Development of Colloidal Gold Immunochromatographic Strips for Detection of Riemerella anatipestifer

Riemerella anatipestifer is one of the most important bacterial pathogen of ducks and causes a contagious septicemia. R. anatipestifer infection causes serositis syndromes similar to other bacterial infections in ducks, including infection by Escherichia coli, Salmonella enterica and Pasteurella multocida. Clinically differentiating R. anatipestifer infections from other bacterial pathogen infections is usually difficult. In this study, MAb 1G2F10, a monoclonal antibody against R. anatipestifer GroEL, was used to develop a colloidal gold immunochromatographic strip. Colloidal gold particles were prepared by chemical synthesis to an average diameter of 20±5.26 nm by transmission electron microscope imaging. MAb 1G2F10 was conjugated to colloidal gold particles and the formation of antibody-colloidal gold conjugates was monitored by UV/Vis spectroscopy. Immunochromatographic strips were assembled in regular sequence through different accessories sticked on PVC plate. Strips specifically detected R. anatipestifer within 10 min, but did not detect E. coli, S. enterica and P. multocida. The detection limit for R. anatipestifer was 1×10⁶ colony forming units, which was 500 times higher than a conventional agglutination test. Accuracy was 100% match to multiplex PCR. Assay stability and reproducibility were excellent after storage at 4°C for 6 months. The immunochromatographic strips prepared in this study offer a specific, sensitive, and rapid detection method for R. anatipestifer, which is of great importance for the prevention and control of R. anatipestifer infections.     

A novel protein, TtpC, is a required component of the TonB2 complex for specific iron transport in the pathogens Vibrio anguillarum and Vibrio cholerae

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.     

A second tonB gene in Pseudomonas aeruginosa is linked to the exbB and exbD genes

The exbBD genes of Pseudomonas aeruginosa PAO were cloned by complementation of the growth defect of an Escherichia coli exbB tolQ double mutant on iron-restricted medium. Nucleotide sequence analysis confirmed that these genes are contiguous and preceded by a second tonB gene in this organism, which we have designated tonB2. lacZ promoter fusions confirmed that expression of the tonB2-exbB-exbD genes is increased under conditions of iron limitation. Deletions within any of these genes, in contrast to deletions in the first tonB gene, tonB1, did not adversely affect growth on iron-restricted medium. On the other hand, tonB1 tonB2 double mutants were more compromised as regards growth in an iron-restricted medium than a tonB1 deletion, indicating that TonB2 could partially replace TonB1 in its role in iron acquisition. TonB1 but not TonB2 deletion strains were also compromised as regards the utilization of hemin or hemoglobin as sole iron sources, indicating that heme transport requires TonB1.     

The Use of a Mercury Biosensor to Evaluate the Bioavailability of Mercury-Thiol Complexes and Mechanisms of Mercury Uptake in Bacteria

As mercury (Hg) biosensors are sensitive to only intracellular Hg, they are useful in the investigation of Hg uptake mechanisms and the effects of speciation on Hg bioavailability to microbes. In this study, bacterial biosensors were used to evaluate the roles that several transporters such as the glutathione, cystine/cysteine, and Mer transporters play in the uptake of Hg from Hg-thiol complexes by comparing uptake rates in strains with functioning transport systems to strains where these transporters had been knocked out by deletion of key genes. The Hg uptake into the biosensors was quantified based on the intracellular conversion of inorganic mercury (Hg(II)) to elemental mercury (Hg(0)) by the enzyme MerA. It was found that uptake of Hg from Hg-cysteine (Hg(CYS)₂) and Hg-glutathione (Hg(GSH)₂) complexes occurred at the same rate as that of inorganic complexes of Hg(II) into Escherichia coli strains with and without intact Mer transport systems. However, higher rates of Hg uptake were observed in the strain with a functioning Mer transport system. These results demonstrate that thiol-bound Hg is bioavailable to E. coli and that this bioavailability is higher in Hg-resistant bacteria with a complete Mer system than in non-resistant strains. No difference in the uptake rate of Hg from Hg(GSH)₂ was observed in E. coli strains with or without functioning glutathione transport systems. There was also no difference in uptake rates between a wildtype Bacillus subtilis strain with a functioning cystine/cysteine transport system, and a mutant strain where this transport system had been knocked out. These results cast doubt on the viability of the hypothesis that the entire Hg-thiol complex is taken up into the cell by a thiol transporter. It is more likely that the Hg in the Hg-thiol complex is transferred to a transport protein on the cell membrane and is subsequently internalized.     

ErmF and ereD Are Responsible for Erythromycin Resistance in Riemerella anatipestifer

To investigate the genetic basis of erythromycin resistance in Riemerella anatipestifer, the MIC to erythromycin of 79 R. anatipestifer isolates from China and one typed strain, ATCC11845, were evaluated. The results showed that 43 of 80 (53.8%) of the tested R. anatipestifer strains showed resistance to erythromycin, and 30 of 43 erythromycin-resistant R. anatipestifer strains carried ermF or ermFU with an MIC in the range of 32–2048 μg/ml, while the other 13 strains carrying the ereD gene exhibited an MIC of 4–16 μg/ml. Of 30 ermF + R. anatipestifer strains, 27 (90.0%) carried the ermFU gene which may have been derived from the CTnDOT-like element, while three other strains carried ermF from transposon Tn4351. Moreover, sequence analysis revealed that ermF, ermFU, and ereD were located within the multiresistance region of the R. anatipestifer genome.     

The wild-type allele of tonB in Escherichia coli is dominant over the tonB1 allele,encoding TonBQ160K,which suppresses the btuB451 mutation

The entire coding sequence of the tonB gene, except for nine codons at the 3′ end, was deleted from the chromosome of Escherichia coli. Introduction of the btuB451 suppressor mutant tonB1 into the chromosome of such a tonB deletion strain showed that the tonB1 allele was active as a suppressor in a single copy at 37° C and 42° C but not at 28° C. No temperature dependence was seen when FepA- or FhuA-dependent activities of the tonB1 gene product (TonB_Q160K) were tested. The btuB451 suppressor activity of tonB1 was inhibited by the simultaneous presence within the cells of the tonB ⁺ allele on a multicopy plasmid. This represents the first case of dominance among different tonB alleles. Inhibition of suppression was abolished by overexpression of the btuB451-encoded receptor protein. Competition for binding of TonB⁺ and TonB_Q150K to ExbB was excluded as the cause of dominance. Based on our data we conclude that competition for binding of TonB ⁺ and TonB_Q160K to the btuB451 gene product is the reason for the observed dominance. The implications of these findings for the mechanism of btuB451 suppression by tonB1 are discussed.     

Characterization of TonB Interactions with the FepA Cork Domain and FecA N-terminal Signaling Domain

The mechanism of TonB dependent siderophore uptake through outer membrane transporters in Gram-negative bacteria is poorly understood. In an effort to expand our knowledge of the interaction between TonB and the outer membrane transporters, we have cloned and expressed the FepA cork domain (11–154) from Salmonella typhimurium and characterized its interaction with the periplasmic C-terminal domain of TonB (103–239) by isotope assisted FTIR and NMR spectroscopy. For comparison we also performed similar experiments using the FecA N-terminal domain (1–96) from Escherichia coli which includes the conserved TonB box. The FepA cork domain was completely unfolded in solution, as observed for the E. coli cork domain previously [Usher et al. (2001) Proc Natl Acad Sci USA 98, 10676–10681]. The FepA cork domain was found to bind to TonB, eliciting essentially the same chemical shift changes in TonB C-terminal domain as was observed in the presence of TonB box peptides. The FecA construct did not cause this same structural change in TonB. The binding of the FepA cork domain to TonB-CTD was found to decrease the amount of ordered secondary structure in TonB-CTD. It is likely that the FecA N-terminal domain interferes with TonB-CTD binding to the TonB box. Binding of the FepA cork domain induces a loss of secondary structure in TonB, possibly exposing TonB surface area for additional intermolecular interactions such as potential homodimerization or additional interactions with the barrel of the outer membrane transporter.     

Iron-free pyoverdin binds to its outer membrane receptor FpvA in Pseudomonas aeruginosa: a new mechanism for membrane iron transport

Under iron limitation, Pseudomonas aeruginosa secretes a fluorescent siderophore called pyoverdin, which, after complexing iron, is transported back into the cell via its outer membrane receptor FpvA. Previous studies demonstrated co-purification of FpvA with iron-free PaA and reported similar binding affinities of iron-free pyoverdin and ferric-pyoverdin to purified FpvA. The fluorescence resonance energy transfer between iron-free PaA and the FpvA receptor here reveals the existence of an FpvA-pyoverdin complex in P. aeruginosa in vivo, suggesting that the pyoverdin-loaded FpvA is the normal state of the receptor in the absence of iron. Using tritiated ferric-pyoverdin, it is shown that iron-free PaA binds to the outer membrane but is not taken up into the cell, and that in vitro and, presumably, in vivo ferric-pyoverdin displaces the bound iron-free pyoverdin on FpvA-PaA to form FpvA-PaA-Fe complexes. In vivo, the kinetics of formation of this FpvA-PaA-Fe complex are more than two orders of magnitude faster than in vitro and depend on the presence of TonB. In P. aeruginosa, two tonB genes have been identified (tonB1 and tonB2). TonB1 is directly involved in ferric-pyoverdin uptake, and TonB2 seems to be able partially to replace TonB1 in its role in iron acquisition. However, no effect of TonB1 or TonB2 on the apparent affinity of free pyoverdin to FpvA was observed, and a 17-fold difference was measured between the affinities of the two forms of pyoverdin (PaA and PaA-Fe) to FpvA in the absence of TonB1 or TonB2. The mechanism of iron uptake in P. aeruginosa via the pyoverdin pathway is discussed in view of these new findings.     

A role for TonB1 in biofilm formation and quorum sensing in Pseudomonas aeruginosa

This study revealed that a Pseudomonas aeruginosa tonB1 mutant was unable to produce a mature biofilm and showed reduced swarming and twitching motilities compared with the parent strain. The tonB1 mutant was also found to produce significantly lower cell-free and cell-associated levels of the quorum sensing (QS) signal molecule 3-oxo-C12-AHL. Altered biofilm and motility phenotypes were restored to wildtype with the addition of exogenous N-acylhomoserine lactones. These functions were independent of the role of TonB1 in iron uptake. This is the first time that a link has been established between TonB1 activity and QS.     

Role of TonB1 in Pyoverdine-Mediated Signaling in Pseudomonas aeruginosa

Pyoverdines are siderophores secreted by Pseudomonas aeruginosa. Uptake of ferripyoverdine in P. aeruginosa PAO1 occurs via the FpvA receptor protein and requires the energy-transducing protein TonB1. Interaction of (ferri)pyoverdine with FpvA activates pyoverdine gene expression in a signaling process involving the cytoplasmic-membrane-spanning anti-sigma factor FpvR and the sigma factor PvdS. Here, we show that mutation of a region of FpvA that interacts with TonB1 (the TonB box) prevents this signaling process, as well as inhibiting bacterial growth in the presence of the iron-chelating compound ethylenediamine-di(o-hydroxy-phenylacetic acid). Signaling via wild-type FpvA was also eliminated in strains lacking TonB1 but was unaffected in strains lacking either (or both) of two other TonB proteins in P. aeruginosa, TonB2 and TonB3. An absence of pyoverdine-mediated signaling corresponded with proteolysis of PvdS. These data show that interactions between FpvA and TonB1 are required for (ferri)pyoverdine signal transduction, as well as for ferripyoverdine transport, consistent with a mechanistic link between the signaling and transport functions of FpvA.Pseudomonas aeruginosa is an opportunistic pathogen that is able to cause severe infections in patients with cystic fibrosis and in immunocompromised individuals, such as burn victims. Under conditions of iron limitation, P. aeruginosa secretes an iron-scavenging compound (siderophore) called pyoverdine. Ferripyoverdine is transported back into the bacteria by an outer membrane (OM) receptor protein, FpvA. The transport of ferripyoverdine via FpvA requires energy provided by a TonB complex (36, 42, 50). TonB is an energy-transducing protein that couples the energy of the cytoplasmic membrane (CM) to a variety of OM receptors required for the import of ferrisiderophores and other molecules. TonB acts in a complex with two CM-associated proteins, ExbB and ExbD, both of which are required for full TonB function (5, 37). The TonB-ExbB-ExbD complex has been identified in many gram-negative bacterial species and is thought to be a conserved mechanism for energy transduction to OM receptor proteins (31). TonB-dependent receptors contain a conserved protein motif known as the TonB box (5). Direct interaction between TonB and the TonB box has been demonstrated for several TonB-dependent receptors (8, 26, 33, 35, 47). Mutations of the TonB box, particularly mutations that are likely to affect the secondary structure, can result in a TonB-uncoupled phenotype characterized by loss of TonB-dependent functions (ferrisiderophore transport) with no loss of TonB-independent functions, such as internalization of bacteriophage (37).The P. aeruginosa PAO1 genome contains three tonB genes, tonB1 (PA5531) (36), tonB2 (PA0197) (55), and tonB3 (PA0406) (20), encoding proteins of 342, 270, and 319 amino acids (aa), respectively. The TonB1 and TonB2 amino acid sequences display 31% identity over a section of 187 aa, but otherwise, the three PAO1 TonB proteins show similarity (30 to 40% aa identity) to each other only over short (<70-aa) regions. TonB1 is considered to be the primary TonB protein involved in iron transport in P. aeruginosa. tonB1 mutants are impaired for growth in iron-limited medium and are defective for siderophore-mediated iron transport and heme utilization (36, 50, 55). Moreover, direct interaction between TonB1 and the ferripyoverdine receptor FpvA has been demonstrated in vitro (1). The tonB2 gene is not required for growth in iron-limited medium (55). However, tonB1 tonB2 double mutants grow even less well under iron limitation than tonB1 mutants, indicating that TonB2 may be able to partially complement TonB1 in its role in iron acquisition (55). The tonB3 gene is required for twitching motility and assembly of extracellular pili (20), but it is not known whether TonB3 has a role in iron acquisition. Genes encoding ExbB and ExbD proteins are located directly downstream of tonB2 (55) but are not found in association with tonB1 or tonB3.Besides its role in ferripyoverdine transport, FpvA is part of a signal transduction pathway and thus belongs to a subset of TonB-dependent receptors known as TonB-dependent transducers (reviewed in references 23 and 51). Mutational analysis has shown that the ferripyoverdine transport and signaling roles of FpvA are separate and discrete functions (21, 46). Besides FpvA, the signal transduction pathway involves a CM-spanning anti-sigma factor protein, FpvR, and (ferri)pyoverdine. (It was previously thought that both ferri- and apopyoverdine could bind FpvA (43). However, it was recently reported that only ferripyoverdine is able to form a high-affinity interaction with FpvA (13). The designation (ferri)pyoverdine will be used here to represent the active signaling molecule. FpvA and (ferri)pyoverdine regulate the activity of FpvR, which in turn regulates the activities of two extracytoplasmic function family sigma factors, PvdS and FpvI (3, 25). Upon binding of (ferri)pyoverdine to FpvA, a signal is transmitted to FpvR, resulting in activation of PvdS and FpvI. Activation of PvdS is required for maximal synthesis of pyoverdine itself, as well as two secreted proteins (25). Activation of FpvI leads to increased expression of fpvA (3, 39). In the absence of pyoverdine-mediated signaling, caused by the lack of FpvA or pyoverdine or overexpression of FpvR, suppression of PvdS- and FpvI-dependent gene expression occurs (3, 25), and this is associated with proteolysis of PvdS (49).Analogous siderophore transport and signaling systems involving an OM TonB-dependent transducer, a CM-bound anti-sigma factor, and an extracytoplasmic function family sigma factor have been described in other bacteria, including the ferric citrate (Fec) system in Escherichia coli and the pseudobactin (Pup) system in Pseudomonas putida (reviewed in reference 6). The TonB protein is required for signaling in both the Fec (14, 33) and Pup (24) systems. Similarly, a TonB system is required for hemophore transport and signaling in Serratia marcescens (4). The aim of this study was to investigate whether TonB was required for pyoverdine-mediated signaling in P. aeruginosa, and if so, to identify which of the three TonB proteins was involved.     

Characterisation of Cyanobacterial Bicarbonate Transporters in E. coli Shows that SbtA Homologs Are Functional in This Heterologous Expression System

Cyanobacterial HCO₃ ^- transporters BCT1, SbtA and BicA are important components of cyanobacterial CO₂-concentration mechanisms. They also show potential in applications aimed at improving photosynthetic rates and yield when expressed in the chloroplasts of C3 crop species. The present study investigated the feasibility of using Escherichia coli to assess function of a range of SbtA and BicA transporters in a heterologous expression system, ultimately for selection of transporters suitable for chloroplast expression. Here, we demonstrate that six β-forms of SbtA are active in E. coli, although other tested bicarbonate transporters were inactive. The sbtA clones were derived from Synechococcus sp. WH5701, Cyanobium sp. PCC7001, Cyanobium sp. PCC6307, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, and Synechococcus sp. PCC7002. The six SbtA homologs varied in bicarbonate uptake kinetics and sodium requirements in E. coli. In particular, SbtA from PCC7001 showed the lowest uptake affinity and highest flux rate and was capable of increasing the internal inorganic carbon pool by more than 8 mM relative to controls lacking transporters. Importantly, we were able to show that the SbtB protein (encoded by a companion gene near sbtA) binds to SbtA and suppresses bicarbonate uptake function of SbtA in E. coli, suggesting a role in post-translational regulation of SbtA, possibly as an inhibitor in the dark. This study established E. coli as a heterologous expression and analysis system for HCO₃ ^- transporters from cyanobacteria, and identified several SbtA transporters as useful for expression in the chloroplast inner envelope membranes of higher plants.     

An Escherichia coli Strain,PGB01, Isolated from Feral Pigeon Faeces,Thermally Fit to Survive in Pigeon,Shows High Level Resistance to Trimethoprim

In this study, of the hundred Escherichia coli strains isolated from feral Pigeon faeces, eighty five strains were resistant to one or more antibiotics and fifteen sensitive to all the antibiotics tested. The only strain (among all antibiotic-resistant E. coli isolates) that possessed class 1 integron was PGB01. The dihydrofolate reductase gene of the said integron was cloned, sequenced and expressed in E. coli JM109. Since PGB01 was native to pigeon’s gut, we have compared the growth of PGB01 at two different temperatures, 42°C (normal body temperature of pigeon) and 37°C (optimal growth temperature of E. coli; also the human body temperature), with E. coli K12. It was found that PGB01 grew better than the laboratory strain E. coli K12 at 37°C as well as at 42°C. In the thermal fitness assay, it was observed that the cells of PGB01 were better adapted to 42°C, resembling the average body temperature of pigeon. The strain PGB01 also sustained more microwave mediated thermal stress than E. coli K12 cells. The NMR spectra of the whole cells of PGB01 varied from E. coli K12 in several spectral peaks relating some metabolic adaptation to thermotolerance. On elevating the growth temperature from 37°C to 42°C, susceptibility to kanamycin (both strains were sensitive to it) of E. coli K12 was increased, but in case of PGB01 no change in susceptibility took place. We have also attempted to reveal the basis of trimethoprim resistance phenotype conferred by the dfrA7 gene homologue of PGB01. Molecular Dynamics (MD) simulation study of docked complexes, PGB01-DfrA7 and E. coli TMP-sensitive-Dfr with trimethoprim (TMP) showed loss of some of the hydrogen and hydrophobic interaction between TMP and mutated residues in PGB01-DfrA7-TMP complex compared to TMP-sensitive-Dfr-TMP complex. This loss of interaction entails decrease in affinity of TMP for PGB01-DfrA7 compared to TMP-sensitive-Dfr.     

Functional characterization of the twin ZIP/SLC39 metal transporters, NpunF3111 and NpunF2202 in Nostoc punctiforme

The ZIP family of metal transporters is involved in the transport of Zn²⁺ and other metal cations from the extracellular environment and/or organelles into the cytoplasm of prokaryotes, eukaryotes and archaeotes. In the present study, we identified twin ZIP transporters, Zip11 (Npun_F3111) and Zip63 (Npun_F2202) encoded within the genome of the filamentous cyanobacterium, Nostoc punctiforme PCC73120. Sequence-based analyses and structural predictions confirmed that these cyanobacterial transporters belong to the SLC39 subfamily of metal transporters. Quantitative real-time (QRT)-PCR analyses suggested that the enzymes encoded by zip11 and zip63 have a broad allocrite range that includes zinc as well as cadmium, cobalt, copper, manganese and nickel. Inactivation of either zip11 or zip63 via insertional mutagenesis in N. punctiforme resulted in reduced expression of both genes, highlighting a possible co-regulation mechanism. Uptake experiments using ⁶⁵Zn demonstrated that both zip mutants had diminished zinc uptake capacity, with the deletion of zip11 resulting in the greatest overall reduction in ⁶⁵Zn uptake. Over-expression of Zip11 and Zip63 in an E. coli mutant strain (ZupT736::kan) restored divalent metal cation uptake, providing further evidence that these transporters are involved in Zn uptake in N. punctiforme. Our findings show the functional role of these twin metal uptake transporters in N. punctiforme, which are independently expressed in the presence of an array of metals. Both Zip11 and Zip63 are required for the maintenance of homeostatic levels of intracellular zinc N. punctiforme, although Zip11 appears to be the primary zinc transporter in this cyanobacterium, both ZIP’s may be part of a larger metal uptake system with shared regulatory elements.     

Protozoan Predation of Escherichia coli O157:H7 Is Unaffected by the Carriage of Shiga Toxin-Encoding Bacteriophages

Escherichia coli O157:H7 is a food-borne bacterium that causes hemorrhagic diarrhea and hemolytic uremic syndrome in humans. While cattle are a known source of E. coli O157:H7 exposure resulting in human infection, environmental reservoirs may also be important sources of infection for both cattle and humans. Bacteriophage-encoded Shiga toxins (Stx) carried by E. coli O157:H7 may provide a selective advantage for survival of these bacteria in the environment, possibly through their toxic effects on grazing protozoa. To determine Stx effects on protozoan grazing, we co-cultured Paramecium caudatum, a common ciliate protozoon in cattle water sources, with multiple strains of Shiga-toxigenic E. coli O157:H7 and non-Shiga toxigenic cattle commensal E. coli. Over three days at ambient laboratory temperature, P. caudatum consistently reduced both E. coli O157:H7 and non-Shiga toxigenic E. coli populations by 1–3 log cfu. Furthermore, a wild-type strain of Shiga-toxigenic E. coli O157:H7 (EDL933) and isogenic mutants lacking the A subunit of Stx 2a, the entire Stx 2a-encoding bacteriophage, and/or the entire Stx 1-encoding bacteriophage were grazed with similar efficacy by both P. caudatum and Tetrahymena pyriformis (another ciliate protozoon). Therefore, our data provided no evidence of a protective effect of either Stx or the products of other bacteriophage genes on protozoan predation of E. coli. Further research is necessary to determine if the grazing activity of naturally-occurring protozoa in cattle water troughs can serve to decrease cattle exposure to E. coli O157:H7 and other Shiga-toxigenic E. coli.     

The Vibrio parahaemolyticus pvuA1 gene (formerly termed psuA) encodes a second ferric vibrioferrin receptor that requires tonB2

We previously reported that the Vibrio parahaemolyticus pvsABCDE and psuA-pvuABCDE operons are involved in the biosynthesis and transport of its own siderophore, vibrioferrin (VF). Of these, psuA and pvuA encode TonB-dependent outer-membrane proteins (OMPs). Although pvuA was characterized as the ferric vibrioferrin receptor gene, the role of the psuA product remains unknown. In this study, a growth assay of isogenic psuA, pvuA, and psuA-pvuA double-deletion mutants followed by complementation of the double-deletion mutant with psuA or pvuA was used to identify psuA as a gene encoding an OMP involved in the uptake of ferric VF. Thus, psuA and pvuA were renamed pvuA1 and pvuA2, respectively. Moreover, we clarified the TonB specificities of PvuA1 and PvuA2, because V. parahaemolyticus has three sets of the TonB systems. The triple deletion of pvuA1, tonB1, and tonB2, and the double deletion of pvuA2 and tonB2 resulted in the complete loss of growth promotion by VF. This finding indicates that the energy required for PvuA1 and PvuA2 to transport ferric VF across the outer membrane is provided by the TonB2 system and by both the TonB1 and TonB2 systems, respectively.     

鸭疫里默氏杆菌铁离子代谢机制研究进展

铁离子是大多数细菌生存所必需的一种营养物质,但过多的铁离子会通过芬顿反应产生的活性氧对细菌造成损伤。因此,细菌通过摄取、调控、螯合、外排等机制维持体内铁离子的稳态。鸭疫里默氏杆菌(Riemerella anatipestifer)是一种最新被归类于威克斯菌科里氏杆菌属的革兰氏阴性菌。该菌主要感染禽类,参与该菌的铁离子代谢基因具有特别之处。本文对鸭疫里默氏杆菌铁离子代谢机制研究进展进行了系统总结和阐述,包括该菌的TonB系统、TonB依赖性受体、Fur蛋白及Dps蛋白等在铁离子转运、调控、螯合中的功能,以及以上蛋白在鸭疫里默氏杆菌致病中的作用,以期更全面地理解鸭疫里默氏杆菌铁代谢机制,并为进一步深入研究该菌铁离子代谢提供理论依据和参考。     

Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake

Abstract The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens , or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.     

Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes

Vibrio cholerae was found to have two sets of genes encoding TonB, ExbB and ExbD proteins. The first set ( tonB1, exbB1, exbD1 ) was obtained by complementation of a V. cholerae tonB mutant. In the mutant, a plasmid containing these genes permitted transport via the known V. cholerae high-affinity iron transport systems, including uptake of haem, vibriobactin and ferrichrome. When chromosomal mutations in exbB1 or exbD1 were introduced into a wild-type V. cholerae background, no defect in iron transport was noted, indicating the existence of additional genes that can complement the defect in the wild-type background. Another region of the V. cholerae chromosome was cloned that encoded a second functional TonB/Exb system ( tonB2, exbB2, exbD2 ). A chromosomal mutation in exbB2 also failed to exhibit a defect in iron transport, but a V. cholerae strain that had chromosomal mutations in both the exbB1 and exbB2 genes displayed a mutant phenotype similar to that of an Escherichia coli tonB mutant. The genes encoding TonB1, ExbB1, ExbD1 were part of an operon that included three haem transport genes ( hutBCD ), and all six genes appeared to be expressed from a single Fur-regulated promoter upstream of tonB1 . A plasmid containing all six genes permitted utilization of haem by an E. coli strain expressing the V. cholerae haem receptor, HutA. Analysis of the hut genes indicated that hutBCD, which are predicted to encode a periplasmic binding protein (HutB) and cytoplasmic membrane permease (HutC and HutD), were required to reconstitute the V. cholerae haem transport system in E. coli. In V. cholerae , the presence of hutBCD stimulated growth when haemin was the iron source, but these genes were not essential for haemin utilization in V. cholerae .