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Accepted Manuscript :3-7-2017; Staphylococcus sciuri is considered to be one of the most ancestral species in the natura 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. In this current study, the genome analysis of S. sciuri ATCC 29059 provided 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, depicted 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.

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.

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.

▪ Abstract 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.

Summary Bacterial extra cytoplasmic function (ECF) sigma fac- tors 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. Sigma and anti- sigma genes generally belong to one autoregulated operon. However, ECF sigma and antisigma genes involved in iron acquisition, also called iron starvation ECF, are non-autoregulated exceptions to this rule. We fully reconstituted the has signalling cascade of Serratia marcescens in Escherichia coli . Binding of the haem-loaded haemophore to the outer membrane receptor, HasR, inactivates the antisigma HasS, turn- ing on HasI and thereby allowing has operon tran- scription. Deletion of the HasR N-terminal extension, present in all characterized outer membrane recep- tors endowed with signal transduction capacity, abol- ished the inducing activity but not the transport activity. Induction required the TonB-ExbB-ExbD com- plex. HasI, like the other iron starvation sigma, is iron repressed but not autoregulated. We found an entirely new regulation for the antisigma hasS gene, the tran- scription of which is HasI dependent. We suggest that the has system is both activated and repressed by the availability of external haem. When there is enough haem, the HasS antisigma activity is turned off and HasI induces the transcription of hasS . This leads to the storage of inactive HasS molecules which become active when HasR is not occupied by holo- haemophore ligand molecules: as soon as there is a haem shortage transcription is turned off. Positive autoregulation of ECF sigma and antisigma genes is usually considered as a mechanism for amplifying a perceived signal. However, our findings suggest, on the contrary, that antisigma regulation allows fine- tuning to the external signal. The biological signifi- cance of ECF sigma and antisigma autoregulation may need to be reconsidered.

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. Haemophores are secreted by several Gram-negative bacteria and are able to extract haem (assimilated as an iron source) from haemoproteins and deliver it to specific outer membrane receptors. The Serratia marcescens haemophore (HasA) is folded into a globular form and tyrosine and histidine are involved in haem ligation. Interaction with the receptor is of high affinity (5 nM) and does not involve haem. Identification and study of mutants with altered binding properties led to the description of two regions of the haemophore that bind to the receptor. They consist of residues involved in two beta strands located on the same side of HasA. Each region is sufficient for high affinity binding. The synthetic peptide corresponding to one beta strand competes with the corresponding haemophore region for binding to the receptor, suggesting that the two binding regions are independent binding sites. We propose a model for haem release and transfer to the receptor.

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. Here, we report a study of the regulation of the Serratia marcescens has operon which is involved in haemophore-dependent haem acquisition. We characterized two genes encoding proteins homologous to specific ECF sigma and antisigma factors. We showed that they regulate the synthesis of the haemophore-specific outer membrane receptor, HasR, by a signal transduction mechanism similar to the siderophore surface-signalling systems. We also showed the essential role of HasR itself in this process. Using haem-loaded and haem-free haemophore, we identified the stimulus for the HasR-mediated signal transduction as being the binding of the haem-loaded haemophore to HasR. Thus, unlike siderophore-uptake systems, in which the signalling molecule is the transported substrate itself, in the haemophore-dependent haem uptake system the inducer and the transported substrate are different compounds.

HasA is the secreted hemophore of the heme acquisition system (Has) of Serratia marcescens . It is secreted by a specific ABC transporter apparatus composed of three proteins: HasD, an inner membrane ABC protein; HasE, another inner membrane protein; and HasF, a TolC homolog. Except for HasF, the structural genes of the Has system are encoded by an iron-regulated operon. In previous studies, this secretion system has been reconstituted in Escherichia coli , where it requires the presence of the SecB chaperone, the Sec pathway-dedicated chaperone. We cloned and inactivated the secB gene from S. marcescens . We show that S. marcescens SecB is 93% identical to E. coli SecB and complements the secretion defects of a secB mutant of E. coli for both the Sec and ABC pathways of HasA secretion. In S. marcescens , SecB inactivation affects translocation by the Sec pathway and abolishes HasA secretion. This demonstrates that S. marcescens SecB is the genuine chaperone for HasA secretion in S. marcescens . These results also demonstrate that S. marcescens SecB is bifunctional, as it is involved in two separate secretion pathways. We investigated the effects of secB point mutations in the reconstituted HasA secretion pathway by comparing the translocation of a Sec substrate in various mutants. Two different patterns of SecB residue effects were observed, suggesting that SecB functions may differ for the Sec and ABC pathways.

Version imprimée publiée en 2004 - version en ligne publiée en 2014; International audience; This chapter describes hemophore secretion and its interaction with heme and hemoproteins, specific receptors, mechanisms of heme transfer from hemophore to receptor, TonB function at various steps in the heme acquisition process, and the regulation of the has operon. HasA hemophores are secreted by ABC transporters. One of the four HasA hemophores identified, the Serratia marcescens HasA protein, has been biochemically characterized. The interaction between HasA and its receptor was first investigated by binding experiments with cells expressing HasR immobilized on nitrocellulose membranes. Second, the binding of HasA to whole cells expressing HasR in liquid media was determined. In a wider perspective, it would be interesting to investigate whether hemophore‐like proteins exist in gram‐positive bacteria and whether hemophores are species specific or are used by nonproducing bacteria, similarly to certain siderophores. Finally, many bacterial species have multiple heme acquisition systems, with only one of these being hemophore dependent. Hemophores enhance the efficiency of heme delivery to bacterial cells and enlarge the heme source range.

Chronic bronchitis is associated with airways obstruction and inflammation. In order to determine whether aerosolized beclomethasone can modulate airway inflammation and diminish airway obstruction, subjects with chronic bronchitis performed spirometry and underwent bronchoalveolar lavage (BAL) before and after receiving 6 wk of therapy (five puffs four times a day) with either aerosolized beclomethasone (n = 20) or placebo (n = 10) in a double-blinded, randomized fashion. All subjects received aerosolized albuterol before each use of the study medications. Before BAL, the airways were visually assessed for the appearance of inflammation and assigned a score, the bronchitis index. BAL was performed by instilling five 20-ml aliquots of saline into each of three sites and pooling and separately analyzing the returns from the first aliquots to yield a "bronchial sample." The bronchial lavages were repeated in an additional three sites to increase the volume of fluid available for analysis. The fluid was prepared for cytologic examination by cytocentrifugation. Albumin (as a measure of epithelium permeability) and lactoferrin and lysozyme (as measures of serous cell activity) were measured in unconcentrated BAL fluid by enzyme-linked immunosorbent assay, and concentrations in epithelial lining fluid were estimated using urea as an internal marker for dilution. After treatment, the beclomethasone group, but not the placebo group, showed improvement in FVC (p = 0.02), FEV1 (p = 0.002), and 25 to 75% forced expiratory flow (p = 0.006). Associated with the improvement in spirometry, the bronchitis index fell (13.5 +/- 1.0 versus 10.75 +/- 1.1, p = 0.02) in the beclomethasone-treated group, but not the placebo-treated group.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemophores are secreted by several gram-negative bacteria ( Serratia marcescens , Pseudomonas aeruginosa , Pseudomonas fluorescens , and Yersinia pestis ) and form a family of homologous proteins. Unlike the S. marcescens hemophore (HasA SM ), the P. fluorescens hemophore HasA PF has an additional region of 12 residues located immediately upstream from the C-terminal secretion signal. We show that HasA PF undergoes a C-terminal cleavage which removes the last 21 residues when secreted from P. fluorescens and that only the processed form is able to deliver heme to the S. marcescens outer membrane hemophore-specific receptor, HasR SM . Functional analysis of variants including those with an internal deletion of the extra C-terminal domain show that the secretion signal does not inhibit the biological activity, whereas the 12-amino-acid region located upstream does. This extra domain may inhibit the interaction of the hemophore with HasR SM . To localize the hemophore regions involved in binding to HasR, chimeric HasA PF -HasA SM proteins were tested for biological activity. We show that residues 153 to 180 of HasA PF are necessary for its interaction with the receptor.

The major mechanism by which bacteria acquire free or haemoglobin-bound haem involves direct binding of haem to specific outer membrane receptors. Serratia marcescens and Pseudomonas aeruginosa have an alternative system, which involves an extracellular haemophore, HasA, that captures free or haemoglobin-bound haem and shuttles it to a specific cell surface outer membrane receptor, HasR. Both haem-free (apoprotein) and haem-loaded (holoprotein) HasA bind to HasR, evidence for direct protein-protein interactions between HasA and HasR. HasA binding to HasR takes place in a tonB mutant. TonB is thus required for a step subsequent to HasA binding.

Free iron availability is strongly limited in vertebrate hosts, making the iron acquisition by siderophores inappropriate. Pathogenic bacteria have developed various ways to use the host's iron from iron-containing proteins. Serratia marcescens can use the iron from hemoglobin through the secretion of a hemophore called HasA, which takes up the heme from hemoglobin and shuttles it to the receptor HasR, which in turn, releases heme into the bacterium. We report here the first crystal structure of such a hemophore, bound to a heme group at two different pH values and at a resolution of 1.9 A. The structure reveals a new original fold and suggests a hypothetical mechanism for both heme uptake and release.

The major mechanism by which bacteria acquire free or haemoglobin-bound haem involves direct binding to specific outer membrane receptors. Serratia marcescens also secretes a haem-binding protein, HasA, which functions as a haemophore that catches haem and shuttles it to a cell surface specific outer membrane receptor, HasR. We report the isolation and characterization of hasAp, a gene from Pseudomonas aeruginosa. HasAp is an iron-regulated extracellular haem-binding protein that shares about 50% identity with HasA. HasAp is required for P. aeruginosa utilization of haemoglobin iron. It can replace HasA for HasR-dependent haemoblobin acquisition in a system reconstituted in Escherichia coli. HasAp, like HasA, lacks a signal peptide and is secreted by an ABC transporter. These findings show that haemophore-dependent haem acquisition is not unique to S. marcescens.

The secretion pathways of the heme-binding protein HasA from Serratia marcescens and of the metalloproteases A, B, C and G from Erwinia chrysanthemi have been reconstituted in Escherichia coli. They are secreted in a single step from the cytoplasm across both membranes of the Gram-negative envelope, after recognition of their specific C-terminal secretion signal by their cognate ABC transporter. We report strong evidence that both HasA and the metalloproteases bind the SecB chaperone involved in the export of several envelope proteins via the Sec pathway. We also show that the secretion of the HasA protein is strongly dependent upon SecB in the reconstituted system, whereas that of the proteases is not. HasA secretion in the original host is strongly inhibited by a protein known to interfere with E.coli SecB function. We propose that the proteins secreted by the ABC pathway may have to be unfolded for efficient secretion.

One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells, is sec independent and dedicated to proteins lacking an N-terminal signal peptide. It depends on ABC protein-mediated exporters, which consist of three cell envelope proteins: two inner membrane proteins: an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide. Erwinia chrysanthemi metalloproteinases B and C, and Serratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein. Interaction between the ABC protein and its substrate has also been evidenced by studies on proteinase and HasA hybrid transporters obtained by combining components from each system. Association between hemoprotein HasA and the three exporter/secretion proteins was demonstrated by affinity chromatography on hemin agarose on which the substrate remained bound with the three secretion proteins. The three component association was ordered and substrate binding was required for the formation of this multiprotein complex.

The utilization by Serratia marcescens of heme bound to hemoglobin requires HasA, an extracellular heme-binding protein. This unique heme acquisition system was studied in an Escherichia coli hemA mutant that was a heme auxotroph. We identified a 92-kDa iron-regulated S. marcescens outer membrane protein, HasR, which alone enabled the E. coli hemA mutant to grow on heme or hemoglobin as a porphyrin source. The concomitant secretion of HasA by the HasR-producing hemA mutant greatly facilitates the acquisition of heme from hemoglobin. This is the first report of a synergy between an outer membrane protein and an extracellular heme-binding protein, HasA, acting as a heme carrier, which we termed a hemophore.

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.

The Erwinia chrysanthemi metalloprotease C and the Serratia marcescens haem acquisition protein HasA are both secreted from Gram-negative bacteria by a signal peptide-independent pathway which requires a C-terminal secretion signal and a specific ABC-transporter made up of three proteins: a membrane ATPase (the ABC-protein), a second inner membrane component belonging to the membrane fusion protein family and an outer membrane polypeptide. HasA and protease C transporters are homologous although the secreted polypeptides share no sequence homology. Whereas protease C can use both translocators, HasA is secreted only by its specific transporter. Functional analysis of protease C and HasA secretion through hybrid transporters obtained by combining components from each system demonstrates that the ABC-protein is responsible for the substrate specificity and that inhibition of protease C secretion in the presence of HasA results from a defective interaction between HasA and the ABC-protein. We also show that the outer membrane protein, TolC, can combine with the membrane fusion protein HasE in the presence of either ABC-protein to form a functional transporter but not with the membrane fusion protein, PrtE. This indicates a specific interaction between the outer membrane component and the membrane fusion protein.

We previously identified a Serratia marcescens extracellular protein, HasA, able to bind heme and required for iron acquisition from heme and hemoglobin by the bacterium. This novel type of extracellular protein does not have a signal peptide and does not show sequence similarities to other proteins. HasA secretion was reconstituted in Escherichia coli, and we show here that like many proteins lacking a signal peptide, HasA has a C-terminal targeting sequence and is secreted by a specific ATP binding cassette (ABC) transporter consisting of three proteins, one inner membrane protein with a conserved ATP binding domain, called the ABC; a second inner membrane protein; and a third, outer membrane component. Since the three S. marcescens components of the HasA transporter have not yet been identified, the reconstituted HasA secretion system is a hybrid. It consists of the two S. marcescens inner membrane-specific components, HasD and HasE, associated with an outer membrane component coming from another bacterial ABC transporter, such as the E. coli TolC protein, the outer membrane component of the hemolysin transporter, or the Erwinia chrysanthemi PrtF protein, the outer membrane component of the protease transporter. This hybrid transporter was first shown to allow the secretion of the S. marcescens metalloprotease and the E. chrysanthemi metalloproteases B and C. On account of that, the two S. marcescens components HasD and HasE were previously named PrtDSM and PrtESM, respectively. However, HasA is secreted neither by the PrtD-PrtE-PrtF transporter (the genuine E. chrysanthemi protease transporter) nor by the HlyB-HlhD-TolC transporter (the hemolysin transporter). Moreover, HasA, coexpressed in the same cell, strongly inhibits the secretion of proteases B and C by their own transporter, indicating that the E. chrysanthemi transporter recognizes HasA. Since PrtF could replace TolC in the constitution of the HasA transporter, this indicates that the secretion block does not take place at the level of the outer membrane component but rather at an earlier step of interaction between HasA and the inner membrane components.

International audience; The detailed structure of a 68-residue chimeric peptide encompassing the 56 last C-terminal residues of Erwinia chrysanthemi protease G has been investigated by using circular dichroism and NMR spectroscopies. The peptide which contains the secretion signal of PrtG was solubilized either in aqueous solvent, in trifluoroethanol (TFE)/H2O mixtures, or in dodecyl beta-D-maltoside detergent. The peptide helical content increases upon TFE and detergent additions. A stable conformation is reached at 40% TFE (v:v) and at a micelle to peptide ratio higher than 1. The 1H NMR spectrum has been assigned in TFE/H2O, 2:1 (v:v), and it is shown that residues 26-29 and 50-62 form a relatively stable helix although a conformational equilibrium between a helix and probably a more random structure is observed throughout fragment 13-63. Comparison of the CterG conformation with results obtained by deletion approach could lead to the hypothesis that the C-terminal secretion signal is composed of an alpha-helix located close to the essential C-terminal tetrapeptide D65VIV.

PrtG is an extracellular metalloprotease secreted by the Gram-negative bacterium Erwinia chrysanthemi through a signal peptide-independent secretion pathway. Previous studies showed that the PrtG secretion signal is COOH-terminal and located in the last 56 residues of PrtG. We have now performed a deletion and elongation mapping of a short secretion competent COOH-terminal peptide CterG. This approach allowed us to show that: (i) the smaller COOH-terminal sequence containing the information necessary to promote the secretion of a small polypeptide is contained in the last 29 residues of PrtG; (ii) a low but significant level of secretion can be promoted by the last 15 residues of PrtG when fused to the COOH terminus of a non-secreted PrtG derivative; (iii) the extreme COOH-terminal sequence Dxxx, where xs are hydrophobic residues, is a conserved motif in all constructs that are secreted through the E. chrysanthemi transporter. (vi) This motif has to be COOH terminally exposed since addition of even one amino acid impairs the secretion of CterG. The extent of the secretion defect observed with the COOH terminally extended variants correlates with the length of the extension. These results indicate a key role for the COOH-terminal exposition of the last four amino acids in the secretion of PrtG.

The presence of the alpha-haemolysin secretion genes sensitizes Escherichia coli to vancomycin, a glycopeptide antibiotic that is normally excluded from the Gram-negative envelope (owing to its large size) (M(r) 1400). The selection of vancomycin mutants in strains carrying such genes was found to be a very powerful method for selecting non-haemolytic mutants. In this way, mutations in the known secretion genes, hlyB, hlyD and tolC, were obtained. However additional mutations mapped in genes rfaH and galU which are required for lipopolysaccharide (LPS) biosynthesis. Mutations in rfaH and galU strongly reduced alpha-haemolysin secretion as well as the secretion of Erwinia chrysanthemi proteases in E. coli without affecting their synthesis. These mutations markedly lowered the content of TolC protein, required for haemolysin secretion and also of the PrtF protein necessary for protease secretion. These results raise the possibility that LPS is involved in the correct incorporation of the TolC and PrtF proteins into the cell envelope.

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.

Erwinia chrysanthemi, a Gram-negative phytopathogenic bacterium, was previously shown to secrete 3 related extracellular metalloproteases, A, B and C via a specific signal-peptide-independent pathway. A new gene (prtG) encoding a fourth, 52-kDa metalloprotease was identified on the same recombinant cosmid (pEW1) that carries the genes for the previously described proteases (prtA, prtB and prtC), for the specific secretion factors (prtD, prtE and prtF) and for a protease inhibitor (inh) cloned from E. chrysanthemi B374. The predicted sequence of PrtG was similar to those of PrtA, PrtB and PrtC, its secretion required PrtD, PrtE and PrtF; its secretion signal was located at the C terminus but its proteolytic activity was distinct from that of the 3 other proteases. Results presented here suggest that prtG could be the first gene of an operon that includes inh, prtD, prtE and prtF.

Many bacteria secrete extracellular proteins such as hydrolytic enzymes or toxins. In Gram-negative bacteria, secreted proteins must cross the two membranes that constitute the cell envelope. Recent studies have identified several specific secretion systems that can be classified in three distinct pathways, and related systems have been discovered in a wide range of prokaryotic and eukaryotic cells.

Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide.

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.

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 secretion signal of extracellular metalloprotease B that is secreted without a signal peptide by the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi is shown by deletion and gene fusion analyses to be located within the last 40 C-terminal amino acids. Secretion of a peptide containing only this region of the protease requires the same three secretion factors (PrtD, PrtE, and PrtF) that were previously shown to be required for the secretion of the full-length protease. This secretion signal can also be recognized, albeit inefficiently, by the analogous secretion machinery of alpha-hemolysin, another protein with a C-terminal secretion signal that is secreted by some strains of the Gram-negative bacterium Escherichia coli. The secretion signal was fused to an internal 200-amino acid fragment from the sequence of the cytoplasmic protein amylomaltase to promote its specific secretion by the protease secretion pathway. Almost exactly the same sequence as that identified as the protease B secretion signal was also found at the C terminus of metalloprotease C that is also secreted by E. chrysanthemi.

Secretion of Escherichia coli alpha-hemolysin into the medium does not require the cleavage of an N-terminal signal peptide. The specific secretion apparatus was shown to consist of two proteins, HlyB and HlyD, both located in the inner membrane and encoded by genes contiguous to the hemolysin structural gene (hlyA). It was proposed that these two proteins constitute a membrane-bound translocator for hemolysin [Mackman, N., Nicaud, J. M., Gray, L. & Holland, I. B. (1986) Curr. Top. Microbiol. Immunol. 125, 159-181]. We show here that an E. coli outer membrane protein, the TolC protein, encoded by a gene not located in the hly cluster, is specifically required for hemolysin secretion. This result suggests that an outer membrane protein might be a component of the secretion apparatus allowing a specific interaction between the inner and the outer membrane.

A 5.5 kb DNA fragment carrying the functions necessary for the specific secretion of the extracellular metalloproteases B and C produced by the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi has been sequenced. The fragment contains four transcribed and translated genes: inh, which codes for a protease inhibitor and is not required for protease secretion, and prtD, prtE and prtF, which share significant homology with the hlyB, hlyD and tolC genes required for alpha-haemolysin secretion in Escherichia coli. Mutations in any of the three prt genes abolish protease secretion. The prtD and prtE products (60 and 50 kd) contain at least one hydrophobic segment and the prtF gene product contains a signal sequence.

International audience; Bacterial hemophores are secreted to the extracellular medium, where they scavenge heme from various hemoproteins due to their higher affinity for this compound, and return it to their specific outer membrane receptor. HasR, the outer membrane receptor of the HasA hemophore, assumes multiple functions which require various energy levels. Binding of heme and, of heme-free or heme-loaded hemophores is energy-independent. Heme transfer from the holo-hemophore to the outer membrane receptor is also energy-independent. In contrast, heme transport and hemophore release require basal or high levels of TonB and proton motive force, respectively. In addition, HasR is a component of a signaling cascade, regulating expression of the has operon via specific sigma and anti-sigma factors encoded by genes clustered at the has operon. The signal is the heme landing on HasR in the presence of the hemophore in its apo form. The has system is the only system thus far characterized in which the anti-sigma factor is submitted to the same signaling cascade as the target operon. Specific autoregulation of the has system, combined with negative regulation by the Fur protein, permits bacterial adaptation to the available iron source. In the presence of a heme-loaded hemophore, inactive anti-sigma factor is accumulated and can be activated as soon as the heme source dries up. Hence, the has system, instead of being submitted to amplification like other systems regulated by sigma anti-sigma factors, functions by pulses triggered by heme availability.

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.

International audience; 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, 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-γ- 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 .”

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.

International audience; 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.

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 β-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 β-barrel alone. The protein lacking the N-terminal extension, HasR Δ11-91, was incorporated in the outer membrane and was fully functional for active uptake of free and hemophore-bound heme. The HasR β-barrel, Δ11-192, 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 β-barrel allowed partial plug insertion in the outer membrane, demonstrating that these two HasR domains interact in vivo. The β-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 β-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 Δ11-192 forms a specific heme channel.

In Gram-negative bacteria, the TonB–ExbB–ExbD inner membrane multiprotein complex is required for active transport of diverse molecules through the outer membrane. We present evidence that Serratia marcescens, like several other Gram-negative bacteria, has two TonB proteins: the previously characterized TonBSM, and also HasB, a newly identified component of the has operon that encodes a haemophore-dependent haem acquisition system. This system involves a soluble extracellular protein (the HasA haemophore) that acquires free or haemoprotein-bound haem and presents it to a specific outer membrane haemophore receptor (HasR). TonBSM and HasB are significantly similar and can replace each other for haem acquisition. However, TonBSM, but not HasB, mediates iron acquisition from iron sources other than haem and haemoproteins, showing that HasB and TonBSM only display partial redundancy. The reconstitution in Escherichia coli of the S. marcescens Has system demonstrated that haem uptake is dependent on the E. coli ExbB, ExbD and TonB proteins and that HasB is non-functional in E. coli. Nevertheless, a mutation in the HasB transmembrane anchor domain allows it to replace TonBEC for haem acquisition. As the change affects a domain involved in specific TonBEC–ExbBEC interactions, HasB may be unable to interact with ExbBEC, and the HasB mutation may allow this interaction. In E. coli, the HasB mutant protein was functional for haem uptake but could not complement the other TonBEC-dependent functions, such as iron siderophore acquisition, and phage DNA and colicin uptake. Our findings support the emerging hypothesis that TonB homologues are widespread in bacteria, where they may have specific functions in receptor–ligand uptake systems.

Secretion of the HasA hemophore is mediated by a C-terminal secretion signal as part of an ATP-binding cassette (ABC) pathway in the Gram-negative bacterium Serratia marcescens. We reconstituted the HasA secretion pathway in Escherichia coli. In E. coli, this pathway required three specific secretion functions and SecB, the general chaperone of the Sec pathway that recognizes HasA. The secretion of the isolated C-terminal secretion signal was not SecB-dependent. We have previously shown that intracellular folded HasA can no longer be secreted, and we proposed a step in the secretion process before the recognition of the secretion signal. Here we show that the secretion of a fully functional HasA variant, lacking the first 10 N-terminal amino acids, was less efficient than that of HasA and was SecB-independent. The N terminus of HasA was required, along with SecB, for the efficient secretion of the whole protein. We have also previously shown that HasA inhibits the secretion of metalloproteases from Erwinia chrysanthemi by their specific ABC transporter. Here we show that this abortive interaction between HasA and the E. chrysanthemi metalloprotease ABC transporter required both SecB and the N terminus of HasA. N-terminal fragments of HasA displayed this abortive interaction in vivo and also interacted specifically in vitro with the ABC protein of the Prt system. SecB also interacted specifically in vitro with the ABC protein of the Prt system. Finally, the HasA variant, lacking the first 10 N-terminal amino acids did not display this abortive interaction with the Prt system. We suggest that the N-terminal domain of HasA specifically recognizes the ABC protein in a SecB-dependent fashion, facilitating functional interaction with the C-terminal secretion signal leading to efficient secretion.

International audience; Gram-negative bacterial proteins secreted by ABC exporters carry a secretion signal in their carboxylic extremities. This characteristic suggests that the polypeptide needs to be fully synthesized before it can be secreted and, therefore, presumably may fold at least in part before its secretion. We investigated the relationship between folding and secretion using HasA, a hemoprotein of Serratia marcescens secreted into the extracellular medium by a dedicated Has ABC exporter. We first demonstrated that when HasA is sequestered in the cytoplasm it can acquire its tertiary structure, as assessed from its capacity to bind heme. The cytoplasmic pool of HasA cannot be secreted and inhibits the secretion of newly synthesized molecules. HasA folding in the cytoplasm was independent of either its capacity to bind heme or the presence of SecB, although SecB is essential for HasA secretion. Our findings indicate a strong coupling between synthesis and secretion in the type I secretion pathway.

Multiple lines of evidence indicate that oxidative stress is an integral component of the pathogenesis of Alzheimer disease (AD). The precipitating cause of such oxidative stress may be misregulated iron homeostasis because there are profound alterations in heme oxygenase-1 (HO-1), redox-active iron, and iron regulatory proteins. In this regard, HasA, a recently characterized bacterial protein involved in heme acquisition and iron metabolism, may also be important in the generation of reactive oxygen species (ROS) given its ability to bind heme and render iron available for free radical generation through the Fenton reaction. To study further the role of heme binding and iron metabolism in AD, we show an abnormal localization of anti-HasA to the neurofibrillary pathology of AD, but not in normal-appearing neurons in the brains of cases of AD or in age-matched controls. These results suggest the increased presence in AD of a HasA homologue or protein sharing a common epitope with HasA, which we term HasAh. We conclude that heme binding of HasAh is a potential source of free soluble iron and therefore toxic free radicals in AD and in aging. This furthers the evidence that redox-active iron and subsequent Fenton reaction generating reactive oxygen are critical factors in the pathogenesis of AD.

Serratia marcescens secretes several proteins, such as the lipase LipA, the metalloprotease PrtA, and the heme-binding protein HasA, which is required for heme acquisition, through two N-terminal signal peptide-independent systems that are classified as bacterial ATP-binding cassette (ABC) exporters. One is the ABC exporter for HasA, consisting of the ABC protein HasD, the membrane fusion protein (MFP) HasE, and the outer membrane protein (OMP) HasF. The second, composed of LipB (an ABC protein), LipC (an MFP), and LipD (an OMP), promotes secretion of LipA and PrtA in Escherichia coli recombinant clones. PrtA, which shows homology to the Erwinia chrysanthemi metalloproteases, is efficiently secreted by E. coli cells carrying the E. chrysanthemi ABC exporter PrtD (ABC protein)-PrtE (MFP)-PrtF (OMP). The existence of distinct systems in this bacterium and of various substrates for these systems allowed the study of protein secretion by heterologous Has, Lip, and Prt systems and by Has-Lip and Lip-Prt hybrid exporters in the genuine host as well as in E. coli. For that purpose, lipB-, lipC-, and lipD-deficient mutants were isolated from S. marcescens 8000 and their secretion of LipA and PrtA was analyzed. This demonstrated that a unique exporter, the Lip apparatus, in S. marcescens secretes both LipA and PrtA. Hybrid exporters were tested for secretion of HasA and LipA. The LipB-HasE-HasF exporter allowed secretion of LipA but not HasA, showing that the ABC protein LipB is responsible for the substrate specificity. LipA, HasA, and E. chrysanthemi PrtC were secreted via heterologous exporters and via some hybrid exporters. Analysis of secretion via hybrid exporters showed that specific interactions occur between MFPs and OMPs in these systems. These genetic experiments demonstrated that specific interactions between the ABC protein and the MFP are required for the formation of active exporters.

One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells is sec-independent and dedicated to proteins lacking an N-terminal signal peptide. Most of these proteins display a C-terminal secretion signal located in the last 60 amino acids (aa). Using one Erwinia chrysanthemi protease, PrtG, secreted by such a pathway it was shown that the smallest C-terminal sequence allowing efficient secretion contains the last 29 aa of PrtG and that low but significant secretion can be promoted by the last 15 aa of PrtG. Moreover, the extreme C-terminal motif, consisting of a negatively charged aa followed by several hydrophobic aa must be exposed and is conserved amongst many proteins following this pathway. This secretion system depends on ABC protein-mediated exporters, which consist of three cell envelope proteins: two inner membrane proteins, an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide. These Gram-negative bacterial protein exporters are dedicated to the secretion of one or several closely related proteins belonging to the toxin, protease and lipase families. The genes encoding the three secretion proteins and the exoproteins are usually all linked, consistent with the specificity of the systems. Er. chrysanthemi metalloproteases B and C and Serratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein. Interaction between the ABC protein and its substrate has also been evidenced by studies on protease and HasA hybrid transporters obtained by combining components from each system. Association between hemoprotein HasA and the three exporter secretion proteins was demonstrated by affinity chromatography on hemin agarose on which the substrate remained bound with the three secretion proteins. The three components' association was ordered and substrate binding was required for the formation of this multiprotein complex.