appropriate antibiotics. Antibiotic concentrations used for E. coli were ampicillin at 100 µg/ml, Cm at 30 µg/ml, Km at 100 µg/ml, Tp at 100 µg/ml, and spectinomycin at 100 µg/ml.

General DNA procedures. Plasmid DNA preparations were performed by using the alkaline lysis method (6). Sequence quality plasmid DNA was generated by using the Qiaprep spin miniprep kit (QIAGEN) and Wizard Plus SV Minipreps (Promega). Restriction endonuclease digestion of DNA was performed under the conditions recommended by the suppliers (Invitrogen, Roche, and New England Biolabs). Transformations of plasmid DNA to E. coli strains and other cloning strategies were performed according to standard protocols (32). Plasmids were transferred from E. coli to V. anguillarum and V. cholerae by triparental conjugation as previously described (38). Primers were designed by using Oligo 6.8 primer analysis software and purchased from Invitrogen. DNA and protein sequence analysis were carried out at the National Center for Biotechnology Information using the BLAST network service (3), with the Sequencher program version 4.2 (Genecodes) and also using the Tcoffee server (27).

Nucleotide and protein sequence accession numbers. The nucleotide sequence of the tonB2 cluster of V. anguillarum is deposited in GenBank under accession number AY644719. Accession numbers for additional sequences are referred to in the tables and figure legends.

Construction of plasmids. Plasmid pMS789 was generated by cloning the 1.7-kb PCR product, obtained using primers TonB2L and ExbB2U (Table 2), into vector pCR2.1-TOPO (Invitrogen). The following cycles were used to obtain the PCR product: 3 min at 95°C, followed by 30 cycles of 1 min at 95°C, 45 s at 55°C, and 2 min at 72°C. The XhoI-BamHI fragment from the PCR2.1-TOPO vector containing exbB2, exbD2, and tonB2 was subcloned into pACYC177 digested with XhoI-BamHI so that the exbB2, exbD2, and tonB2 genes are under the control of the constitutive Km resistance (Km^r) gene promoter. Plasmid pMS800 was generated by cloning the PCR product that contains the ttpC, exbB2, exbD2, and tonB2 genes in pCR2.1-TOPO using the TOPO cloning strategy from Invitrogen. The PCR product was obtained using total DNA from V. anguillarum 775 and the primers TtpCvaU and ExbD2vaL (Table 2) for 3 min at 95°C, followed by 30 cycles of 1 min at 95°C, 45 s at 55°C, and 2 min at 72°C. From the pCR2.1 construct, the 2-kb XhoI-BamHI fragment was cloned into pACYC177 digested with XhoI-BamHI, so that the four V. anguillarum genes are under the control of the constitutive Km^r gene promoter. To generate plasmids pMS800-exbB2 and pMS800-exbD2, we used inverse PCR with the primers exbB2invU and exbB2invL for exbB2 and exbD2invU and exbD2invL for exbD2 (Table 2) and pMS800 as template DNA. All four primers were engineered to contain an NcoI site. The same PCR cycles were used as described above with an extension time of 5 min. The PCR product was then digested with NcoI and ligated prior to electroporation into HB101 competent cells. The plasmids pMS789 and pMS800 and its mutant derivatives were all sequenced to assure that no mutation was induced during the PCR and cloning steps. Plasmid pMS989 was generated by first creating a construct containing an internal deletion of the ttpC open reading frame of V. anguillarum from two separate PCR products amplified with primers TtpCmutU1 and TtpCmutL1 for the first 500 bp and primers TtpCmutU2 and TtpCmutL2 (Table 2) for the last 500 bp. These two products were then used in a second PCR with primers TtpCmutU1 and TtpCmutL2 to obtain the ligated product of the first and last 500 bp of ttpC. This product was then cloned into pCR2.1-TOPO using the TOPO cloning strategy from Invitrogen. The Tp resistance (Tp^r) gene from p34E-TP (14) was then inserted as a blunt EcoRI product in the unique SspI site within the PCR product cloned in pCR2.1. The deleted ttpC gene, including the Tp^r gene, was cloned as a SpeI-XbaI product into the SpeI site of pDM4, resulting in pMS989. To generate pMS1029, a PCR product made with primers TtpCvcU and TtpCvcL (Table 2), containing the complete ttpC gene of V. cholerae was cloned in pCR8 by using the TOPO cloning strategy from Invitrogen. The Km^r gene from pUC4K (37) was cloned as a SalI fragment into the unique SalI site within the ttpC open reading frame. The ttpC gene with the Km^r gene inserted was then removed by restriction endonuclease digestion with EcoRI, followed by treatment with the Klenow fragment of DNA polymerase I, and cloned in the EcoRV site of pTW-MEV (39), resulting in pMS1029.

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TABLE 2. Primers used in this study

Construction of V. anguillarum and V. cholerae strains by conjugation and allelic exchange. To generate the nonpolar mutant in ttpC strain MS995, the plasmid pMS989, harboring the ttpC deletion and insertion of the Tp cassette, was transferred to V. anguillarum 775 by conjugation. Exconjugants were plated on plates containing rifampin (selection for V. anguillarum 775), Tp (selection for mutant ttpC gene), and 5% sucrose. Only cells that have recombined and have the Tp cassette in ttpC inserted at the wild-type locus will be able to grow on 5% sucrose. The mutants were confirmed by PCR to assure that the wild-type copy was replaced by the deletion containing the Tp^r cassette (data not shown). Strain MS1033 was generated by conjugating the suicide plasmid pMS1029, containing the ttpC gene of V. cholerae with the Km^r gene inserted in the SalI site, to V. cholerae CA401. Exconjugants were selected on TCBS agar plates supplemented with Km to obtain colonies that have the whole pM1029 integrated in the chromosome of V. cholerae. These strains were then streaked on plates containing 10% sucrose and Km to select for colonies that lost the vector but maintained the Km^r insertion. Loss of the vector was verified by PCR (data not shown).

RNA isolation. A 1:100 inoculum from an overnight culture was grown in minimal medium with appropriate antibiotics. Cultures were grown either with 4 µg of ferric ammonium citrate/ml (iron-rich) or with 2 µM EDDA (iron limiting). Total RNA was prepared when the culture reached an optical density at 600 nm (OD₆₀₀) of 0.3 to 0.5 using the RNAwiz (Ambion) isolation kit according to the manufacturer's recommendations.

RT-PCR analysis. One microgram of total RNA was reverse-transcribed to generate cDNA, using Moloney murine leukemia virus-reverse transcriptase (RT) from Ambion according to the manufacturer's recommendations. Prior to reverse transcription, total RNA was made DNA free by using TURBO DNA-free (Ambion) according to the manufacturer's recommendation. As a control, parallel samples were run in which RT was omitted from the reaction mixture. The primers used for the RT reaction were, RT-PCRvaTonB2L for V. anguillarum tonB2 and RT-PCRvcTonB2L for tonB2 of V. cholerae (Table 2). The cDNA was diluted five times, and a 10% aliquot was used in the subsequent PCR with the primers RT-PCRvaTonB2U and RT-PCRvaTonB2L for V. anguillarum and RT-PCRvcTonB2U and RT-PCRvcTonB2L for V. cholerae as listed in Table 2. For the PCR, Taq polymerase from New England Biolabs was used according to the manufacturer's recommendations.

Bioassays. A 50-µl portion of an overnight culture of each strain to be tested was seeded in a plate containing minimal medium, 0.7% agarose, and the iron chelator EDDA. The purified compounds spotted on top of the bioassay plates to determine the functionality of the TonB cluster genes in V. anguillarum and V. cholerae were anguibactin (1 mg/ml; our laboratory), enterobactin (1 mg/ml; Biophore Research), heme (20 µM; Sigma), ferrichrome (1 mg/ml; Sigma), and ferric ammonium citrate (500 µg/ml; Sigma). From all iron sources, 5 µl was spotted onto the bioassay plate.

Antibody generation. One rabbit per protein was immunized with a peptide for either TtpC-CSVYNKEQNRSVEALE- or TonB2-EQEHDLQRRQRSVPEC- of V. anguillarum. The peptides and antibodies were generated by Lampire Biological Laboratories according to their standard protocols.

Protein isolation. V. anguillarum cells grown in minimal medium were pelleted and resuspended in phosphate buffer (pH 6.7). Cells were lysed by six 10-s sonications at 10-s intervals. The lysates were centrifuged for 1 min at 13,000 rpm to remove whole cells. These samples were used as total proteins. To obtain total membrane proteins, the total protein suspension was pelleted for 40 min at 30,000 x g. The pellet contains the total membrane fraction, and the supernatant was used as a soluble fraction containing the periplasmic and cytoplasmic proteins. To obtain outer and inner membrane proteins, the total membrane fraction was extracted with a 1.5% final concentration of Sarkosyl. After centrifugation of 30 min at 30,000 x g, the outer membranes are in the pellet while the inner membrane proteins are in the supernatant.

Sucrose density gradients. V. anguillarum cells were grown in minimal medium, and whole-cell lysates were extracted as described above, with the exception that 10 mM HEPES, 25% sucrose, and 5 mM EDTA buffer was used. A 1-ml portion of the cell lysate was pipetted on top of a sucrose gradient in Ultra-Clear centrifuge tubes from Beckman (14 by 89 mm). Sucrose gradients were made by pipetting 2 ml of sucrose solutions in 5 mM EDTA at 55, 50, 45, 40, 35, and 30%. Sucrose gradients were run in a Beckman ultracentrifuge at 37,000 x g for 24 h. A total of 24 0.5-ml fractions were collected from each tube, and 20 to 80 µl was used for Western blot analysis.

In vivo formaldehyde cross-linking. These experiments were performed as described by Skare et al. (34). In brief, V. anguillarum cells were grown in minimal medium to an OD₆₀₀ of 0.8. Cells were washed twice in phosphate buffer (pH 6.7) and resuspended in the same buffer to an OD₆₀₀ of 0.5. A one-seventh aliquot was taken for the total membrane preparation, and formaldehyde was added to a 1% final concentration to the remaining cells suspension. Immediately after the addition of formaldehyde a sample was taken (0.5-min time point), and samples of equal volume were then taken at 5, 10, 20, and 30 min. The 30-min time point was done in duplicate, and one these samples was used for the boiled sample. From all samples total membrane proteins were obtained for Western blot analysis.

Western blot analysis. Protein samples were mixed with 2x Laemmli buffer (32), and the proteins were separated on 10% Bis-Tris criterion XT precast gels in 1x XT morpholinepropanesulfonic acid buffer (Bio-Rad). Gels were soaked for 15 min in transfer buffer (0.3% Tris, 1.44% glycine, and 20% methanol) and transferred to Protran nitrocellulose membrane (Schleicher & Schuell) in transfer buffer for 4 h at 300 mA in a Transblot cell (Bio-Rad). Membranes were blocked for 1 h in 5% skim milk in phosphate-buffered saline containing 0.5% Tween 20 (PBST). After the blocking step, membranes were washed for 10 min in PBST and then incubated with the primary antibody diluted in PBST for 16 h. The antibody concentrations used were as follows: TtpC, 1/4,000; TonB2, 1/4,000; FatA, 1/20,000; and FatB, 1/10,000. Membranes were washed three times for 10 min in PBST and incubated for 1 h in the secondary antibody goat anti-rabbit horseradish peroxidase. Membranes were washed six times for 10 min each time and then incubated for 5 min in SuperSignal West Pico chemiluminescent substrate (Pierce) according to the manufacturer's recommendations. Membranes were then exposed to Kodak X-Omat Blue XB-1 film and developed in a Kodak M35A X-Omat processor.

Top Abstract Introduction Materials and Methods Results Discussion References

 
A novel protein is essential for iron transport mediated by the TonB2 system. We recently reported that a transposon-generated mutant of a gene upstream of exbB2 in V. anguillarum resulted in an impairment of ferric-anguibactin transport (36). Sequence analysis indicated that this mutation occurred in an open reading frame that in V. cholerae was reported to encode a predicted protein with homology to the V. cholerae TolR, a designation that we adopted for this gene in V. anguillarum, and it has also now been used for other Vibrio spp. We also demonstrated that this mutation is polar on the transcription of the downstream genes that include exbB2, exbD2, and tonB2 (36). In the annotation of the V. cholerae genome there are two tolR genes. One of the tolR genes is part of the tolQRAB operon and encodes a predicted protein of 142 amino acids that shares homology with the tolR gene of E. coli. The second tolR gene of V. cholerae is located upstream of exbB2 and encodes a predicted protein of 457 amino acids that shares no significant general homology with the sequence of the other tolR gene in V. cholerae. On the other hand, this second tolR gene product shares 65% identity with the 444 amino acids of the open reading frame we identified in V. anguillarum by transposition mutagenesis, and as a consequence we named it TolR (36). However, these assignations were incorrect, likely because of the difficulty in assessing the divergence between these genes in the vibrios and in E. coli. The taxonomic issue is complicated even further because the homology of this open reading frame to Exb-related proteins is confined to just the carboxy-terminal region that inserts in the membrane, with three transmembrane segments. The remaining part of the protein has no homology to this family of proteins. Thus, although this open reading frame has a domain with homology to the MotA/TolQ/ExbB family, it falls into a class of its own. Based on our results in the present study we propose that the tolR gene found upstream of exbB2 in both V. anguillarum and V. cholerae be renamed ttpC, encoding TtpC (for TonB2 complex-associated transport protein C).

To investigate the possible function of the ttpC gene product in iron transport, we used the double mutant strain MS570 (Fig. 1A) that is affected in the expression of TonB1 (Cm cassette insertion in the tonB1 gene) and harbors the polar insertion in ttpC that also affects the expression of exbB2, exbD2, and tonB2 (exbB2-exbD2-tonB2) (36). This strain cannot transport any of the TonB-dependent ferric-iron sources tested, including ferric-anguibactin. Ferric ammonium citrate is an iron source that does not require TonB for internalization and is used as a positive control for the viability of the cells. Table 3 shows that when this strain was complemented with the cluster exbB2, exbD2, and tonB2 harbored by plasmid pMS789 (Fig. 1B), none of the TonB-dependent iron sources tested were positive in bioassays with this strain. Transport of ferric-anguibactin and other iron sources could only be restored by using plasmid pMS800 harboring ttpC in addition to exbB2-exbD2-tonB2 (Fig. 1B). This result demonstrates that TtpC is required for TonB2-mediated iron transport. To determine whether ExbB2 and ExbD2 are also required for transport by the TonB2 system, we complemented strain MS570 harboring the polar ttpC and the tonB1::Cm^r mutation with two pMS800 plasmid derivatives with internal deletions in either exbB2 (pMS800-exbB2) or exbD2 (pMS800-exbD2). Table 3 shows the results of the bioassays performed with these strains, and it can be concluded that besides TtpC, both ExbB2 and ExbD2 are essential for TonB2-mediated iron transport. To assess whether TtpC is also necessary for TonB1-mediated iron transport, we used a strain containing the wild-type tonB1 gene in which the ttpC gene was disrupted by a Tp cassette, 775 ttpC::Tp, resulting in a knockout of ttpC (Fig. 1A). This mutation was nonpolar on the expression of exbB2-exbD2-tonB2, as determined by RT-PCR analysis of the tonB2 transcripts in this strain (Fig. 1C). Table 3 clearly illustrates that this ttpC mutant is still able to transport heme and ferrichrome, but not anguibactin and enterobactin, which are only transported when a functional TonB2 system is present. This demonstrates that TonB1-mediated iron transport does not require TtpC and emphasizes the need for TtpC for the TonB2 system.

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FIG. 1. Schematic representation of the TonB2 cluster genes in V. anguillarum and plasmids used. (A) Schematic of the mutants used. Also shown is the site of insertion of the Tn10Kmr transposon. (B) Schematics of the complementing clones pMS789 and pMS800 used throughout the present study. (C) RT-PCR results of the ttpC::Tp mutant. Lane 1, molecular weight marker (1-kb ladder; New England Biolabs); lane 2, no RT enzyme used; lane 3, RT enzyme used in the RT reaction.

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TABLE 3. Bioassay results

Subcellular localization and cross-linking studies of TtpC. TtpC is necessary for TonB2-mediated iron uptake and, since in E. coli TonB forms a complex with ExbB and ExbD, we hypothesize that in V. anguillarum the TonB2 system might form a complex consisting of TonB2, ExbB2, ExbD2, and TtpC. To test our hypothesis, we used in vivo formaldehyde cross-linking (see Materials and Methods and reference 34). However, before performing the cross-linking experiments we needed to ascertain the subcellular localization of the TonB2 and TtpC proteins in V. anguillarum cells.

To determine the subcellular localization of these proteins, we prepared total cell lysates from V. anguillarum strain 775 and isolated total, outer, inner, and cytoplasmic/periplamic proteins (see Materials and Methods). Using polyclonal antibodies generated against TtpC- and TonB2-specific peptides, we performed Western blot analysis to assess the localization of these proteins and their complexes. Figure 2A shows that both the TonB2 and the TtpC antisera are specific for the respective proteins since we do not see a reaction in the mutant, although the TonB2 antiserum shows a cross-reaction with another protein of higher molecular weight. When the cells are fractionated and the total membranes are treated with Sarkosyl we found both TonB2 and TtpC in the total membrane fraction and in the inner membrane fraction (Fig. 2B, lanes 4 and 8). We do not find any TtpC or TonB2 in the cytoplasm or periplasm or in the outer membrane fraction. We did see a reaction with the TonB2 antiserum with a larger protein in the cytoplasm or periplasm; we do not know the nature of this protein. When the same fractions were probed with antiserum to the highly abundant outer membrane receptor FatA, we found that FatA is present in the total and outer membrane fraction (Fig. 2C, lanes 1 and 3) and in minor amounts in the inner membrane fraction (Fig. 2C, lane 4), indicating that the fractions have little contamination from other cellular compartments. However, from this experiment alone we cannot conclude that both the TonB2 and the TtpC proteins are exclusively inner membrane proteins, since extraction with Sarkosyl can solubilize some outer membrane proteins (11). It should be noted that in silico prediction programs predict that both the TonB2 and the TtpC proteins are located in the inner membrane. To determine the exact cellular localization of these two proteins, we sedimented the proteins from the whole-cell lysates in sucrose density gradients (see Materials and Methods and reference 24) and used the specific antibodies for their detection in the fractions. We also used specific antibodies to FatA (1) as a control for the outer membrane fractions. The status of the sucrose gradients was determined by measuring the refractive index from a sucrose gradient in which no protein sample was added (not shown). After we collected the fractions, the proteins were transferred to nitrocellulose membranes, and the subsequent Western blots show that TonB2 is found in fractions 2 to 12 (Fig. 3A), whereas TtpC is found in fractions 3 to 23 (Fig. 3B). FatA spans fractions 8 to 23 with its peak from fraction 17 to 20 (Fig. 3C). Figure 3D shows the quantification of these results by densitometry. Both TtpC and TonB2 show a curve that is shifted to the left (top of gradient), where there is no or little FatA. Consequently, from these results together with the fractionation experiments in Fig. 2, we can conclude that TonB2 and TtpC must be inner membrane proteins, although it seems that TtpC can also be found in a small proportion in the outer membrane, as indicated by the TtpC second peak that overlaps with the region where FatA is found in large amounts.

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FIG. 2. Western blot analysis of the cellular localization of TonB2 and TtpC. Proteins were isolated as described in Materials and Methods. (A) Western blot with antibodies against TtpC (above) and TonB2 (below) on wild-type V. anguillarum and the respective mutant strains. (B) Western blot with TonB2 antibodies (lanes 1 to 4) and TtpC antibodies (lanes 5 to 8). Cellular fractions loaded are indicated on top of the figure. (C) Western blot using the same samples as in panel B but antibodies to FatA were used to indicate the purity of the fractionation. MW, molecular weight (in thousands).

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FIG. 3. Sucrose density gradients. (A to C) Western blot analysis on fractions from the sucrose gradients using antibodies to TonB2 (A), TtpC (B), and FatA (C). (D) Density of each band in panels A to C plotted against the sample number.

In the cross-linking experiments we used the TtpC antiserum, rather than the TonB2 antiserum, because the latter has a cross-reaction with another membrane protein of higher molecular weight, as indicated by Western blot analysis (Fig. 2). Although there are several bands that can be detected with anti-TtpC in Western blots (Fig. 2), none of these bands can be detected in a ttpC mutant; we hypothesize that these bands result from oligomerization of TtpC, perhaps due to disulfide bonding of the sole cysteine in the inner membrane domain of this protein. To perform the cross-linking experiment, wild-type V. anguillarum was incubated in phosphate buffer (pH 6.7) containing 1% formaldehyde prior to the isolation of total membrane proteins. The Western blot of cross-linked samples shown in Fig. 4, allowed us to identify four complexes that contain TtpC. These four complexes disappear when the cross-linked sample is boiled. Based on their size, the two bands that can still be detected in the boiled sample are probably TtpC dimers and tetramers even though 2-mercaptoethanol was added to the loading buffer since they are not present in a cross-linked sample (30 min boiled) of the TtpC mutant strain MS570 (Fig. 4A, lane 8). One of the cross-linked complexes (complex 1) forms after a few seconds but disappears with time (compare lane 2 with lane 5), possibly shifting to higher-molecular-weight complexes by the addition of other proteins, whereas complexes 2 to 4 form later, and some complexes stay for longer periods of time. These four complexes are also detected when the polar TtpC mutant (ttpC::Tn10Km) is complemented with the wild-type cluster ttpC, exbB2, exbD2, and tonB2 cloned in plasmid pMS800 that restores the wild-type phenotype (Fig. 4B). The in vivo formaldehyde cross-linking experiment was also carried out with mutants of tonB2, exbB2, and exbD2. In cross-linking experiments with cells deficient in TonB2 (strain MS801) none of the four complexes could be found (Fig. 5A). Since we do see less free TtpC in the membrane of the tonB2 mutant, it is possible that the complexes are not abundant enough to be detected. In both the exbB2 and the exbD2 mutant stains (MS570/pMS800-exbB2 and strain MS570/pMS800-exbD2, respectively, only complex 4 could be detected (Fig. 5B and C). Complex 4 that forms after extended incubation with formaldehyde possibly consists of TtpC and TonB2. Since we do find this complex in the exbB2 and exbD2 mutant strains, we can conclude that the proteins encoded by these genes are not part of complex 4. However, the resolution of the area where complex 4 runs is not very good due to the large size of this complex. It could be that complex 4 consists of several complexes and that some do contain ExbD2 or ExbB2. Complex 4 could also include an outer membrane receptor such as the ferric-anguibactin receptor FatA. We tried cross-linking experiments with a strain lacking FatA, but in this strain complex 4 is still present (data not shown). This could be simply explained by the fact that the TonB2 complex can interact with several receptors, such as those for heme, ferrichrome, enterobactin, and vanchrobactin, and these receptors are all expressed in the FatA-deficient strain. From the cross-linking experiment it can be concluded that TtpC is found in at least four distinct complexes; these complexes are all absent in a TonB2-deficient strain, and complexes 1 to 3 are also not present in the exbB2 and exbD2 mutants.

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FIG. 4. Western blot on total membrane proteins from formaldehyde cross-linked cells. The TtpC antibody was used to detect TtpC containing complexes. (A) Cross-linking of wild-type V. anguillarum strain 775. Marker (M) is shown schematically on the left hand side (Kaleidoscope Bio-Rad). Different complexes are indicated on the right-hand side. Lane 1, no formaldehyde used (more sample was loaded than lanes 2 to 6); lanes 2 to 6, 1% formaldehyde for 0.5 to 30 min as indicated on top of each lane; lane 7, 1% formaldehyde for 30 min, followed by 10 min of incubation at 100°C; lane 8, cross-linking of the ttpC mutant MS570 for 30 min, followed by 10 min of incubation at 100°C. (B) Same as panel A but with strain MS570 complemented with plasmid pMS800.

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FIG. 5. Western blot of total membrane proteins from formaldehyde cross-linked samples of different mutant V. anguillarum strains. Cells were incubated for various times in 1% formaldehyde as indicated at the top of each lane. The complexes present are indicated on the right-hand side using the same numbering used for the wild-type strain in Fig. 4. (A) tonB2 mutant (MS801); (B) exbB2 mutant (MS570/pMS800-exbB2); (C) exbD2 mutant (MS570/pMS800-exbD2).

We found TtpC in the membrane in all mutant strains used, but it seemed to be present in lower amounts in the tonB2 mutant (Fig. 5A). Although the same amounts of cells as measured by OD₆₀₀ were loaded in each lane, the Western blots cannot be compared directly. Perhaps TonB2 is in some way stabilizing TtpC in the membrane, whereas TtpC is not required to stabilize TonB2, as can be seen by the fact that we could detect wild-type levels of TonB2 in the TtpC mutant strain (data not shown).

Complementation of E. coli and V. cholerae tonB mutants with the tonB2 cluster genes from V. anguillarum. The genes exbB2, exbD2, and tonB2 of V. anguillarum cannot complement the tonB mutant strain KP1032 of E. coli (36). Because in the present study we showed that TtpC is necessary for TonB2-mediated iron uptake, we complemented E. coli KP1032 with a construct that expresses ttpC, exbB2, exbD2, and tonB2. The resulting E. coli strain is still not able to transport any of the TonB-dependent iron sources tested (Table 4) and was still resistant to phage 80.

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TABLE 4. Bioassay results with complemented V. cholerae and E. coli strains

We therefore investigated whether the TonB2 cluster genes can complement the TonB2 mutant strains of the more closely related V. cholerae. As shown in Table 4, the V. cholerae exbB2 polar mutant DOV221 (26) cannot be complemented with the exbB2, exbD2, and tonB2 genes from V. anguillarum (expressed from plasmid pMS789), even though the V. anguillarum TonB2_Va protein is expressed in this V. cholerae strain, as detected by Western blot analysis (Fig. 6, lane 1). Surprisingly, the antibody against TonB2_Va of V. anguillarum does not react with the TonB2_Vc of V. cholerae (Fig. 6, lane 2). When the plasmid pMS800 that includes the V. anguillarum ttpC was used, the V. cholerae strain recovered the ability to transport enterobactin, a siderophore whose transport is mediated by the TonB2 complexes of V. cholerae and V. anguillarum (33, 36). Results similar to those obtained for the single V. cholerae exbB2 mutant were obtained with the V. cholerae exbB2 polar and exbB1 nonpolar mutant strain DOV300 (26). In the latter double exbB1 and exbB2 knockouts of V. cholerae we could demonstrate that both heme and ferrichrome could also be transported by the TonB2 system of V. anguillarum as long as TtpC of V. anguillarum is present (Table 4). Since the V. cholerae mutants used have the wild-type ttpC_Vc gene of V. cholerae, it is clear that the TonB2 system of V. anguillarum needs its own TtpC_Va, even though the ttpC genes of V. anguillarum and V. cholerae share 65% identity. An alignment of the two TtpC proteins is shown in Fig. 7 with domain features identified by the program PSORT (31) underlined.

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FIG. 6. Western blot analysis of TonB2 of V. anguillarum expressed in V. cholerae. Lane 1, V. cholerae DOV221 expressing TonB2 from V. anguillarum from plasmid pMS789; lane 2, wild-type V. cholerae CA401.

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FIG. 7. Amino acid sequence alignment of TtpC from V. anguillarum and V. cholerae. Va, Vibrio anguillarum; Vc, Vibrio cholerae. Gray-shaded amino acids indicate identical residues, and light gray amino acids indicate similar amino acids. Domains identified by using the computer program PSORT are underlined.

TtpC-containing bacteria and correlation with the presence of TonB2. TtpC homologues are found in all Vibrio species sequenced thus far and in a few other bacteria not only limited to the -proteobacteria (Table 5). Among the vibrios, the TtpC proteins share high homology, ranging from 57 to 66% identity and from 73 to 80% similarity. However, there is a species specificity for the TtpC protein since the ttpC gene from V. cholerae cannot replace the V. anguillarum ttpC in the TonB2 system of V. anguillarum. In general, microorganisms in which we could identify a TtpC homologue also have at least two TonB systems, and the ttpC gene is part of the tonB2 cluster. It is quite possible that ttpC is only found associated with the TonB2 systems, but some of the bacterial genomes are not sufficiently annotated to easily identify all of the TonB systems present. To understand some of the evolutionary possibilities, we aligned known TonB2 proteins sequences and TonB from E. coli (Fig. 8). We did not include the TonB2 proteins of Microbulbifer degradans, Methylococcus capsulatus, Desulfovibrio desulfuricans, and Desulfotalea psychrophila since these TonB proteins are not annotated as such.

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TABLE 5. Similarity of V. anguillarum TtpC with the TtpC encoded by other bacteria

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FIG. 8. Amino acid sequence alignment of most TonB2 proteins and TonB from E. coli. The suffixes _1 and _2 are randomly assigned to bacteria that harbor more than one TonB2. The bacterial species (and GenBank accession numbers) of the TonB sequences used for the alignment are as follows: Ec, Escherichia coli (AAB59066); Pa, Pseudomonas aeruginosa (AAF04082); So, Shewanella oneidensis (NP_720081 and AAN54880); Il, Idiomarina loihiensis (YP_156236 and YP_156496); Vp, Vibrio parahaemolyticus (NP_799665 and BAC58426); Vv, Vibrio vulnificus (NP_759823 and AAO09350); Pd, Photobacterium damselae (CAD68981); Pp, Photobacterium profundum (CAG23089); Vf, Vibrio fischeri (YP_206734 AND YP_206154); Va, Vibrio anguillarum (AAV48777); Vc, Vibrio cholerae (AAC69456); As, Actinobacillus suis (AAW21313); and Ap, Actinobacillus pleuropneumoniae (AAR95695).

Figure 8 shows that the TonB2 sequences from Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae, and A. suis are generally longer than the Vibrio TonB2 proteins and are like the E. coli TonB, containing the proline-rich domain that spans amino acids 64 to 100. Interestingly, these TonB2 systems do not have a TtpC homologue, and there seems to be a correlation between the presence of TtpC and the absence of the proline-rich region in TonB2.

The V. cholerae TtpC is essential for TonB2-mediated transport of enterobactin in this bacterium. To explore whether TtpC is also essential for TonB2-mediated transport in other bacteria, we mutated the ttpC gene of V. cholerae CA401 by inserting the Km^r gene cassette in the unique SalI site. This insertion is nonpolar, since we can still detect tonB2 transcripts in this mutant by RT-PCR (Fig. 9). Seliger et al. (33) showed that in V. cholerae the transport of enterobactin is exclusively dependent on TonB2. We therefore used the ability to transport enterobactin to measure whether the V. cholerae TtpC is necessary for TonB2-mediated transport. The ttpC mutant and wild-type V. cholerae strains were then used in a bioassay with ferric ammonium citrate, heme, and enterobactin as iron sources. As shown in Table 6, the ttpC mutant fails to transport enterobactin, whereas ferric ammonium citrate is still transported, indicating that, as in V. anguillarum, TonB2-mediated iron uptake in V. cholerae also requires TtpC. We included heme as an iron source since heme transport depends on either the TonB1 or the TonB2 system. As expected, the bioassay is positive for heme uptake in the ttpC mutant, suggesting that transport mediated by the TonB1 system in V. cholerae does not require TtpC.

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FIG. 9. RT-PCR analysis of the ttpC::Km mutant of V. cholerae. Lane 1, marker (1-kb ladder; New England Biolabs); lane 2, RT-PCR of tonB2 in the ttpC::Km mutant; lane 3, no RT enzyme used.

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TABLE 6. Bioassays results for the V. cholerae ttpC mutant

Top Abstract Introduction Materials and Methods Results Discussion References

 
The siderophore anguibactin is synthesized via a nonribosomal peptide synthetase mechanism that is entirely encoded on the virulence plasmid pJM1 of the fish pathogen V. anguillarum (15). The secreted siderophore is, once bound to ferric-iron, transported back into the cells cytosol via a specific transport system that starts with the outer membrane receptor FatA (1). FatA binds the ferric-siderophore and transports it to the periplasm, where it must interact with the lipoprotein FatB. Once in the periplasm, the iron complex is then further internalized by the integral membrane proteins FatC and FatD (21). It was demonstrated in E. coli that transport across the outer membrane requires energy from the proton motive force of the inner membrane that is transduced to the outer membrane (9). The energy-transducing complex consists of the proteins TonB, ExbB, and ExbD. The TonB protein is the actual energy transducer, whereas the ExbB and ExbD proteins are necessary for the stability of TonB (2). TonB systems have been found in almost all bacteria sequenced thus far, and some bacteria have more than one TonB system. Studies of TonB proteins in other systems have focused mainly on their importance in iron uptake and virulence, and many similarities with E. coli TonB have been observed (5, 17, 20, 33).

V. anguillarum harbors two TonB systems (36), TonB1 and TonB2, and these systems are redundant with respect to the transport of heme and ferrichrome. Only TonB2, however, can also transport anguibactin, enterobactin, and vanchrobactin (36). In the present study we have shown that just the classic TonB2 system (TonB2, ExbB2, and ExbD2) is not sufficient for the transport of the TonB2-specific iron compounds in V. anguillarum. A fourth protein, TtpC, was identified that is essential for iron transport mediated by the TonB2 system. The ttpC gene encodes a polypeptide of 49 kDa with three predicted transmembrane domains. TtpC, however, is not essential for transport mediated by the TonB1 system. Furthermore, the cross-linking experiments show that TtpC can be cross-linked in at least four complexes, and some of these complexes are missing in mutant strains of TonB2, ExbB2, and ExbD2, suggesting possible complex formation of TtpC, TonB2, ExbB2, and ExbD2 in V. anguillarum. This differs from the E. coli TonB system, where the complex consists of only TonB, ExbB, and ExbD. Unlike the E. coli TonB, TonB2 from V. anguillarum does not seem to shuttle between the membranes and is solely found in the inner membrane.

We identified ttpC gene homologues in many bacteria, including all Vibrio species sequenced thus far, with the TtpC homologues sharing high similarity. Even though the TtpC proteins from V. anguillarum and V. cholerae share 66% identity and 80% similarity, the TtpC_Vc protein from V. cholerae cannot complement a mutation in the ttpC_Va gene of V. anguillarum in V. cholerae. However, the V. cholerae TtpC_Vc is necessary for TonB2_Vc-mediated transport in V. cholerae, since a ttpC_Vc mutant in V. cholerae fails to transport enterobactin. We speculate that each specific TtpC plays an essential role in the specific TonB2-mediated transport in all bacteria that harbor this protein. The dissection of the mechanism by which TtpC operates together with the TonB2 cluster in specific iron transport will contribute to our understanding of the mechanism of energy transduction in bacteria other than E. coli.

 
This study was supported by grant AI19018 and GM64600 from the National Institute of Health to J.H.C.

We thank S. M. Payne for the V. cholerae mutant strains DOV221 and DOV300 and K. Postle for the E. coli TonB mutant strain KP1032.

 
* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, L-220, Oregon Health and Science University, 3181 SW Sam Jackson Park Rd., Portland, OR 97239-3098. Phone: (503) 494-7583. Fax: (503) 494-6862. E-mail: crosajor{at}ohsu.edu.

Published ahead of print on 22 December 2006.

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Journal of Bacteriology, March 2007, p. 1803-1815, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.00451-06
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