Sylvie Létoffé

Isolation and characterization of an extracellular haem-binding protein from Pseudomonas aeruginosa that shares function and sequence similarities with the Serratia marcescens HasA haemophore

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.

Heme acquisition by hemophores.

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.

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.

Identification and characterization of the hemophore-dependent heme acquisition system of Yersinia pestis

ABSTRACT Yersinia pestis possesses a heme-protein acquisition system (Hmu) that allows it to utilize heme and heme-protein complexes as the sole sources of iron. Analysis of the Y. pestisCO92 genomic sequence revealed a second heme-protein acquisition gene cluster that shares homology with the hemophore-dependent heme acquisition system (Has system) of Serratia marcescens. This locus consisted of thehasRyp receptor gene, thehasAyp hemophore gene, and genes encoding components of the HasAyp dedicated ABC transporter factor (hasDEyp), as well as atonB homologue (hasByp). By using a reconstituted secretion system in Escherichia coli, we showed that HasAyp is a secreted heme-binding protein and that expression of HasAyp is iron regulated in E. coli. The use of a transcriptional reporter fusion showed that the hasRADEB promoter is Fur regulated and has increased activity at 37°C. Hemoglobin utilization via the Hasyp system was studied with both E. coli and Y. pestis, for which hasand has hmu mutant strains were used. No contribution of the Has system to heme utilization was observed in either E. coli or Y. pestis under the conditions we tested. Previously it was shown that a deletion of the Hmu system had no effect on the virulence of Y. pestis in a mouse model of bubonic plague. An Hmu− Has− double mutant also retained full virulence in this model of infection. This report constitutes the first attempt to investigate the contribution of the hemophore-dependent heme acquisition system in bacterial pathogenicity.

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-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.”

Biogenic ammonia modifies antibiotic resistance at a distance in physically separated bacteria.

Bacteria release low‐molecular‐weight by‐products called secondary metabolites, which contribute to bacterial ecology and biology. Whereas volatile compounds constitute a large class of potential infochemicals, their role in bacteria–bacteria interactions remains vastly unexplored. Here we report that exposure to gaseous ammonia released from stationary‐phase bacterial cultures modifies the antibiotic resistance spectrum of all tested Gram‐negative and Gram‐positive bacteria. Using Escherichia coli K12 as a model organism, and increased resistance to tetracycline as the phenotypic read‐out, we demonstrate that exposure to ammonia generated by the catabolism of l‐aspartate increases the level of intracellular polyamines, in turn leading to modifications in membrane permeability to different antibiotics as well as increased resistance to oxidative stress. We show that the inability to import ammonia via the Amt gas channel or to synthesize polyamines prevent modification in the resistance profile of aerially exposed bacteria. We therefore provide here the first detailed molecular characterization of widespread, long‐range chemical interference between physically separated bacteria.

Haemophore‐mediated bacterial haem transport: evidence for a common or overlapping site for haem‐free and haem‐loaded haemophore on its specific outer membrane receptor

Bacterial extracellular haemophores also named HasA for haem acquisition system form an independent family of haemoproteins that take up haem from host haeme carriers and shuttle it to specific receptors (HasR). Haemophore receptors are required for the haemophore‐dependent haem acquisition pathway and alone allow free or haemoglobin‐bound haem uptake, but the synergy between the haemophore and its receptor greatly facilitates this uptake. The three‐dimensional structure of the Serratia marcescens holo‐haemophore (HasASM) has been determined previously and revealed that the haem iron atom is ligated by tyrosine 75 and histidine 32. The phenolate of tyrosine 75 is also tightly hydrogen bonded to the Nδ atom of histidine 83. Alanine mutagenesis of these three HasASM residues was performed, and haem‐binding constants of the wild‐type protein, the three single mutant proteins, the three double mutant proteins and the triple mutant protein were compared by absorption spectrometry to probe the roles of H32, Y75 and H83 in haem binding. We show that one axial iron ligand is sufficient to ligate haem efficiently and that H83 may become an alternative iron ligand in the absence of Y75 or both H32 and Y75. All the single mutant proteins retained the ability to stimulate haemophore‐dependent haem uptake in vivo. Thus, the residues H32, Y75 and H83 are not individually necessary for haem delivery to the receptor. The binding of haem‐free and haem‐loaded HasASM proteins to HasRSM‐producing strains was studied. Both proteins bind to HasRSM with similar apparent Kd. The double mutant H32A‐Y75A competitively inhibits binding to the receptor of both holo‐HasASM and apo‐HasASM, showing that there is a unique or overlapping site on HasRSM for the apo‐ and holo‐haemophores. Thus, we propose a new mechanism for haem uptake, in which haem is exchanged between haem‐loaded haemophores and unloaded haemophores bound to the receptor without swapping of haemophores on the receptor.

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. This second mechanism is much more efficient and requires a higher TonB-ExbB-ExbD (TonB complex) concentration than does free or hemoglobin-bound heme uptake. This requirement for more of the TonB complex is associated with a higher energy requirement. Indeed, the sensitivity of heme-hemophore uptake to the protonophore carbonyl cyanide m-chlorophenyl hydrazone is higher than that of heme uptake from hemoglobin. We show that a higher TonB complex concentration is required for hemophore dissociation from the receptor. This dissociation is concomitant with heme uptake. We propose that increasing the TonB complex concentration drives more energy to the outer membrane receptor and speeds up the release of empty hemophores, which, if they remained on receptors, would inhibit heme transport.

Similarity between the bacterial histone-like protein HU and a protein from spinach chloroplasts

The histone‐like protein HU isolated from E. coli is well conserved in prokaryotes. We show here that antiserum prepared against bacterial HU cross‐reacts with a DNA‐binding protein co‐sedimenting with the nucleoid of spinach chloroplasts. Antibodies prepared against cyanobacterial HU are more reactive than those raised against E. coli HU. The chloroplast protein resembles HU in that both appear to be composed of two related subunits.

Characterization of a protein inhibitor of extracellular proteases produced by Erwinia chrysanthemi

Erwinia chrysanthemi, a phytopathogenic bacterium, produces a protease inhibitor which is a low‐molecu‐lar‐weight, heat‐stable protein. In addition to its action on the three E. chrysanthemi extracellular proteases A, B and C, it also strongly inhibits the 50 kD extracellu‐lar protease of Serratia marcescens. Its structural gene (inh) was subcloned and expressed in Escher‐ichia coli, in which it encodes an active inhibitor which was purified. The nucleotide sequence of the inh gene shows an open reading frame of 114 codons. The N‐terminal amino acid sequence of the purified inhibi‐tor was also determined. It indicated the existence of an amino‐terminal signal peptide absent from the mature protein. The inhibitor is entirely periplasmic in E. chrysanthemi and partially periplasmic in E. coli.