scholarly journals Vibrio cholerae VciB Promotes Iron Uptake via Ferrous Iron Transporters

2008 ◽  
Vol 190 (17) ◽  
pp. 5953-5962 ◽  
Author(s):  
Alexandra R. Mey ◽  
Elizabeth E. Wyckoff ◽  
Lindsey A. Hoover ◽  
Carolyn R. Fisher ◽  
Shelley M. Payne
Keyword(s):  
Vibrio Cholerae ◽  
Transport System ◽  
Ferrous Iron ◽  
Iron Uptake ◽  
Iron Transport ◽  
Growth Enhancement ◽  
Outer Membrane Receptor ◽  
E Coli

ABSTRACT Vibrio cholerae uses a variety of strategies for obtaining iron in its diverse environments. In this study we report the identification of a novel iron utilization protein in V. cholerae, VciB. The vciB gene and its linked gene, vciA, were isolated in a screen for V. cholerae genes that permitted growth of an Escherichia coli siderophore mutant in low-iron medium. The vciAB operon encodes a predicted TonB-dependent outer membrane receptor, VciA, and a putative inner membrane protein, VciB. VciB, but not VciA, was required for growth stimulation of E. coli and Shigella flexneri strains in low-iron medium. Consistent with these findings, TonB was not needed for VciB-mediated growth. No growth enhancement was seen when vciB was expressed in an E. coli or S. flexneri strain defective for the ferrous iron transporter Feo. Supplying the E. coli feo mutant with a plasmid encoding either E. coli or V. cholerae Feo, or the S. flexneri ferrous iron transport system Sit, restored VciB-mediated growth; however, no stimulation was seen when either of the ferric uptake systems V. cholerae Fbp and Haemophilus influenzae Hit was expressed. These data indicate that VciB functions by promoting iron uptake via a ferrous, but not ferric, iron transport system. VciB-dependent iron accumulation via Feo was demonstrated directly in iron transport assays using radiolabeled iron. A V. cholerae vciB mutant did not exhibit any growth defects in either in vitro or in vivo assays, possibly due to the presence of other systems with overlapping functions in this pathogen.

scholarly journals Vibrio cholerae VibF Is Required for Vibriobactin Synthesis and Is a Member of the Family of Nonribosomal Peptide Synthetases

2000 ◽  
Vol 182 (6) ◽  
pp. 1731-1738 ◽  
Author(s):  
Joan R. Butterton ◽  
Michael H. Choi ◽  
Paula I. Watnick ◽  
Patricia A. Carroll ◽  
Stephen B. Calderwood
Keyword(s):  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Consensus Sequence ◽  
Membrane Receptor ◽  
Transcriptional Level ◽  
Outer Membrane Receptor ◽  
Chromosomal Dna ◽  
E Coli ◽  
Binding Consensus Sequence

ABSTRACT A 7.5-kbp fragment of chromosomal DNA downstream of theVibrio cholerae vibriobactin outer membrane receptor,viuA, and the vibriobactin utilization gene,viuB, was recovered from a Sau3A lambda library of O395 chromosomal DNA. By analogy with the genetic organization of the Escherichia coli enterobactin gene cluster, in which the enterobactin biosynthetic and transport genes lie adjacent to the enterobactin outer membrane receptor, fepA, and the utilization gene, fes, the cloned DNA was examined for the ability to restore siderophore synthesis to E. coli entmutants. Cross-feeding studies demonstrated that an E. coli entF mutant complemented with the cloned DNA regained the ability to synthesize enterobactin and to grow in low-iron medium. Sequence analysis of the cloned chromosomal DNA revealed an open reading frame downstream of viuB which encoded a deduced protein of greater than 2,158 amino acids, homologous to Yersinia sp. HMWP2, Vibrio anguillarum AngR, and E. coliEntF. A mutant with an in-frame deletion of this gene, namedvibF, was created with classical V. choleraestrain O395 by in vivo marker exchange. In cross-feeding studies, this mutant was unable to synthesize ferric vibriobactin but was able to utilize exogenous siderophore. Complementation of the mutant with a cloned vibF fragment restored vibriobactin synthesis to normal. The expression of the vibF promoter was found to be negatively regulated by iron at the transcriptional level, under the control of the V. cholerae fur gene. Expression ofvibF was not autoregulatory and neither affected nor was affected by the expression of irgA or viuA. The promoter of vibF was located by primer extension and was found to contain a dyad symmetric nucleotide sequence highly homologous to the E. coli Fur binding consensus sequence. A footprint of purified V. cholerae Fur on the vibFpromoter, overlapping the Fur binding consensus sequence, was observed using DNase I footprinting. The protein product of vibF is homologous to the multifunctional nonribosomal protein synthetases and is necessary for the biosynthesis of vibriobactin.

scholarly journals Vibrio cholerae FeoB hydrolyzes ATP and GTP in vitro in the absence of stimulatory factors

Metallomics ◽  
2020 ◽  
Author(s):  
Camilo Gómez-Garzón ◽  
Shelley M. Payne
Keyword(s):  
Vibrio Cholerae ◽  
Ferrous Iron ◽  
Iron Uptake ◽  
Molar Ratio

V. cholerae FeoA, FeoB, and FeoC work at a 1 : 1 : 1 molar ratio to mediate ferrous iron uptake through a mechanism driven by FeoB NTP hydrolysis without requiring stimulatory factors.

scholarly journals Characterization of Ferric and Ferrous Iron Transport Systems in Vibrio cholerae

2006 ◽  
Vol 188 (18) ◽  
pp. 6515-6523 ◽  
Author(s):  
Elizabeth E. Wyckoff ◽  
Alexandra R. Mey ◽  
Andreas Leimbach ◽  
Carolyn F. Fisher ◽  
Shelley M. Payne
Keyword(s):  
Vibrio Cholerae ◽  
Transport System ◽  
Iron Transport ◽  
Iron Acquisition ◽  
Shigella Flexneri ◽  
Transport Systems ◽  
Infant Mouse ◽  
Mouse Intestine ◽  
Dependent Transport

ABSTRACT Vibrio cholerae has multiple iron acquisition systems, including TonB-dependent transport of heme and of the catechol siderophore vibriobactin. Strains defective in both of these systems grow well in laboratory media and in the infant mouse intestine, indicating the presence of additional iron acquisition systems. Previously uncharacterized potential iron transport systems, including a homologue of the ferrous transporter Feo and a periplasmic binding protein-dependent ATP binding cassette (ABC) transport system, termed Fbp, were identified in the V. cholerae genome sequence. Clones encoding either the Feo or the Fbp system exhibited characteristics of iron transporters: both repressed the expression of lacZ cloned under the control of a Fur-regulated promoter in Escherichia coli and also conferred growth on a Shigella flexneri mutant that has a severe defect in iron transport. Two other ABC transporters were also evaluated but were negative by these assays. Transport of radioactive iron by the Feo system into the S. flexneri iron transport mutant was stimulated by the reducing agent ascorbate, consistent with Feo functioning as a ferrous transporter. Conversely, ascorbate inhibited transport by the Fbp system, suggesting that it transports ferric iron. The growth of V. cholerae strains carrying mutations in one or more of the potential iron transport genes indicated that both Feo and Fbp contribute to iron acquisition. However, a mutant defective in the vibriobactin, Fbp, and Feo systems was not attenuated in a suckling mouse model, suggesting that at least one other iron transport system can be used in vivo.

scholarly journals Vibrio cholerae FeoA, FeoB, and FeoC Interact To Form a Complex

2016 ◽  
Vol 198 (7) ◽  
pp. 1160-1170 ◽  
Author(s):  
Begoña Stevenson ◽  
Elizabeth E. Wyckoff ◽  
Shelley M. Payne
Keyword(s):  
Complex Formation ◽  
Vibrio Cholerae ◽  
Ferrous Iron ◽  
Iron Transport ◽  
Active Form ◽  
Integral Membrane Protein ◽  
Content Type ◽  
Membrane Complex ◽  
Ferrous Iron Transport

ABSTRACTFeo is the major ferrous iron transport system in prokaryotes. Despite having been discovered over 25 years ago and found to be widely distributed among bacteria, Feo is poorly understood, as its structure and mechanism of iron transport have not been determined. Thefeooperon inVibrio choleraeis made up of three genes, encoding the FeoA, FeoB, and FeoC proteins, which are all required for Feo system function. FeoA and FeoC are both small cytoplasmic proteins, and their function remains unclear. FeoB, which is thought to function as a ferrous iron permease, is a large integral membrane protein made up of an N-terminal GTPase domain and a C-terminal membrane-spanning region. To date, structural studies of FeoB have been carried out using a truncated form of the protein encompassing only the N-terminal GTPase region. In this report, we show that full-length FeoB forms higher-order complexes when cross-linkedin vivoinV. cholerae. Our analysis of these complexes revealed that FeoB can simultaneously associate with both FeoA and FeoC to form a large complex, an observation that has not been reported previously. We demonstrate that interactions between FeoB and FeoA, but not between FeoB and FeoC, are required for complex formation. Additionally, we identify amino acid residues in the GTPase region of FeoB that are required for function of the Feo system and for complex formation. These observations suggest that this large Feo complex may be the active form of Feo that is used for ferrous iron transport.IMPORTANCEThe Feo system is the major route for ferrous iron transport in bacteria. In this work, theVibrio choleraeFeo proteins, FeoA, FeoB, and FeoC, are shown to interact to form a large inner membrane complexin vivo. This is the first report showing an interaction among all three Feo proteins. It is also determined that FeoA, but not FeoC, is required for Feo complex assembly.

Active transport of iron by intestine: mucosal iron pools

1964 ◽  
Vol 207 (4) ◽  
pp. 893-900 ◽  
Author(s):  
James Manis ◽  
David Schachter
Keyword(s):  
Active Transport ◽  
Hemagglutination Inhibition ◽  
Iron Uptake ◽  
Iron Transport ◽  
Iron Absorption ◽  
Duodenal Mucosa ◽  
Serosal Surface ◽  
Normal Rat

Methods were developed for the specific estimation of Fe++, Fe+++, Fe59++ and Fe59+++ in homogenates of rat duodenal mucosa and applied to studies of iron transport by everted gut sacs in vitro and duodenal loops in vivo. In the course of active transport in vitro Fe++ is absorbed into a mucosal ferrous pool which turns over relatively rapidly by a) transport of Fe++ to the serosal surface and b) formation of a mucosal Fe+++ pool. The latter turns over more slowly and appears to be a storage depot for excess mineral. When rats are pretreated with oral iron, uptake of Fe++ at the mucosal surface is decreased, diversion of mucosal Fe++ to mucosal Fe+++ is increased, and transport toward the serosal surface is reduced. Corresponding effects, which tend to limit iron absorption in vivo, are observed with duodenal loops. A sensitive hemagglutination-inhibition method was developed to estimate apoferritin in normal rat mucosa, and this protein was detected and quantified. Ferritin could account for only 8.0% of the trivalent mucosal pool and appeared to play an insignificant role in the present studies.

scholarly journals The Tail Sheath of Bacteriophage N4 Interacts with the Escherichia coli Receptor

2008 ◽  
Vol 191 (2) ◽  
pp. 525-532 ◽  
Author(s):  
Jennifer McPartland ◽  
Lucia B. Rothman-Denes
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Outer Membrane Receptor ◽  
E Coli ◽  
Hexahistidine Tag ◽  
C Terminus ◽  
Successful Infection ◽  
Tail Sheath

ABSTRACT Unlike other characterized phages, the lytic coliphage N4 must inject the 360-kDa virion RNA polymerase (vRNAP), in addition to its 72-kbp genome, into the host for successful infection. The process of adsorption to the host sets up and elicits the necessary conformational changes in the virion to allow genome and vRNAP injection. Infection of suppressor and nonsuppressor strains, Escherichia coli W3350 supF and E. coli W3350, with a mutant N4 isolate (N4am229) harboring an amber mutation in Orf65 yielded virions containing (N4gp65+) and lacking (N4gp65−) gp65, respectively. N4gp65+ but not N4gp65− phage was able to adsorb to the host. Recombinant gp65 with a hexahistidine tag at the N terminus or hexahistidine and c-myc tags at the C terminus was able to complement N4gp65− virions in vivo and in vitro. Immunogold detection of gp65 in vivo complemented virions revealed its localization at the N4 tail. Finally, we show both in vitro and in vivo that gp65 interacts with the previously determined N4 outer membrane receptor, NfrA.

scholarly journals In Vivo Expression and Immunoadjuvancy of a Mutant of Heat-Labile Enterotoxin of Escherichia coli in Vaccine and Vector Strains of Vibrio cholerae

1999 ◽  
Vol 67 (4) ◽  
pp. 1694-1701 ◽  
Author(s):  
Edward T. Ryan ◽  
Thomas I. Crean ◽  
Manohar John ◽  
Joan R. Butterton ◽  
John D. Clements ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Vibrio Cholerae ◽  
Supernatant Fraction ◽  
E Coli ◽  
Heat Labile ◽  
In Vivo Expression ◽  
Heterologous Antigens

ABSTRACT Vibrio cholerae secretes cholera toxin (CT) and the closely related heat-labile enterotoxin (LT) of Escherichia coli, the latter when expressed in V. cholerae. Both toxins are also potent immunoadjuvants. Mutant LT molecules that retain immunoadjuvant properties while possessing markedly diminished enterotoxic activities when expressed by E. coli have been developed. One such mutant LT molecule has the substitution of a glycine residue for arginine-192 [LT(R192G)]. Live attenuated strains of V. cholerae that have been used both as V. cholerae vaccines and as vectors for inducing mucosal and systemic immune responses directed against expressed heterologous antigens have been developed. In order to ascertain whether LT(R192G) can act as an immunoadjuvant when expressed in vivo by V. cholerae, we introduced a plasmid (pCS95) expressing this molecule into three vaccine strains ofV. cholerae, Peru2, ETR3, and JRB14; the latter two strains contain genes encoding different heterologous antigens in the chromosome of the vaccine vectors. We found that LT(R192G)was expressed from pCS95 in vitro by both E. coli andV. cholerae strains but that LT(R192G) was detectable in the supernatant fraction of V. choleraecultures only. In order to assess potential immunoadjuvanticity, groups of germfree mice were inoculated with the three V. cholerae vaccine strains alone and compared to groups inoculated with the V. cholerae vaccine strains supplemented with purified CT as an oral immunoadjuvant or V. choleraevaccine strains expressing LT(R192G) from pCS95. We found that mice continued to pass stool containing V. cholerae strains with pCS95 for at least 4 days after oral inoculation, the last day evaluated. We found that inoculation withV. cholerae vaccine strains containing pCS95 resulted in anti-LT(R192G) immune responses, confirming in vivo expression. We were unable to detect immune responses directed against the heterologous antigens expressed at low levels in any group of animals, including animals that received purified CT as an immunoadjuvant. We were, however, able to measure increased vibriocidal immune responses against vaccine strains in animals that receivedV. cholerae vaccine strains expressing LT(R192G) from pCS95 compared to the responses in animals that received V. cholerae vaccine strains alone. These results demonstrate that mutant LT molecules can be expressed in vivo by attenuated vaccine strains of V. cholerae and that such expression can result in an immunoadjuvant effect.

scholarly journals Contributions of Escherichia coli and its motility to the formation of dual-species biofilms with Vibrio cholerae

2021 ◽  
Author(s):  
Hui Wang ◽  
Feiyu Li ◽  
Li Xu ◽  
Hyuntae Byun ◽  
JinMing Fan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Life Cycle ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Type I ◽  
Flagellar Motility ◽  
E Coli ◽  
Aggregation Formation

Biofilm formation is important in both the environmental and intestinal phases of the Vibrio cholerae life cycle. Nevertheless, most studies of V. cholerae biofilm formation focus on mono-species cultures, whereas nearly all biofilm communities found in nature consist of a variety of microorganisms. Multi-species biofilms formed between V. cholerae and other bacteria in the environment and the interactions that exist between these species are still poorly understood. In this study, the influence of Escherichia coli on the biofilm formation of V. cholerae was studied in the context of both in vitro coculture and in vivo coinfection. To understand the underlying synergistic mechanisms between these two species and to investigate the role of E. coli in V. cholerae biofilm formation, different pathotypes of E. coli and corresponding deletion mutants lacking genes that influence flagella motility, curli fibers, or type I pili were cocultured with V. cholerae . Our findings demonstrate that the presence of commensal E. coli increases biofilm formation at the air-liquid interface in vitro and the generation of biofilm-like multicellular clumps in the mice feces. Examination of laboratory E. coli flagellar-motility mutants Δ fliC and Δ motA in the dual-species biofilm formation suggests that flagellar motility plays an important role in the synergistic interaction and co-aggregation formation between V. cholerae and E. coli . This study facilitates a better understanding of how V. cholerae resides in harsh environments and colonizes the intestine. IMPORTANCE Biofilms play an important role in the V. cholerae life cycle. Until now, mono-species biofilm formation of V. cholerae has been well studied. However, in nature, bacteria live in complex microbial communities, where biofilm is mostly composed of multiple microbial species that interact to cooperate with or compete against each other. Uncovering how V. cholerae forms multi-species biofilm is critical for furthering our understanding of how V. cholerae survives in the environment and transitions to infecting the human host. In this work, the dual-species biofilm between V. cholerae and E. coli was investigated. We demonstrate that the presence of commensal E. coli increased overall biofilm formation. Furthermore, we demonstrate that the motility of E. coli flagella is important for V. cholerae and E. coli to form co-aggregation clumps in dual-species biofilm. These results shed light on a new mechanism for understanding the survival and pathogenesis of V. cholerae .

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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