Lipopolysaccharide O-antigen prevents phagocytosis of Vibrio anguillarum by rainbow trout (Oncorhynchus mykiss) skin epithelial cells

Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues.     

Chemotactic motility is required for invasion of the host by the fish pathogen Vibrio anguillarum

The role of the flagellum and motility in the virulence of the marine fish pathogen Vibrio anguillarum was examined. Non-motile mutants were generated by transposon mutagenesis. Infectivity studies revealed that disruption of the flagellum and subsequent loss of motility correlated with an approximate 500-fold decrease in virulence when fish were inoculated by immersion in bacteria-containing water. However, the flagellar filament and motility were not required for pathogenicity following intraperitoneal injection of fish. The transposon-insertion site for six mutants was determined by cloning and sequencing of the Vibrio DNA flanking the transposon. V. anguillarum genes whose products showed strong homology to proteins with an established role in flagellum biosynthesis were identified. One of the aflagellate mutants had a transposon insertion in the rpoN gene of V. anguillarum . This rpoN mutant failed to grow at low concentrations of available iron and was avirulent by both the immersion and intraperitoneal modes of inoculation. A chemotaxis gene, cheR , was located upstream of one transposon insertion and an in-frame deletion was constructed in the coding region of this gene. The resulting non-chemotactic mutant exhibited wild-type pathogenicity when injected intraperitoneally into fish but showed a decrease in virulence similar to that seen for the non-motile aflagellate mutants following immersion infection. Hence, chemotactic motility is a required function of the flagellum for the virulence of V. anguillarum     

The molecular weight distribution of succinoglycan produced by Sinorhizobium meliloti is influenced by specific tyrosine phosphorylation and ATPase activity of the cytoplasmic domain of the ExoP protein

It is thought that in the gram-negative soil bacterium Sinorhizobium meliloti the protein ExoP is involved in biosynthesis of the acidic exopolysaccharide succinoglycan (EPS I). The amounts and compositions of EPS I produced by mutants expressing ExoP proteins characterized by specific amino acid substitutions in the C-terminal cytoplasmic domain were analyzed. The cytoplasmic domain of the ExoP protein was shown to have ATPase activity. Mutations in the highly conserved Walker A ATP-binding motif prevented ATPase activity of the ExoP protein. Phenotypically, these mutations resulted in much lower levels of succinoglycan which consisted only of monomers of the octasaccharide repeating unit. The ExoP protein has similarities to proteins with autophosphorylating protein tyrosine kinase activity. We found that ExoP was phosphorylated on tyrosine and that site-directed mutagenesis of specific tyrosine residues in the cytoplasmic domain of ExoP resulted in an altered ratio of low-molecular-weight succinoglycan to high-molecular-weight succinoglycan.     

The constant gene orf14.9, which belongs to the variable eps (exopolysaccharide) cluster, is involved in the cell growth of Streptococcus thermophilus

In Streptococcus thermophilus, the eps clusters involved in exopolysaccharide (EPS) biosynthesis are very polymorphic, nevertheless they all contain a highly conserved sequence corresponding to that of orf14.9. This open reading frame (ORF) is transcribed in a reverse direction with respect to eps genes. Amino acid sequence analysis showed a possible transmembrane location of the putative Orf14.9 protein but did not permit a proposed function. Insertional mutants of orf14.9 were obtained in strains NST2280 and A054 of S. thermophilus. EPS yields of these mutants are similar to those of their respective wild strains, suggesting that orf14.9 does not modify the quantity of produced EPS. Growth parameter determination for wild strains and their respective mutants showed that orf14.9 is involved in the cell growth of S. thermophilus.     

The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139

Throughout most of history, epidemic and pandemic cholera was caused by Vibrio cholerae of the serogroup O1. In 1992, however, a V. cholerae strain of the serogroup O139 emerged as a new agent of epidemic cholera. Interestingly, V. cholerae O139 forms biofilms on abiotic surfaces more rapidly than V. cholerae O1 biotype El Tor, perhaps because regulation of exopolysaccharide synthesis in V. cholerae O139 differs from that in O1 El Tor. Here, we show that all flagellar mutants of V. cholerae O139 have a rugose colony morphology that is dependent on the vps genes. This suggests that the absence of the flagellar structure constitutes a signal to increase exopolysaccharide synthesis. Furthermore, although exopolysaccharide production is required for the development of a three-dimensional biofilm, inappropriate exopolysaccharide production leads to inefficient colonization of the infant mouse intestinal epithelium by flagellar mutants. Thus, precise regulation of exopolysaccharide synthesis is an important factor in the survival of V. cholerae O139 in both aquatic environments and the mammalian intestine.     

Isolation and characterization of turbot (Scophtalmus maximus)-associated bacteria with inhibitory effects against Vibrio anguillarum.

More than 400 isolates from the intestine and the external surface of farmed Scophtalmus maximus as well as from fish food and hatchery water were screened for inhibitory effects against the fish pathogen Vibrio anguillarum HI 11345 and seven other fish pathogens. The bacteria with inhibitory effects were then characterized with regard to their sites of colonization, especially the intestinal regions and sites within each region. Of the total number of bacterial isolates from the intestine, 28% were inhibitory against V. anguillarum HI 11345. A marine biochemical assay was used to order the inhibitory strains into different phena. Most inhibitory bacteria were found in the rinse and mucus fractions of the gastrointestinal tract. No correlations among the different phena, site of colonization, and inhibitory effect could be found; however, a biochemical diversity was noted in the strains with an inhibitory effect. Of the isolates with an inhibitory effect against V. anguillarum HI 11345, 60% had an inhibitory effect on five other fish-pathogenic serotypes of V. anguillarum. Inhibitory effects of the isolates were also shown against Aeromonas salmonicida and Aeromonas hydrophila.     

Multiple determinants influence root colonization and induction of induced systemic resistance by Pseudomonas chlororaphis O6

Colonization of the roots of tobacco by Pseudomonas chlororaphis O6 induces systemic resistance to the soft-rot pathogen, Erwinia carotovora ssp. carotovara SCC1. A screen of the transposon mutants of P. chlororaphis O6 showed mutants with about a fivefold reduction in ability to induce systemic resistance to the soft-rot disease. These mutations disrupted genes involved in diverse functions: a methyl-accepting chemotaxis protein, biosynthesis of purines, phospholipase C, transport of branched-chain amino acids and an ABC transporter. Additional mutations were detected in the intergenic spacer regions between genes encoding a GGDEF protein and fumarate dehydratase, and in genes of unknown function. The mutants in the ABC transporters did not display reduced root colonization. However, the other mutants had up to 100-fold reduced colonization levels. Generally the production of metabolites important for interactions in the rhizosphere, phenazines and siderophores, was not altered by the mutations. A reduced induction of systemic resistance by a purine biosynthesis mutant with a disrupted purM gene correlated with poor growth rate, lesser production of phenazines and siderophore and low levels of root colonization. These studies showed that multiple determinants are involved in the induction of systemic resistance, with there being a requirement for strong root colonization.     

Isolation and characterization of turbot (Scophtalmus maximus)-associated bacteria with inhibitory effects against Vibrio anguillarum.

More than 400 isolates from the intestine and the external surface of farmed Scophtalmus maximus as well as from fish food and hatchery water were screened for inhibitory effects against the fish pathogen Vibrio anguillarum HI 11345 and seven other fish pathogens. The bacteria with inhibitory effects were then characterized with regard to their sites of colonization, especially the intestinal regions and sites within each region. Of the total number of bacterial isolates from the intestine, 28% were inhibitory against V. anguillarum HI 11345. A marine biochemical assay was used to order the inhibitory strains into different phena. Most inhibitory bacteria were found in the rinse and mucus fractions of the gastrointestinal tract. No correlations among the different phena, site of colonization, and inhibitory effect could be found; however, a biochemical diversity was noted in the strains with an inhibitory effect. Of the isolates with an inhibitory effect against V. anguillarum HI 11345, 60% had an inhibitory effect on five other fish-pathogenic serotypes of V. anguillarum. Inhibitory effects of the isolates were also shown against Aeromonas salmonicida and Aeromonas hydrophila.     

Sinorhizobium meliloti ExoR and ExoS proteins regulate both succinoglycan and flagellum production

The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinoglycan and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum.

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protective immunity could be recorded 1 week after bath vaccination with 10(7) bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonas salmonicida.     

Complete sequence of virulence plasmid pEIB1 from the marine fish pathogen Vibrio anguillarum strain MVM425 and location of its replication region

AIMS: The aim of this study was to determine the whole DNA sequence of pEIB1, one pJM1-like virulence plasmid from Vibrio anguillarum MVM425 and locate the replication region. METHODS AND RESULTS: DNA sequence of virulence plasmid pEIB1 from V. anguillarum MVM425 was determined using the methods of restriction endonuclease digestion, subcloning, and primer walking. The whole nucleotide sequence of pEIB1 comprises 66,164 bp, encoding 44 open reading frames (>400 bp) containing the genes of DNA replication, biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes. With no demonstrated replication origin, the Sau3AI partial digested plasmid DNA fragments of pEIB1 were ligated into the BamHI-fragment containing the kanamycin-resistance gene (Kmr). For there is no effective transformation in V. anguillarum, the ligated DNA was first introduced into E. coli JM83, and the transfomants were selected for resistance to kanamycin. It was demonstrated with southern blotting and DNA sequencing that plasmid pEIB7 containing the Sau3AI DNA fragment of pEIB1 (from 12516 to 13957) has the ability to replicate in E. coli JM83 and V. anguillarum MVM425sh. The segregational stability of plasmid pEIB7 kept in 100 and 4% in E. coli JM83 and V. anguillarum MVM425sh respectively when the cells were cultured in 200th generation. In following experiments, we also found that plasmid pEIB7 replicated at a middle-copy number of 10-40 in JM83, while at a high-copy number of 100-300 in MVM425sh. Moreover, pEIB7 can survive in V. alginolyticus, another fish pathogenic. CONCLUSIONS: With the whole DNA sequence of pEIB1 determining, it was found that pEIB1 showed microheterogeneity in its restriction endonuclease patterns with pJM1 though their DNA sequences had slight difference. According to the complete DNA sequence of pEIB1, its replication region was located from 12516 to 13957. And this replication region is compatible to pUC18 (pMB1), pKA3 (pSC101) and p15A: caiE (p15A). SIGNIFICANCE AND IMPACT OF THE STUDY: The worldwide vibriosis marine pathogen V. anguillarum strains contain common virulence, pJM1-like plasmids, independent on the geographical source. The pEIB1 was the second common virulence plasmid, which sequence was determined. Its sequence is highly homologous to pJM1 as they both encode biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes. Some interesting features as in pJM1 were also identified, such as transposon-like structures. So it can be deferred that the whole DNA sequences of virulent plasmid pEIB1 will be great helpful to future revealing these V. anguillarum virulence-related genes derived during evolution from transposition events or horizontal transfer of genes potentially originating in other organisms. Another result, replication region of pEIB1 locating is the first report about replication of pJM1-like plasmid. This work will be useful for researching pJM1-like plasmid replication mechanism in V. anguillarum.     

Overcoming a defect in generalized recombination in the marine fish pathogen Vibrio anguillarum 775: construction of a recA mutant by marker exchange.

A defect in generalized recombination has prevented the use of marker exchange for the construction of specific chromosomal mutations in the marine fish pathogen Vibrio anguillarum 775. Through the use of large segments of homologous DNA, we were successful in overcoming this defect and used marker exchange to construct a recA mutant of V. anguillarum H775-3. A recombinant cosmid carrying the recA gene of V. anguillarum 775 in the center of a 25-kb cloned DNA insert was isolated by complementation of methyl methanesulfonate (MMS) sensitivity in Escherichia coli HB101. The recA gene was inactivated by inserting a kanamycin resistance gene into recA, and the mutant gene was subsequently introduced into V. anguillarum H775-3 by conjugal mobilization. Isolation of recombinants between cosmid-borne recA::kan sequences and chromosomal DNA was facilitated by the introduction of an incompatible plasmid, and Southern hybridization was used to verify the presence of recA::kan in the chromosomal DNA of the recA mutant. V. anguillarum carrying recA::kan was considerably more sensitive to UV radiation and to MMS than was its parent, and near wild-type levels of resistance to MMS and UV light were restored by introduction of cloned recA genes from both E. coli and V. anguillarum. These results indicate that recA is required for DNA repair in V. anguillarum and demonstrate the utility of this modified marker exchange technique for the construction of mutations in this economically important fish pathogen.     

Intestinal colonization potential of turbot (Scophthalmus maximus)- and dab (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum.

Of more than 400 bacteria isolated from turbot (Scophthalmus maximus), 89 have previously been shown to inhibit the in vitro growth of the fish pathogen Vibrio anguillarum. The aim of the present study was to investigate the potential of seven of these strains, as well as of intestinal isolates (four strains) from a closely related fish, dab (Limanda limanda), for colonizing farmed turbot as a means of protecting the host from infection by V. anguillarum. In addition, the inhibitory effect of these strains on the pathogen was further studied. Colonization potential was measured by the capacity of the strains to adhere to and grow in turbot intestinal mucus. These parameters were also used to investigate the potential of V. anguillarum to amplify in the turbot intestinal tract. Because of the observed rapid growth of V. anguillarum in intestinal mucus, it can be proposed that the intestinal tract is a site for V. anguillarum multiplication. Strains isolated from the intestine showed greater capacity for adhesion to and growth in fish intestinal mucus than did the pathogen and the skin mucus isolates. All of the isolates released metabolites into the culture medium that had inhibitory effects against V. anguillarum. The results are discussed with emphasis on administering bacteria of host origin to farmed turbot in order to control V. anguillarum-induced disease.     

Chromosome-mediated iron uptake system in pathogenic strains of Vibrio anguillarum.

We describe in this work a new iron uptake system encoded by chromosomal genes in pathogenic strains of Vibrio anguillarum. This iron uptake system differs from the plasmid-encoded anguibactin-mediated system present in certain strains of V. anguillarum in several properties. The siderophore anguibactin is not utilized as an external siderophore, and although characteristic outer membrane proteins are synthesized under iron-limiting conditions, these are not related to the plasmid-mediated outer membrane protein OM2 associated with ferric anguibactin transport. Furthermore, the siderophore produced by the plasmidless strains may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants, although its behavior under various chemical treatments suggested major differences from that siderophore. Hybridization experiments suggested that the V. anguillarum chromosome-mediated iron uptake system is unrelated genetically to either the anguibactin or enterobactin-associated iron assimilation systems.     

Two replication regions in the pJM1 virulence plasmid of the marine pathogen Vibrio anguillarum

Vibrio anguillarum is a fish pathogen that causes vibriosis, a serious hemorrhagic septicemia, in wild and cultured fish. Many serotype O1 strains of this bacterium harbor the 65kb plasmid pJM1 carrying the majority of genes encoding the siderophore anguibactin iron transport system that is one of the most important virulence factors of this bacterium. We previously identified a replication region of the pJM1 plasmid named ori1. In this work we determined that ori1 can replicate in Escherichia coli and that the chromosome-encoded proteins DnaB, DnaC and DnaG are essential for its replication whereas PolI, IHF and DnaA are not required. The copy number of the pJM1 plasmid is 1-2, albeit cloned smaller fragments of the ori1 region replicate with higher copy numbers in V. anguillarum while in E. coli we did not observe an obvious difference of the copy numbers of these constructs which were all high. Furthermore, we were able to delete the ori1 region from the pJM1 plasmid and identified a second replication region in pJM1 that we named ori2. This second replication region is located on ORF25 that is within the trans-acting factor (TAFr) region, and showed that it can only replicate in V. anguillarum.     

Intestinal colonization potential of turbot (Scophthalmus maximus)- and dab (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum.

Of more than 400 bacteria isolated from turbot (Scophthalmus maximus), 89 have previously been shown to inhibit the in vitro growth of the fish pathogen Vibrio anguillarum. The aim of the present study was to investigate the potential of seven of these strains, as well as of intestinal isolates (four strains) from a closely related fish, dab (Limanda limanda), for colonizing farmed turbot as a means of protecting the host from infection by V. anguillarum. In addition, the inhibitory effect of these strains on the pathogen was further studied. Colonization potential was measured by the capacity of the strains to adhere to and grow in turbot intestinal mucus. These parameters were also used to investigate the potential of V. anguillarum to amplify in the turbot intestinal tract. Because of the observed rapid growth of V. anguillarum in intestinal mucus, it can be proposed that the intestinal tract is a site for V. anguillarum multiplication. Strains isolated from the intestine showed greater capacity for adhesion to and growth in fish intestinal mucus than did the pathogen and the skin mucus isolates. All of the isolates released metabolites into the culture medium that had inhibitory effects against V. anguillarum. The results are discussed with emphasis on administering bacteria of host origin to farmed turbot in order to control V. anguillarum-induced disease.     

Isolation of mutants of<Emphasis Type="Italic"> Vibrio anguillarum</Emphasis> defective in haeme utilisation and cloning of<Emphasis Type="Italic"> huvA</Emphasis>, a gene coding for an outer membrane protein involved in the use of haeme as iron source

The isolation of Vibrio anguillarum mutants lacking the ability to use haemin and haemoglobin as the only iron sources, as well as the identification of a gene involved in haeme utilisation are described. One of the isolated mutants defective in haeme utilisation lacked an iron-regulated outer membrane protein of 79-kDa. Although growth on haeme as iron source was completely abolished, the haemin and haemoglobin binding activities remained intact in the mutant, suggesting that the absent protein is not the only one involved in haeme binding. The wild-type phenotype in this mutant was restored by transformation with a cosmid clone (pML1) containing a 21-kb DNA fragment isolated from a gene library derived from the parental strain of V. anguillarum. Sequence analysis of pML1 subclones led to the finding of an ORF, huvA, that codes for a 79-kDa protein (HuvA) and whose sequence shows high identity with haeme receptors from Vibrio choleare (HutA) and Vibrio vulnificus (HupA). The sequence of huvA from the V. anguillarum haeme-utilisation mutant revealed a single mutation, leading to the synthesis of a truncated HuvA protein of 70 kDa. The parental strain and the cosmid-complemented mutant showed a higher degree of virulence for fish than the mutant strain in experimental infections in which fish were previously overloaded with haemin. This finding suggests that haeme uptake plays an important role in V. anguillarum multiplication in fish tissues when free haeme is available.     

Molecular characterization of the iron transport system mediated by the pJM1 plasmid in Vibrio anguillarum 775.

Complementation of insertion mutants showed that the polypeptides FatD, FatC, FatB, and FatA are essential for the iron-transport process encoded by pJM1. Sequence analysis followed by homology studies indicated that transport of ferric anguibactin into Vibrio anguillarum 775 follows the same mechanism as reported for transport of Fe(3+)-hydroxamates, Fe(3+)-catecholates, ferric dicitrate, and vitamin B12 into Escherichia coli. Homology of FatA, part of the receptor complex, to seven E. coli receptor proteins involved in uptake of siderophores and vitamin B12 supports the idea of a common ancestral gene. A "TonB-Box" was found in FatA suggesting the existence of a TonB-like protein function in V. anguillarum. A high homology in the primary structure of FatB to FhuD, FecB, FepB, and BtuE suggests that FatB is the anguibactin-binding protein located in the periplasmic space. FatD and FatC are polytopic integral membrane proteins. According to their homologies to other proteins from other transport systems, they may be involved in the translocation of ferric anguibactin across the cytoplasmic membrane.     

Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum

In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine replacements affecting the final six amino acids of TonB2. Deletions of more than two amino acids of the TonB2 C-terminus abolished ferric-anguibactin transport, whereas replacement of the last three residues resulted in a protein with wild-type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121-206) has an alphabetabetaalphabeta structure, whereas residues 76-120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain, albeit with two differences: the beta4 strand found at the C-terminus of TonB is absent in TonB2, and loop 3 is extended by 9 A (0.9 nm) in TonB2. By examining several mutants, we determined that a complete loop 3 is not essential for TonB2 activity. Our results indicate that the beta4 strand of E. coli TonB is not required for activity of the TonB system across Gram-negative bacterial species. We have also determined, through NMR chemical-shift-perturbation experiments, that the E. coli TonB binds in vitro to the TonB box from the TonB2-dependent outer membrane transporter FatA; moreover, it can substitute in vivo for TonB2 during ferric-anguibactin transport in V. anguillarum. Unexpectedly, TonB2 did not bind in vitro to the FatA TonB-box region, suggesting that additional factors may be required to promote this interaction. Overall our results indicate that TonB2 is a representative of a different class of TonB proteins.