Abstract
Vibrio alginolyticus, Vibrio fluvialis, and Vibrio parahaemolyticus utilized heme and hemoglobin as iron sources and contained chromosomal DNA similar to several Vibrio cholerae heme iron utilization genes. A V. parahaemolyticus gene that performed the function of V. cholerae hutA was isolated. A portion of the tonB1 locus of V. parahaemolyticus was sequenced and found to encode proteins similar in amino acid sequence to V. cholerae HutW, TonB1, and ExbB1. A recombinant plasmid containing the V. cholerae tonB1 and exbB1D1 genes complemented a V. alginolyticus heme utilization mutant. These data suggest that the heme iron utilization systems of the pathogenic vibrios tested, particularly V. parahaemolyticus and V. alginolyticus, are similar at the DNA level, the functional level, and, in the case of V. parahaemolyticus, the amino acid sequence or protein level to that of V. cholerae.
Vibrios are gram-negative marine bacteria which often cause disease in humans. Infections by Vibrio cholerae (7), Vibrio fluvialis (4, 15), and Vibrio parahaemolyticus (12) are acquired through consumption of contaminated water or seafood, and they lead to excessive watery diarrhea (V. cholerae and V. fluvialis) or to acute gastroenteritis (V. parahaemolyticus). Vibrio alginolyticus (5, 20) causes extraintestinal infections, such as wound infections.
Bacterial pathogens must acquire iron inside the host to multiply to numbers sufficient to cause disease (for reviews, see references 14 and 19). V. cholerae and V. parahaemolyticus acquire iron by at least two methods. Under low-iron conditions, they produce the siderophores vibriobactin (9) and vibrioferrin (25), respectively. These low-molecular-weight compounds bind iron with high affinity and are transported back into the cell. Both Vibrio species also acquire iron from heme or hemoglobin (22, 23, 26). V. fluvialis and V. alginolyticus produce siderophores (2), but neither has been tested for the ability to utilize heme or hemoglobin as an iron source.
V. cholerae heme iron utilization involves the following genes: hutA, which encodes the heme receptor (10, 11); tonB1, which encodes an inner membrane protein required for the transport of heme into the periplasm; exbB1D1, which encodes the inner membrane proteins required for TonB function; and hutB and hutCD, which encode a periplasmic binding protein and a cytoplasmic membrane permease, respectively, which are involved in the transport of heme to the cytoplasm (18). A second V. cholerae TonB system encoded by tonB2 and exbB2D2, which also may be involved in heme transport, recently has been identified (18). To date, no other gram-negative bacterium has been found to contain two TonB systems.
Heme iron utilization systems have been studied for V. cholerae and Vibrio vulnificus (13), but heme iron utilization systems in other vibrios have not been well characterized. The goals of this study were to (i) identify vibrios that can acquire iron from heme or hemoglobin, (ii) determine if these species have heme utilization and tonB-like genes similar to those in V. cholerae, and (iii) determine if heme utilization proteins of V. cholerae and other heme-utilizing vibrios have similar amino acid sequences and whether they are functionally interchangeable.
Testing Vibrio species for heme and hemoglobin iron utilization.
The ability of various strains to use several iron-containing compounds was tested. In the assay, cultures were seeded into Luria (L) agar containing the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDA), and 5 μl of each iron-containing compound was spotted onto the media. All of the wild-type vibrios tested, including both clinical and environmental isolates, exhibited substantial zones of growth around the heme and hemoglobin spots (Table 1). V. cholerae DHH-11, a heme iron utilization deletion mutant (Table 2), exhibited no detectable growth around the heme or hemoglobin spots, and it served as the negative control (Table 1). All of the strains tested could utilize FeSO4 as an iron source.
TABLE 1.
Growth of different Vibrio species with heme and hemoglobin as iron sources
| Bacterial strain | Zone of growth (mm) with:
|
||||
|---|---|---|---|---|---|
| Heme (20 μM) | Hemoglobin (5 μM) | FeSO4 (10 mM) | BE2-542 | DTH-1 | |
| V. cholerae | |||||
| CA401a | 21 | 12 | 21.0 | NDb | ND |
| DHH-11c | NGd | NG | 10.0 | ND | ND |
| V. parahaemolyticuse | |||||
| 474801 | 22.5 | 22.5 | 27.5 | ND | ND |
| M47314 | 25.0 | 25.0 | 30.0 | ND | ND |
| 115 | 22.5 | 20.0 | 25.0 | ND | ND |
| V. fluvialisc BE2-819 | 32.5 | 35.0 | 40.0 | ND | ND |
| V. alginolyticuse | |||||
| BE2-542 | 20.0 | 17.0 | 26.0 | 18.0 | 19.0 |
| DTH-1 | NG | NG | 26.0 | 22.0 | 21.0 |
1 × 104 bacteria/ml seeded into L-EDDA agar.
ND, not determined.
5 × 103 bacteria/ml seeded into L-EDDA agar.
NG, no growth.
1 × 105 bacteria/ml seeded into L-EDDA agar.
TABLE 2.
Bacterial strains and plasmids
| Strain or plasmid | Description or relevant phenotype | Source or reference |
|---|---|---|
| Strains | ||
| V. parahaemolyticus | ||
| 474801 | Clinical isolate | Texas Department of Health |
| M47314 | Clinical isolate | Texas Department of Health |
| 115 | Environmental isolate | P. Baumann |
| V. alginolyticus | ||
| BE2-542 | Clinical isolate; Ampr | Texas Department of Health |
| DTH-1 | Heme utilization mutant of BE2-542 | This study |
| V. fluvialis BE2-819 | Clinical isolate | Texas Department of Health |
| V. cholerae | ||
| DHH-11 | TonB− Vib− mutant of CA401 | 10 |
| CA401 | Classical strain | 8 |
| E. coli 1017 | Ent::Tn5 mutant of HB101 | S. M. Payne |
| Plasmids | ||
| pHUT2 | Tetr; 16-kb Sau3A fragment of V. cholerae CA401 DNA cloned into pLAFR3; encodes outer membrane receptor protein HutA | 10 |
| pHUT3 | Ampr; 3-kb HindIII-SalI fragment of pHUT2 cloned into pAT153; encodes outer membrane receptor protein HutA | 10 |
| pHUT10 | Cmr; 10.3-kb HindIII fragment of V. cholerae CA401 DNA cloned into pACYC184; contains tonB1, exbB1D1, and hutBCD | 10 |
| pHUT7 | Cmr; 6.7-kb SalI-HindIII fragment from pHUT10::Tn5f (10) cloned into pACYC184; contains tonB1, exbB1D1, and hutBCD | 18 |
| pHUT11 | Cmr; 3.1-kb HpaI fragment from pHUT7 cloned into the EcoRV site of pACYC184; contains tonB1 and exbB1D1 | This study |
| pOUT11 | Ampr; 10.5-kb HindIII fragment of V. cholerae CA401 cloned into pWSK29; encodes TonB2 system | 18 |
| pTONB1 | Cmr; 1.1-kb PCR fragment containing V. cholerae tonB1 cloned into pACYC184 | 18 |
| pTEE1 | Cmr; 2.1-kb PCR fragment containing V. cholerae tonB1 and exbB1D1 cloned into pACYC184 | 18 |
| pPHU1 | Tetr; 7-kb Sau3A fragment of V. parahaemolyticus 474801 DNA cloned into pLAFR3; contains hutA-like gene | This study |
| pPHU2 | Cmr; 2.8-kb HindIII fragment of V. parahaemolyticus cloned into pACYC184; contains tonB1 and part of exbB1 and phuW | This study |
Determining if the Vibrio species contain DNA similar to V. cholerae heme iron utilization genes.
Chromosomal preparations (17) were digested with HindIII, electrophoresed on agarose gels, and subjected to Southern blotting on charged nylon membranes (Boehringer Mannheim, Indianapolis, Ind.) under low-stringency conditions (21). Chromosomal DNA from the noncholera strains was loaded onto the gels at a threefold-higher concentration than that of the V. cholerae chromosomal DNA. The Genius DIG DNA labeling and detection system with CDP Star (Boehringer Mannheim) was used to label the probes and detect hybridization. The hutA probe contained a 1.8-kb EcoRI fragment from pHUT3 (10). The probes for tonB1, exbB1, and hutC and for tonB2 were generated by PCR from pHUT7 and pOUT11 (18), respectively, and were internal fragments of each gene. All of the strains tested contained DNA sequences similar to tonB2 (Fig. 1; Table 3), exbB1, and hutC (Table 3), whereas all the strains except V. fluvialis contained DNA sequences similar to hutA (Table 3) and tonB1 (Fig. 1; Table 3). The hybridization signal generated with the tonB2 probe in V. fluvialis DNA (Fig. 1) was barely detectable, but upon prolonged exposure of the blot to X-ray film, a distinct signal at 5.2 kb was detected (Table 3). The tonB1 probe hybridized to different-sized HindIII fragments than the tonB2 probe in V. alginolyticus and the three strains of V. parahaemolyticus, suggesting that these strains contained two distinct tonB-like genes (Fig. 1; Table 3). The hutA probe hybridized to two different fragments in all the strains in which a signal was generated (Table 3). This may reflect the presence of an internal HindIII site in each gene. In all the Southern blots, the hybridization signal of each probe which hybridized to DNA from the V. cholerae chromosome was significantly more intense than those obtained with the other Vibrio species.
FIG. 1.
Autoradiograms of Southern blots of Vibrio strains probed with V. cholerae tonB1 (A) and tonB2 (B). The following strains were tested: V. cholerae CA401 (lane 1); V. parahaemolyticus 474801, M47314, and 115 (lanes 2 to 4, respectively); V. alginolyticus BE2-542 (lane 5); and V. fluvialis BE2-819 (lane 6). Size markers are indicated on the left.
TABLE 3.
Presence of heme iron utilization genes in Vibrio species
| V. cholerae DNA probe | Fragment size(s) (kb) obtained in hybridization to chromosomal DNA froma:
|
||||
|---|---|---|---|---|---|
|
V. parahaemolyticus
|
V. alginolyticus BE2-542 | V. fluvialis BE2-819 | |||
| 474801 | M47314 | 115 | |||
| hutA | 2.9, 0.6 | 2.9, 0.6 | 2.9, 0.6 | 6.0, 2.0 | NDHb |
| hutC | 3.5 | 4.4 | 3.5 | 9.0 | 2.9 |
| tonB1 | 2.8 | 2.8 | 2.8 | 9.0 | NDH |
| exbB1 | 2.8 | 2.8 | 2.8 | 9.0 | 6.1 |
| tonB2 | 4.4 | 3.8 | 3.8 | 1.6 | 5.2c |
Values are approximate sizes of the HindIII fragments to which the probes hybridized.
NDH, no detectable hybridization.
Hybridization signal was weaker than that observed with other species.
Isolation of a heme iron utilization mutant of V. alginolyticus.
To isolate a heme iron utilization mutant of V. alginolyticus BE2-542, diethylsulfate mutagenesis and nalidixic acid enrichment were performed as previously described (10). Colonies were screened for growth on L-EDDA-hemin agar, and the heme utilization mutant, DTH-1, was isolated. When assayed as described above, DTH-1 exhibited no detectable growth around the spots containing heme or hemoglobin (Table 1). To determine if DTH-1 produced and/or utilized the V. alginolyticus siderophore, fully grown cultures of the mutant and its parent strain were spotted onto L-EDDA agar seeded with either bacterial strain. Significant zones of growth occurred around the spots of the mutant and the wild type on both plates, indicating that DTH-1 both produces and utilizes its siderophore. These data suggest that the heme utilization defect did not affect the siderophore synthesis or transport system.
Complementation of V. alginolyticus DTH-1 with the V. cholerae TonB1 system.
To determine if the defect in DTH-1 was in a TonB system, the mutant was transformed by electroporation (18) with pHUT11, which contains V. cholerae tonB1 and exbB1D1, or with the vector pACYC184. DTH-1/pHUT11 grew as well as the parent strain, BE2-542, in L-EDDA-hemin broth (Table 4), suggesting that DTH-1 has a defective TonB, ExbB, or ExbD protein and that the comparable V. cholerae protein is functionally interchangeable. Combined with the data in Table 1 showing that the mutation in DTH-1 had no effect on siderophore production or transport, these data indicate that the V. alginolyticus siderophore transport system may use a second TonB system. Occhino et al. (18) recently determined that mutations in either the TonB1 system or the TonB2 system in V. cholerae had no impact on heme iron or vibriobactin uptake in V. cholerae. However, when both TonB systems in V. cholerae were defective, both heme iron and vibriobactin utilization were disrupted. Thus, either TonB system in V. cholerae can function in both siderophore and heme uptake. This does not appear to be the case in V. alginolyticus, where apparent disruption of one of the TonB systems leads to the loss of only heme iron utilization, not siderophore uptake. Thus, V. alginolyticus may contain one TonB system that plays a role in heme uptake and another that plays a role in siderophore uptake.
TABLE 4.
Growth of V. alginolyticus DTH-1 and E. coli 1017 transformed with various recombinant heme iron utilization plasmids
| Bacterial strain | Absorbancea of cultures grown in:
|
||
|---|---|---|---|
| L broth | L-EDDA broth | L-EDDA-hemin broth | |
| V. alginolyticus | |||
| BE2-542 | 1.40 | 0.12 | 1.27 |
| DTH-1/pHUT11 | 1.40 | 0.08 | 1.20 |
| DTH-1/pTEE1 | 1.53 | 0.04 | 1.30 |
| DTH-1/pTONB1 | 1.30 | 0.03 | 0.12 |
| DTH-1/pACYC184 | 1.50 | 0.12 | 0.12 |
| E. coli | |||
| 1017/pHUT10/pPHU1 | 1.88 | 0.14 | 1.56 |
| 1017/pHUT10/pHUT2 | 2.40 | 0.13 | 1.42 |
| 1017/pPHU1 | 2.47 | 0.46 | 0.29 |
| 1017/pHUT10 | 2.32 | 0.24 | 0.29 |
Absorbance at 600 nm after 7 h of growth for V. alginolyticus and 18 h of growth for E. coli 1017.
Additional work was performed to determine if the defect in one of the DTH-1 TonB systems was in a tonB gene. DTH-1 was transformed with pTONB1 (containing V. cholerae tonB1) or with pTEE1 (containing V. cholerae tonB1 and exbB1D1). DTH-1/pTONB1 failed to grow in L-EDDA-hemin broth, whereas DTH-1/pTEE1 grew as well as DTH-1/pHUT11 (Table 4). These data suggest that while the mutation in DTH-1 is in a tonB locus, it is not in a tonB gene. It is not clear whether the defect in DTH-1 is in an exbBD gene(s) or a promoter that controls expression of all three genes or is a polar mutation in tonB.
Isolation of a V. parahaemolyticus gene that is functionally interchangeable with V. cholerae hutA.
A cosmid library of V. parahaemolyticus 474801 DNA was constructed (10) and transferred by triparental mating to Escherichia coli 1017 containing pHUT10 (10), which contains all the V. cholerae heme iron utilization genes except hutA. Bacteria were plated on L-EDDA-hemin agar, and a heme utilization-positive isolate was identified. The cosmid was named pPHU1 (parahaemolyticus heme utilization), and E. coli 1017/pHUT10/pPHU1 grew to a density similar to that of E. coli 1017/pHUT10/pHUT2 (pHUT2 contains the V. cholerae hutA gene) when tested for growth in L-EDDA-hemin broth (Table 4). These data suggested that pPHU1 contains the V. parahaemolyticus hutA equivalent and that it is functionally interchangeable with V. cholerae hutA. E. coli 1017 transformed with pPHU1 alone grew poorly in L-EDDA-hemin medium, indicating that it needs tonB1 and accessory genes present on pHUT10. To confirm that pPHU1 contained the V. parahaemolyticus hutA equivalent, pPHU1 was digested with HindIII, electrophoresed on a gel, and probed with the hutA probe described above. The probe annealed to cosmid clone fragments of 2.9 and 0.6 kb, which are the same size as the fragments observed in Southern blots of genomic DNA (Table 3 and data not shown).
Cloning and sequencing of a portion of the tonB1 locus from V. parahaemolyticus.
Additional work was performed to confirm that V. parahaemolyticus has a TonB1 system similar to that in V. cholerae. As indicated in Table 3, both the tonB1 and exbB1 probes hybridized to a 2.8-kb HindIII fragment from V. parahaemolyticus, suggesting that these two genes are linked, as they are in V. cholerae (18). To clone the tonB1 and exbB1 genes, 2.5- to 3.4-kb HindIII fragments from V. parahaemolyticus 474801 chromosomal preparations were isolated, ligated into pACYC184, and transformed into E. coli DH5α. Tetracycline-sensitive colonies were pooled into groups of 25, and plasmids were screened by Southern hybridization with the V. cholerae tonB1 probe. A clone was identified (pPHU2) to which the V. cholerae tonB1 and exbB1 probes, but not the tonB2 or hutC probes, hybridized. This indicated that pPHU2 contained tonB1 and exbB1, but not hutC or tonB2.
The DNA sequence of both strands of the insert in pPHU2 was determined with a ABI Prism 377 DNA sequencer from Applied Biosystems and was analyzed with the DNA Strider program (16). The BLAST program of the National Center for Biotechnology Information (1) was used to determine homologies of the deduced amino acid sequences, and MacVector Clustal W was used to determine protein identity and similarity. Our analyses of pPHU2 indicated that the cloned DNA contained three open reading frames (ORFs) (Fig. 2). ORFs 1 and 3 are missing the region encoding the carboxy termini of the respective proteins, as no stop codon was identified in either ORF. ORFs 1 to 3 encoded proteins that are homologous to the V. cholerae HutW (18a), TonB1, and ExbB1 proteins, respectively (18) (Fig. 2; Table 5).
FIG. 2.
Genetic map of cloned DNA from pPHU2. The putative Fur box is indicated by an open box. The arrows labeled P1 and P2 indicate the locations of the proposed promoters for phuW and for tonB1 and exbB1, respectively. The arrows beneath the filled boxes denote the direction of transcription. Below the map is the DNA sequence containing the predicted divergent promoters and the proposed Fur box, which is marked with a thick black line above the DNA.
TABLE 5.
Proteins with highest homology to products of V. parahaemolyticus phuW, tonB1, and exbB1
V. parahaemolyticus TonB1 (predicted molecular weight [MW], 27,100; 247 amino acids; pI 9.20) has 66% amino acid similarity with V. cholerae TonB1 (18), and it has weaker homology to numerous TonB proteins in other organisms, most notably to that in Pseudomonas putida (3) (Table 5). The similarity between V. cholerae TonB1 and V. parahaemolyticus TonB1 is greatest in the 103 amino acids at the carboxy terminus, where the similarity is 87%.
V. parahaemolyticus ExbB1 (predicted MW, 25,000; 231 amino acids; pI 8.32) is 81% similar to V. cholerae ExbB1 (18), and it exhibits weak homology to other ExbB proteins, such as that from Haemophilus ducreyi (Table 5). The incomplete ExbB1 protein contains three more amino acids than V. cholerae ExbB1 (228 amino acids).
V. cholerae HutW is a protein that has weak homology with a number of putative coproporphyrinogen oxidases in other organisms (18a). PhuW (predicted MW, 40,400; 420 amino acids; pI 6.62), the HutW homologue in V. parahaemolyticus, has 82% similarity to HutW and has weak homology with a number of putative coproporphyrinogen oxidases, such as HemN in Bacillus subtilis (Table 5).
The arrangement of genes in pPHU2 (Fig. 2) is similar to that in the V. cholerae tonB1 locus, which contains hutW, tonB1, and exbB1, in that order (18, 18a), with hutW being transcribed in the opposite direction of tonB1 and exbB1. The proposed promoters for V. parahaemolyticus phuW and for tonB1 and exbB1 contain a sequence similar to the E. coli consensus Fur box sequence (Fig. 2) (6). This sequence overlaps the predicted −35 region of the phuW promoter and the predicted −10 region of the promoter for tonB1 and exbB1, suggesting that expression of the genes is iron regulated.
We have shown that the heme iron utilization systems of V. parahaemolyticus, V. alginolyticus, and V. fluvialis are similar at the DNA level to that of V. cholerae and that some of the heme utilization proteins of V. cholerae, V. parahaemolyticus, and V. alginolyticus are functionally interchangeable. V. fluvialis can use heme and hemoglobin as iron sources, but our data suggest that its heme utilization system has diverged from that of V. cholerae. The gene encoding the V. fluvialis heme receptor is not sufficiently similar to V. cholerae hutA to be detected by Southern hybridization. Since a tonB2-like gene, but not a tonB1-like gene, was detected in V. fluvialis, its heme receptor may function with a TonB2-like protein. Or V. fluvialis may have a TonB1-like protein, but the gene may not be similar enough to the V. cholerae tonB1 gene to be detected by Southern hybridization.
This study indicates that V. parahaemolyticus and V. alginolyticus contain two tonB-like genes similar in DNA sequence to V. cholerae tonB1 and tonB2. Our data suggest that heme utilization in V. alginolyticus requires one TonB system and that the other TonB system can function in siderophore uptake but not in heme iron uptake. This is contrary to what occurs in V. cholerae, where the TonB systems appear to be redundant in that either system can support both heme and siderophore uptake (18). Future work will center on confirming that V. alginolyticus uses each TonB system to support a different function and on determining if V. parahaemolyticus is more similar to V. cholerae or V. alginolyticus in this regard.
Our sequencing data for pPHU2 supported our Southern blotting data concerning the presence of tonB1- and exbB1-like genes in V. parahaemolyticus. In addition, the sequencing data indicated that V. parahaemolyticus, like V. cholerae, contains a coproporphyrinogen oxidase-like gene linked to the TonB1 system genes. It is not clear at this time if HutW and PhuW are involved in heme iron utilization in their respective organisms. Shigella dysenteriae and E. coli O157:H7 also contain coprophorphyrinogen oxidase-like genes linked to heme iron utilization genes (24). Future work will be done to construct a V. parahaemolyticus phuW mutant that can be tested for heme iron utilization.
Nucleotide sequence accession number.
The nucleotide and amino acid sequences corresponding to this region can be found under GenBank/EMBL accession no. AF119047.
Acknowledgments
This study was supported by Grant Development Funds from the University of Texas of the Permian Basin and Alliance for Minority Participation funds from the University of Texas System.
We thank Elizabeth Wyckoff for critical reading of the manuscript.
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