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

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.

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Vibrio cholerae VciB Mediates Iron Reduction

Journal of Bacteriology ◽

10.1128/jb.00874-16 ◽

2017 ◽

Vol 199 (12) ◽

Cited By ~ 6

Author(s):

Eric D. Peng ◽

Shelley M. Payne

Keyword(s):

Electron Transport ◽

Vibrio Cholerae ◽

Ferrous Iron ◽

Electron Transport Chain ◽

Iron Reduction ◽

Iron Transport ◽

Ferric Iron ◽

Transport Systems ◽

Content Type ◽

Transport Chain

ABSTRACT Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. V. cholerae thrives within the human host, where it replicates to high numbers, but it also persists within the aquatic environments of ocean and brackish water. To survive within these nutritionally diverse environments, V. cholerae must encode the necessary tools to acquire the essential nutrient iron in all forms it may encounter. A prior study of systems involved in iron transport in V. cholerae revealed the existence of vciB, which, while unable to directly transport iron, stimulates the transport of iron through ferrous (Fe2+) iron transport systems. We demonstrate here a role for VciB in V. cholerae in which VciB stimulates the reduction of Fe3+ to Fe2+, which can be subsequently transported into the cell with the ferrous iron transporter Feo. Iron reduction is independent of functional iron transport but is associated with the electron transport chain. Comparative analysis of VciB orthologs suggests a similar role for other proteins in the VciB family. Our data indicate that VciB is a dimer located in the inner membrane with three transmembrane segments and a large periplasmic loop. Directed mutagenesis of the protein reveals two highly conserved histidine residues required for function. Taken together, our results support a model whereby VciB reduces ferric iron using energy from the electron transport chain. IMPORTANCE Vibrio cholerae is a prolific human pathogen and environmental organism. The acquisition of essential nutrients such as iron is critical for replication, and V. cholerae encodes a number of mechanisms to use iron from diverse environments. Here, we describe the V. cholerae protein VciB that increases the reduction of oxidized ferric iron (Fe3+) to the ferrous form (Fe2+), thus promoting iron acquisition through ferrous iron transporters. Analysis of VciB orthologs in Burkholderia and Aeromonas spp. suggest that they have a similar activity, allowing a functional assignment for this previously uncharacterized protein family. This study builds upon our understanding of proteins known to mediate iron reduction in bacteria.

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Secreted Pyomelanin of Legionella pneumophila Promotes Bacterial Iron Uptake and Growth under Iron-Limiting Conditions

Infection and Immunity ◽

10.1128/iai.00858-13 ◽

2013 ◽

Vol 81 (11) ◽

pp. 4182-4191 ◽

Cited By ~ 33

Author(s):

Huaixin Zheng ◽

Christa H. Chatfield ◽

Mark R. Liles ◽

Nicholas P. Cianciotto

Keyword(s):

Legionella Pneumophila ◽

Ferrous Iron ◽

Iron Uptake ◽

Iron Transport ◽

Ferric Iron ◽

Iron Acquisition ◽

Ferric Hydroxide ◽

Supernatant Fraction ◽

Content Type ◽

Ferrous Iron Transport

ABSTRACTIron acquisition is critical to the growth and virulence ofLegionella pneumophila. Previously, we found thatL. pneumophilauses both a ferrisiderophore pathway and ferrous iron transport to obtain iron. We now report that two molecules secreted byL. pneumophila, homogentisic acid (HGA) and its polymerized variant (HGA-melanin, a pyomelanin), are able to directly mediate the reduction of various ferric iron salts. Furthermore, HGA, synthetic HGA-melanin, and HGA-melanin derived from bacterial supernatants enhanced the ability ofL. pneumophilaand other species ofLegionellato take up radiolabeled iron. Enhanced iron uptake was not observed with a ferrous iron transport mutant. Thus, HGA and HGA-melanin mediate ferric iron reduction, with the resulting ferrous iron being available to the bacterium for uptake. Upon further testing ofL. pneumophilaculture supernatants, we found that significant amounts of ferric and ferrous iron were associated with secreted HGA-melanin. Importantly, a pyomelanin-containing fraction obtained from a wild-type culture supernatant was able to stimulate the growth of iron-starved legionellae. That the corresponding supernatant fraction obtained from a nonpigmented mutant culture did not stimulate growth demonstrated that HGA-melanin is able to both promote iron uptake and enhance growth under iron-limiting conditions. Indicative of a complementary role in iron acquisition, HGA-melanin levels were inversely related to the levels of siderophore activity. Compatible with a role in the ecology and pathogenesis ofL. pneumophila, HGA and HGA-melanin were effective at reducing and releasing iron from both insoluble ferric hydroxide and the mammalian iron chelates ferritin and transferrin.

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Vibrio cholerae VciB Promotes Iron Uptake via Ferrous Iron Transporters

Journal of Bacteriology ◽

10.1128/jb.00569-08 ◽

2008 ◽

Vol 190 (17) ◽

pp. 5953-5962 ◽

Cited By ~ 15

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.

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MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽

10.1128/mbio.00236-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 28

Author(s):

Ryan W. Bogard ◽

Bryan W. Davies ◽

John J. Mekalanos

Keyword(s):

Amino Acid ◽

Vibrio Cholerae ◽

Virulence Factor ◽

Current Knowledge ◽

Intestinal Colonization ◽

Wild Type ◽

Pathogenic Potential ◽

Content Type ◽

Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

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Toward a mechanistic understanding of Feo-mediated ferrous iron uptake

Metallomics ◽

10.1039/c8mt00097b ◽

2018 ◽

Vol 10 (7) ◽

pp. 887-898 ◽

Cited By ~ 14

Author(s):

Alexandrea E. Sestok ◽

Richard O. Linkous ◽

Aaron T. Smith

Keyword(s):

Ferrous Iron ◽

Iron Uptake ◽

Iron Transport ◽

Structure And Function ◽

Ferrous Iron Transport ◽

Mechanistic Understanding ◽

And Function

The ferrous iron transport (Feo) system is the predominant mode of bacterial Fe2+import. Advancements in the structure and function of FeoB provide glimpses into the mechanism of Fe2+uptake.

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Ins and Outs: Recent Advancements in Membrane Protein-Mediated Prokaryotic Ferrous Iron Transport

Biochemistry ◽

10.1021/acs.biochem.1c00586 ◽

2021 ◽

Author(s):

Janae B. Brown ◽

Mark A. Lee ◽

Aaron T. Smith

Keyword(s):

Membrane Protein ◽

Ferrous Iron ◽

Iron Transport ◽

Ferrous Iron Transport

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Dynamic Membrane Localization of RNase Y in Bacillus subtilis

mBio ◽

10.1128/mbio.03337-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 3

Author(s):

Lina Hamouche ◽

Cyrille Billaudeau ◽

Anna Rocca ◽

Arnaud Chastanet ◽

Saravuth Ngo ◽

...

Keyword(s):

Bacillus Subtilis ◽

Active Form ◽

Model Organism ◽

Living Cells ◽

Cell Periphery ◽

Rnase E ◽

Content Type ◽

Endonucleolytic Cleavage ◽

Rnase Y

ABSTRACT Metabolic turnover of mRNA is fundamental to the control of gene expression in all organisms, notably in fast-adapting prokaryotes. In many bacteria, RNase Y initiates global mRNA decay via an endonucleolytic cleavage, as shown in the Gram-positive model organism Bacillus subtilis. This enzyme is tethered to the inner cell membrane, a pseudocompartmentalization coherent with its task of initiating mRNA cleavage/maturation of mRNAs that are translated at the cell periphery. Here, we used total internal reflection fluorescence microscopy (TIRFm) and single-particle tracking (SPT) to visualize RNase Y and analyze its distribution and dynamics in living cells. We find that RNase Y diffuses rapidly at the membrane in the form of dynamic short-lived foci. Unlike RNase E, the major decay-initiating RNase in Escherichia coli, the formation of foci is not dependent on the presence of RNA substrates. On the contrary, RNase Y foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. The Y-complex of three proteins (YaaT, YlbF, and YmcA) has previously been shown to play an important role for RNase Y activity in vivo. We demonstrate that Y-complex mutations have an effect similar to but much stronger than that of depletion of RNA in increasing the number and size of RNase Y foci at the membrane. Our data suggest that the Y-complex shifts the assembly status of RNase Y toward fewer and smaller complexes, thereby increasing cleavage efficiency of complex substrates like polycistronic mRNAs. IMPORTANCE All living organisms must degrade mRNA to adapt gene expression to changing environments. In bacteria, initiation of mRNA decay generally occurs through an endonucleolytic cleavage. In the Gram-positive model organism Bacillus subtilis and probably many other bacteria, the key enzyme for this task is RNase Y, which is anchored at the inner cell membrane. While this pseudocompartmentalization appears coherent with translation occurring primarily at the cell periphery, our knowledge on the distribution and dynamics of RNase Y in living cells is very scarce. Here, we show that RNase Y moves rapidly along the membrane in the form of dynamic short-lived foci. These foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. This contrasts with RNase E, the major decay-initiating RNase in E. coli, where it was shown that formation of foci is dependent on the presence of RNA substrates. We also show that a protein complex (Y-complex) known to influence the specificity of RNase Y activity in vivo is capable of shifting the assembly status of RNase Y toward fewer and smaller complexes. This highlights fundamental differences between RNase E- and RNase Y-based degradation machineries.

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Ferrous iron transport in Streptococcus mutans.

Journal of Bacteriology ◽

10.1128/jb.168.3.1096-1099.1986 ◽

1986 ◽

Vol 168 (3) ◽

pp. 1096-1099 ◽

Cited By ~ 40

Author(s):

S L Evans ◽

J E Arceneaux ◽

B R Byers ◽

M E Martin ◽

H Aranha

Keyword(s):

Streptococcus Mutans ◽

Ferrous Iron ◽

Iron Transport ◽

Ferrous Iron Transport

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Ferrous Iron Is a Significant Component of Bioavailable Iron in Cystic Fibrosis Airways

mBio ◽

10.1128/mbio.00557-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 86

Author(s):

Ryan C. Hunter ◽

Fadi Asfour ◽

Jozef Dingemans ◽

Brenda L. Osuna ◽

Tahoura Samad ◽

...

Keyword(s):

Cystic Fibrosis ◽

Ferrous Iron ◽

Ferric Iron ◽

Relative Proportion ◽

Iron Oxidation ◽

The United States ◽

Environmental Parameter ◽

Content Type

ABSTRACTChronic, biofilm-like infections by the opportunistic pathogenPseudomonas aeruginosaare a major cause of mortality in cystic fibrosis (CF) patients. While much is known aboutP. aeruginosafrom laboratory studies, far less is understood about what it experiencesin vivo. Iron is an important environmental parameter thought to play a central role in the development and maintenance ofP. aeruginosainfections, for both anabolic and signaling purposes. Previous studies have focused on ferric iron [Fe(III)] as a target for antimicrobial therapies; however, here we show that ferrous iron [Fe(II)] is abundant in the CF lung (~39 µM on average for severely sick patients) and significantly correlates with disease severity (ρ = −0.56,P= 0.004), whereas ferric iron does not (ρ = −0.28,P= 0.179). Expression of theP. aeruginosagenesbqsRS, whose transcription is upregulated in response to Fe(II), was high in the majority of patients tested, suggesting that increased Fe(II) is bioavailable to the infectious bacterial population. Because limiting Fe(III) acquisition inhibits biofilm formation byP. aeruginosain various oxicin vitrosystems, we also tested whether interfering with Fe(II) acquisition would improve biofilm control under anoxic conditions; concurrent sequestration of both iron oxidation states resulted in a 58% reduction in biofilm accumulation and 28% increase in biofilm dissolution, a significant improvement over Fe(III) chelation treatment alone. This study demonstrates that the chemistry of infected host environments coevolves with the microbial community as infections progress, which should be considered in the design of effective treatment strategies at different stages of disease.IMPORTANCEIron is an important environmental parameter that helps pathogens thrive in sites of infection, including those of cystic fibrosis (CF) patients. Ferric iron chelation therapy has been proposed as a novel therapeutic strategy for CF lung infections, yet until now, the iron oxidation state has not been measured in the host. In studying mucus from the infected lungs of multiple CF patients from Europe and the United States, we found that ferric and ferrous iron change in concentration and relative proportion as infections progress; over time, ferrous iron comes to dominate the iron pool. This information is relevant to the design of novel CF therapeutics and, more broadly, to developing accurate models of chronic CF infections.

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