Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli.

In most organisms, heme biosynthesis is strictly controlled so as to avoid heme and heme precursor accumulation, which is toxic. Escherichia coli regulates heme biosynthesis by a feedback loop involving heme-induced proteolytic cleavage of HemA, glutamyl-tRNA reductase, which is the first enzyme in the heme biosynthetic pathway. We show here that heme homeostasis can be disrupted by overproduction of YfeX, a cytoplasmic protein that captures iron from heme that we named deferrochelatase.

The structure of HasB reveals a new class of TonB protein fold.

TonB is a key protein in active transport of essential nutrients like vitamin B12 and metal sources through the outer membrane transporters of Gram-negative bacteria. This inner membrane protein spans the periplasm, contacts the outer membrane receptor by its periplasmic domain and transduces energy from the cytoplasmic membrane pmf to the receptor allowing nutrient internalization. Whereas generally a single TonB protein allows the acquisition of several nutrients through their cognate receptor, in some species one particular TonB is dedicated to a specific system.

Staphylococcus aureus FepA and FepB proteins drive heme iron utilization in Escherichia coli.

EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined.

Haemophore functions revisited.

Haem is the major iron source for bacteria that develop in higher organisms. In these hosts, bacteria have to cope with nutritional immunity imposed by the host, since haem and iron are tightly bound to carrier and storage proteins. Siderophores were the first recognized fighters in the battle for iron between bacteria and host.

Multiple signals direct the assembly and function of a type 1 secretion system.

Type 1 secretion systems (T1SS) are present in a wide range of Gram-negative bacteria and are involved in the secretion of diverse substrates such as proteases, lipases, and hemophores. T1SS consist of three proteins: an inner membrane ABC (ATP binding cassette) protein, a periplasmic adaptor, and an outer membrane channel of the TolC family. Assembly of the tripartite complex is transient and induced upon binding of the substrate to the ABC protein.

Bacteria capture iron from heme by keeping tetrapyrrol skeleton intact.

Because heme is a major iron-containing molecule in vertebrates, the ability to use heme-bound iron is a determining factor in successful infection by bacterial pathogens. Until today, all known enzymes performing iron extraction from heme did so through the rupture of the tetrapyrrol skeleton. Here, we identified 2 Escherichia coli paralogs, YfeX and EfeB, without any previously known physiological functions.

Heme uptake across the outer membrane as revealed by crystal structures of the receptor-hemophore complex.

Gram-negative bacteria use specific heme uptake systems, relying on outer membrane receptors and excreted heme-binding proteins (hemophores) to scavenge and actively transport heme. To unravel the unknown molecular details involved, we present 3 structures of the Serratia marcescens receptor HasR in complex with its hemophore HasA. The transfer of heme over a distance of 9 A from its high-affinity site in HasA into a site of lower affinity in HasR is coupled with the exergonic complex formation of the 2 proteins.

Functional differences between heme permeases: Serratia marcescens HemTUV permease exhibits a narrower substrate specificity (restricted to heme) than the Escherichia coli DppABCDF peptide-heme permease.

Serratia marcescens hemTUV genes encoding a potential heme permease were cloned in Escherichia coli recombinant mutant FB827 dppF::Km(pAM 238-hasR). This strain, which expresses HasR, a foreign heme outer membrane receptor, is potentially capable of using heme as an iron source. However, this process is invalidated due to a dppF::Km mutation which inactivates the Dpp heme/peptide permease responsible for heme, dipeptide, and delta-aminolevulinic (ALA) transport through the E.

Comparative analysis of structural and dynamic properties of the loaded and unloaded hemophore HasA: functional implications.

A heme-acquisition system present in several Gram-negative bacteria requires the secretion of hemophores. These extracellular carrier proteins capture heme and deliver it to specific outer membrane receptors. The Serratia marcescens HasA hemophore is a monodomain protein that binds heme with a very high affinity.

HasB, the Serratia marcescens TonB paralog, is specific to HasR.

Serratia marcescens possesses two functional TonB paralogs, TonB(Sm) and HasB, for energizing TonB-dependent transport receptors (TBDT). Previous work had shown that HasB is specific to heme uptake in the natural host and in Escherichia coli expressing the S. marcescens TBDT receptor HasR, whereas the S.

Probing the in vivo dynamics of type I protein secretion complex association through sensitivity to detergents.

The Serratia marcescens hemophore is secreted by a type I secretion system consisting of three proteins: a membrane ABC protein, an adaptor protein, and the TolC-like outer membrane protein. Assembly of these proteins is induced by substrate binding to the ABC protein. Here we show that a hemophore mutant lacking the last 14 C-terminal amino acids is not secreted but rather interacts with the ABC protein and promotes a stable multiprotein complex.

The crystal structure of the secreted dimeric form of the hemophore HasA reveals a domain swapping with an exchanged heme ligand.

To satisfy their iron needs, several Gram-negative bacteria use a heme uptake system involving an extracellular heme-binding protein called hemophore. The function of the hemophore is to acquire free or hemoprotein-bound heme and to transfer it to HasR, its specific outer membrane receptor, by protein-protein interaction. The hemophore HasA secreted by Serratia marcescens, an opportunistic pathogen, was the first to be identified and is now very well characterized.

The housekeeping dipeptide permease is the Escherichia coli heme transporter and functions with two optional peptide binding proteins.

Heme, a major iron source, is transported through the outer membrane of Gram-negative bacteria by specific heme/hemoprotein receptors and through the inner membrane by heme-specific, periplasmic, binding protein-dependent, ATP-binding cassette permeases. Escherichia coli K12 does not use exogenous heme, and no heme uptake genes have been identified. Nevertheless, a recombinant E.

The heme transfer from the soluble HasA hemophore to its membrane-bound receptor HasR is driven by protein-protein interaction from a high to a lower affinity binding site.

HasA is an extracellular heme binding protein, and HasR is an outer membrane receptor protein from Serratia marcescens. They are the initial partners of a heme internalization system allowing S. marcescens to scavenge heme at very low concentrations due to the very high affinity of HasA for heme (Ka = 5,3 x 10(10) m(-1)).

Heme and a five-amino-acid hemophore region form the bipartite stimulus triggering the has signaling cascade.

Bacterial cells sense the extracellular environment and adapt to that environment by activating gene regulation circuits, often by means of signaling molecules. The Serratia marcescens hemophore is a signaling molecule that acts as an extracellular heme-scavenging protein. The heme-loaded hemophore interacts with its cognate receptor (HasR), triggering transmembrane signaling and turning on transcription of hemophore-dependent heme uptake genes.

Purification, crystallization and preliminary X-ray analysis of the outer membrane complex HasA-HasR from Serratia marcescens.

Serratia marcescens is able to acquire iron using its haem-acquisition system (;has'), which contains an outer membrane receptor HasR and a soluble haemophore HasA. After secretion, HasA binds free haem in the extracellular medium or extracts it from haemoproteins and delivers it to the receptor. Here, the crystallization of a HasA-HasR complex is reported.

Activities of the Serratia marcescens heme receptor HasR and isolated plug and beta-barrel domains: the beta-barrel forms a heme-specific channel.

The Serratia marcescens hemophore-specific outer membrane receptor HasR is a member of the TonB-dependent family of autoregulated receptors. It can transport either heme itself or heme bound to the hemophore HasA. On the basis of sequence and functional similarities with other TonB-dependent outer membrane receptors whose three-dimensional structures have been determined, a HasR structure model was proposed.

Bacterial iron sources: from siderophores to hemophores.

Iron is an essential element for most organisms, including bacteria. The oxidized form is insoluble, and the reduced form is highly toxic for most macromolecules and, in biological systems, is generally sequestrated by iron- and heme-carrier proteins. Thus, despite its abundance on earth, there is practically no free iron available for bacteria whatever biotope they colonize.

Haemophore-mediated signalling in Serratia marcescens: a new mode of regulation for an extra cytoplasmic function (ECF) sigma factor involved in haem acquisition.

Bacterial extra cytoplasmic function (ECF) sigma factors control a wide range of cell envelope activities including iron and haem uptake systems. Sigma activity is usually inhibited by membrane-bound antisigma. An extra cytoplasmic signal modulates sigma-antisigma interactions and thereby leads to the transcription of the target operon.

Free and hemophore-bound heme acquisitions through the outer membrane receptor HasR have different requirements for the TonB-ExbB-ExbD complex.

Many gram-negative bacteria have specific outer membrane receptors for free heme, hemoproteins, and hemophores. Heme is a major iron source and is taken up intact, whereas hemoproteins and hemophores are not transported: the iron-containing molecule has to be stripped off at the cell surface, with only the heme moiety being taken up. The Serratia marcescens hemophore-specific outer membrane receptor HasR can transport either heme itself or heme bound to the hemophore HasA.

Ligand delivery by haem carrier proteins: the binding of Serratia marcescens haemophore to its outer membrane receptor is mediated by two distinct peptide regions.

Haem is involved in essential processes. It is toxic and thus is not found free in living organisms but almost entirely sequestered by haem carrier proteins. We investigated the mechanisms of haem transfer between the proteins of a bacterial haem acquisition system involving haemophores.

Thermodynamics of heme binding to the HasA(SM) hemophore: effect of mutations at three key residues for heme uptake.

HasA(SM) secreted by the Gram-negative bacterium Serratia marcescens belongs to the hemophore family. Its role is to take up heme from host heme carriers and to shuttle it to specific receptors. Heme is linked to the HasA(SM) protein by an unusual axial ligand pair: His32 and Tyr75.

Haemophore-mediated signal transduction across the bacterial cell envelope in Serratia marcescens: the inducer and the transported substrate are different molecules.

Numerous bacteria are able to use free and haemoprotein-bound haem as iron sources because of the action of small secreted proteins called haemophores. Haemophores have very high affinity for haem, and can therefore extract haem from the haem-carrier proteins and deliver it to the cells by means of specific cell surface receptors. Haem is then taken up and the empty haemophores are recycled.