Manual and expert annotation of the nearly complete genome sequence of Staphylococcus sciuri strain ATCC 29059: A reference for the oxidase-positive staphylococci that supports the atypical phenotypic features of the species group.

Staphylococcus sciuri is considered to be one of the most ancestral species in the natural history of the Staphylococcus genus that consists of 48 validly described species. It belongs to the basal group of oxidase-positive and novobiocin-resistant staphylococci that diverged from macrococci approximately 250 million years ago. Contrary to other groups, the S. sciuri species group has not developed host-specific colonization strategies. Genome analysis of S. sciuri ATCC 29059 provides here the first genetic basis for atypical traits that would support the switch between the free-living style and the infective state in animals and humans. From among the most remarkable features, it was noticed in this extensive study that there were a number of phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS), almost twice as many as any other staphylococci, and the co-occurrence of mevalonate and non-mevalonate pathways for isoprenoid synthesis. The sequenced strain was devoid of the main virulence factors present in Staphylococcus aureus, although it exhibited numerous heme and iron acquisition systems, as well as crt and aldH genes necessary for gold pigment synthesis. The sensing and signaling networks, exemplified by a large and typical repertoire of two-component regulatory systems and a complete panel of master regulators, such as agr, rex, mgrA, rot, sarA and sarR genes, depict the background in which S. aureus virulence genes were later acquired. An additional sigma factor, a distinct set of electron transducer elements and many gene operons similar to those found in Bacillus spp. would constitute the most visible remnant links with Bacillaceae organisms.

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. We also show that it is disrupted by iron chelation, which reduces the intracellular iron concentration necessary for loading iron into protoporphyrin IX (PPIX, the immediate heme precursor). In both cases, we established that there is an increased PPIX concentration and we demonstrate that this compound is expelled by the MacAB-TolC pump, an efflux pump involved in E. coli and Salmonella for macrolide efflux. The E. coli macAB and tolC mutants accumulate PPIX and are sensitive to photo-inactivation. The MacAB-TolC pump is required for Salmonella typhimurium survival in macrophages. We propose that PPIX is an endogenous substrate of the MacAB-TolC pump in E. coli and S. typhimurium and that this compound is produced inside bacteria when natural heme homeostasis is disrupted by iron shortage, as happens when bacteria invade the mammalian host.

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. Despite a considerable amount of data available, the molecular mechanism of TonB-dependent active transport is still poorly understood. In this work, we present a structural study of a TonB-like protein, HasB dedicated to the HasR receptor. HasR acquires heme either free or via an extracellular heme transporter, the hemophore HasA. Heme is used as an iron source by bacteria. We have solved the structure of the HasB periplasmic domain of Serratia marcescens and describe its interaction with a critical region of HasR. Some important differences are observed between HasB and TonB structures. The HasB fold reveals a new structural class of TonB-like proteins. Furthermore, we have identified the structural features that explain the functional specificity of HasB. These results give a new insight into the molecular mechanism of nutrient active transport through the bacterial outer membrane and present the first detailed structural study of a specific TonB-like protein and its interaction with the receptor.

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. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems.

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. They are non-proteinaceus organic molecules having an extremely high affinity for Fe(3+) and able to extract it from host proteins. Haemophores, that display functional analogy with siderophores, were more recently discovered. They are a class of secreted proteins with a high affinity for haem; they are able to extract haem from host haemoproteins and deliver it to specific receptors that internalize haem. In the past few years, a wealth of data has accumulated on haem acquisition systems that are dependent on surface exposed/secreted bacterial proteins. They promote haem transfer from its initial source (in most cases, a eukaryotic haem binding protein) to the transporter that carries out the membrane crossing step. Here we review recent discoveries in this field, with particular emphasis on similar and dissimilar mechanisms in haemophores and siderophores, from the initial host source to the binding protein/receptor at the cell surface.

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. It is generally accepted that T1SS-secreted proteins have a C-terminal secretion signal required for secretion and that this signal interacts with the ABC protein. However, we have previously shown that for the Serratia marcescens hemophore HasA, interactions with the ABC protein and subsequent T1SS assembly require additional regions. In this work, we characterize these regions and demonstrate that they are numerous, distributed throughout the HasA polypeptide, and most likely linear. Together with the C-terminal signal, these elements maximize the secretion of HasA. The data also show that the C-terminal signal of HasA triggers HasD-driven ATP hydrolysis, leading to disassembly of the complex. These data support a model of type 1 secretion involving a multistep interaction between the substrate and the ABC protein that stabilizes the assembled secretion system until the C terminus is presented. This model also supports tight coupling between synthesis and secretion.

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. YfeX and EfeB promote iron extraction from heme preserving the tetrapyrrol ring intact. This novel enzymatic reaction corresponds to the deferrochelation of the heme. YfeX and EfeB are the sole proteins able to provide iron from exogenous heme sources to E. coli. YfeX is located in the cytoplasm. EfeB is periplasmic and enables iron extraction from heme in the periplasm and iron uptake in the absence of any heme permease. YfeX and EfeB are widespread and highly conserved in bacteria. We propose that their physiological function is to retrieve iron from heme.

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. Upon docking to the receptor, 1 of the 2 axial heme coordinations of the hemophore is initially broken, but the position and orientation of the heme is preserved. Subsequently, steric displacement of heme by a receptor residue ruptures the other axial coordination, leading to heme transfer into the receptor.

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. Its alpha/beta structure, as that of its binding pocket, has no common features with other iron- or heme-binding proteins. Heme is held by two loops L1 and L2 and coordinated to iron by an unusual ligand pair, H32/Y75. Two independent regions of the hemophore beta-sheet are involved in HasA-HasR receptor interaction. Here, we report the 3-D NMR structure of apoHasA and the backbone dynamics of both loaded and unloaded hemophore. While the overall structure of HasA is very similar in the apo and holo forms, the hemophore presents a transition from an open to a closed form upon ligand binding, through a large movement, of up to 30 A, of loop L1 bearing H32. Comparison of loaded and unloaded HasA dynamics on different time scales reveals striking flexibility changes in the binding pocket. We propose a mechanism by which these structural and dynamic features provide the dual function of heme binding and release to the HasR receptor.

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. coli inner membrane. We show here that hemTUV genes complement the Dpp permease for heme utilization as an iron source and thus are functional in E. coli. However, hemTUV genes do not complement the Dpp permease for ALA uptake, indicating that the HemTUV permease does not transport ALA. Peptides do not inhibit heme uptake in vivo, indicating that, unlike Dpp permease, HemTUV permease does not transport peptides. HemT, the periplasmic binding protein, binds heme. Heme binding is saturable and not inhibited by peptides that inhibit heme uptake by the Dpp system. Thus, the S. marcescens HemTUV permease and, most likely, HemTUV orthologs present in many gram-negative pathogens form a class of heme-specific permeases different from the Dpp peptide/heme permease characterized in E. coli.

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. marcescens TonB and E. coli TonB proteins function equally well with various TBDT receptors for heme and siderophores. This has raised the question of the target of this specificity. HasB could be specific either to heme TBDT receptors or only to HasR. To resolve this question, we have cloned in E. coli another S. marcescens heme receptor, HemR, and we show here that this receptor is TonB dependent and does not work with HasB. This demonstrates that HasB is not dedicated to heme TBDT receptors but rather forms a specific pair with HasR. This is the first reported case of a specific TonB protein working with only one TBDT receptor in one given species. We discuss the occurrence, possible molecular mechanisms, and selective advantages of such dedicated TonB paralogs.

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. HasA is a monomer that binds one b heme with strong affinity. The heme in HasA is highly exposed to solvent and coordinated by an unusual pair of ligands, a histidine and a tyrosine. Here, we report the identification, the characterization and the X-ray structure of a dimeric form of HasA from S. marcescens: DHasA. We show that both monomeric and dimeric forms are secreted in iron deficient conditions by S. marcescens. The crystal structure of DHasA reveals that it is a domain swapped dimer. The overall structure of each monomeric subunit of DHasA is very similar to that of HasA but formed by parts coming from the two different polypeptide chains, involving one of the heme ligands. Consequently DHasA binds two heme molecules by residues coming from both polypeptide chains. We show here that, while DHasA can bind two heme molecules, it is not able to deliver them to the receptor HasR. However, DHasA can efficiently transfer its heme to the monomeric form that, in turn, delivers it to HasR. We assume that DHasA can function as a heme reservoir in the hemophore system.

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. Strains expressing the transporter and the mutant protein are sensitive to detergents (sodium dodecyl sulfate [SDS]). TolC is trapped in the transporter jammed by the truncated substrate and therefore is not present at sufficient concentrations to allow the efflux pumps to expel detergents. Using an SDS sensitivity assay, we showed that the hemophore interacts with the ABC protein via two nonoverlapping sites. We also demonstrated that the C-terminal peptide, which functions as an intramolecular signal sequence in the complete substrate, may also have intermolecular activity and triggers complex dissociation in vivo when it is provided as a distinct peptide. The SDS sensitivity test on plates enables workers to study type I secretion protein association and dissociation independent of the secretion process itself.

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. coli strain expressing just one foreign heme outer membrane receptor can use exogenous heme as an iron source. This result suggests either that heme might be able to cross the cytoplasmic membrane in the absence of specific carrier or that there is a functional inner membrane heme transporter. Here, we show that to use heme iron E. coli requires the dipeptide inner membrane ATP-binding cassette transporter (DppBCDF) and either of two periplasmic binding proteins: MppA, the L-alanyl-gamma-D-glutamyl-meso-diaminopimelate binding protein, or DppA, the dipeptide binding protein. Thus, wild-type E. coli has a peptide/heme permease despite being unable to use exogenous heme. DppA, which shares sequence similarity with the Haemophilus influenzae heme-binding protein HbpA, and MppA are functional heme-binding proteins. Peptides compete with heme for binding both "in vitro" and "in vivo."

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 is then transferred to HasR, which has a lower affinity for heme. The mechanism of the heme transfer between HasA and HasR is largely unknown. HasR has been overexpressed and purified in holo and apo forms. It binds one heme molecule with a Ka of 5 x 10(6) m(-1) and shows the characteristic absorbance spectrum of a low spin heme iron. Both holoHasA and apoHasA bind tightly to apoHasR in a 1:1 stoichiometry. In this study we show that heme transfer occurs in vitro in the purified HasA.HasR complex, demonstrating that heme transfer is energy- and TonB complex-independent and driven by a protein-protein interaction. We also show that heme binding to HasR involves two conserved histidine residues.

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. We investigated the features of the holo-hemophore, the only HasR ligand known to act as an inducer. We used a hemophore mutant that does not deliver its heme and a HasR mutant that does not bind heme, and we showed that heme transfer from the hemophore to the receptor is necessary for induction. Using a hemophore mutant that does not bind heme and that blocks heme transport, we demonstrated that two molecules that do not interact (heme and the mutant hemophore) may nonetheless induce this system. These findings suggest that hemophore-mediated induction and heme transport involve different mechanisms. The hemophore region important for induction was precisely localized to amino acids 50 to 55, which lie in one of the two HasR-binding hemophore regions. This bipartite stimulus probably corresponds to a physiological process because heme is transferred to the receptor before apo-hemophore release. This bipartite regulation mechanism may allow the bacterium to adjust its heme transport mechanism to the perceived environmental heme concentration.

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. HasA and HasR have been overexpressed in Escherichia coli and the complex formed and crystallized. Small platelets and bunches of needles of dimensions 0.01 x 0.1 x 1 mm were obtained. A native data set has been collected to 6.8 A.

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. The mature HasR protein comprises a 99-residue amino-terminal extension necessary for hasR transcription, followed by a plug domain of 139 amino acids and a beta-barrel domain inserted in the outer membrane, the lumen of which is closed by the plug. This model was used to generate hasR deletions encoding HasR proteins with the native signal peptides but lacking either the N-terminal regulatory extension or encoding the plug or the beta-barrel alone. The protein lacking the N-terminal extension, HasR delta11-91, was incorporated in the outer membrane and was fully functional for active uptake of free and hemophore-bound heme. The HasR beta-barrel, delta11-192, was also incorporated in the outer membrane and bound the hemophore but expressed no active heme transport properties. The HasR plug remained in the periplasm. Coexpression of the plug and the beta-barrel allowed partial plug insertion in the outer membrane, demonstrating that these two HasR domains interact in vivo. The beta-barrel and the plug also interact in vitro. Nevertheless, the two domains did not complement each other to reconstitute an active TonB-dependent receptor for free or hemophore-bound heme uptake. Production of the beta-barrel alone selectively increased passive diffusion of heme but not of other exogenous compounds. A mutation at histidine 603, which is required for heme uptake through the wild-type receptor, abolished heme diffusion, showing that HasR delta11-192 forms a specific heme channel.

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. To fulfill their iron needs, bacteria have multiple iron acquisition systems, reflecting the diversity of their potential biotopes. The iron/heme acquisition systems in bacteria have one of two general mechanisms. The first involves direct contact between the bacterium and the exogenous iron/heme sources. The second mechanism relies on molecules (siderophores and hemophores) synthesized and released by bacteria into the extracellular medium; these molecules scavenge iron or heme from various sources. Recent genetic, biochemical, and crystallographic studies have allowed substantial progress in describing molecular mechanisms of siderophore and hemophore interactions with the outer membrane receptors, transport through the inner membrane, iron storage, and regulation of genes encoding biosynthesis and uptake proteins.