RESUMEN
Xenon is frequently used as a general anesthetic in humans, but the mechanism remains an issue of debate. While for some membrane proteins, a direct interaction of xenon with the protein has been shown to be the inhibitory mechanism, other membrane protein functions could be affected by changes of membrane properties due to partitioning of the gas into the lipid bilayer. Here, the effect of xenon on a mechanosensitive ion channel and a copper ion-translocating ATPase was compared under different conditions. Xenon inhibited spontaneous gating of the Escherichia coli mechano-sensitive mutant channel MscL-G22E, as shown by patch-clamp recording techniques. Under high hydrostatic pressure, MscL-inhibition was reversed. Similarly, the activity of the Enterococcus hirae CopB copper ATPase, reconstituted into proteoliposomes, was inhibited by xenon. However, the CopB ATPase activity was also inhibited by xenon when CopB was in a solubilized state. These findings suggest that xenon acts by directly interacting with these proteins, rather than via indirect effects by altering membrane properties. Also, inhibition of copper transport may be a novel effect of xenon that contributes to anesthesia.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Escherichia coli/antagonistas & inhibidores , Canales Iónicos/antagonistas & inhibidores , Xenón/farmacología , Proteínas Transportadoras de Cobre , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Mecanotransducción Celular/efectos de los fármacos , Técnicas de Placa-ClampRESUMEN
Desulfovibrio sp. A2 is a novel Gram-negative sulfate-reducing bacterium that was isolated from sediments of the Norilsk mining/smelting area in Russia. The organism possesses a monocistronic operon encoding a 71 kDa periplasmic multicopperoxidase, which we call DA2_CueO. Histidine-tagged DA2_CueO expressed from a plasmid in Escherichia coli and purified by Ni-NTA affinity chromatography oxidizes Cu+ and Fe2+, and exhibits phenol oxidase activity with 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,3-dihydroxybenzoic acid and 2,6-dimethoxyphenol as substrates, using O2 as the oxidant. When expressed in an E. coli cueO knock-out strain, DA2_CueO exhibits phenol oxidase activity in vivo and enhances the copper tolerance of the strain. These findings indicate that the DA2_CueO gene of Desulfovibrio sp. A2 encodes a multicopperoxidase with a role in metal ion resistance. The enzyme displays some novel structural features, which are discussed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cobre/metabolismo , Desulfovibrio/enzimología , Compuestos Ferrosos/metabolismo , Oxidorreductasas/metabolismo , Fenol/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Desulfovibrio/química , Desulfovibrio/genética , Desulfovibrio/aislamiento & purificación , Sedimentos Geológicos/microbiología , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/aislamiento & purificaciónRESUMEN
The killing of bacteria on metallic copper surfaces in minutes to hours is referred to as contact killing. Why copper possesses such strong antimicrobial activity has remained enigmatic. Based on the physicochemical properties of metals, it was recently predicted that cadmium should also be active in contact killing [Hans et al., Biointerphases 11, 018902 (2010)]. Here, the authors show that cadmium is indeed antimicrobial. It kills three logs of bacteria in 9 h, compared to copper which kills eight logs of bacteria. Metallic silver kills less than one log of bacteria in 9 h. These findings support the novel concept whereby oxide formation, metal ion dissolution, and a Pearson soft character are the key factors for a metal to be antibacterial. Based on these parameters, copper and cadmium are expected to be the two most antibacterial metals.
Asunto(s)
Antibacterianos/farmacología , Cadmio/farmacología , Fenómenos Químicos , Cobre/farmacología , Viabilidad Microbiana/efectos de los fármacos , Plata/farmacología , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Acero Inoxidable/farmacología , Propiedades de SuperficieRESUMEN
The active transport of ions across biological membranes requires their hydration shell to interact with the interior of membrane proteins. However, the influence of the external lipid phase on internal dielectric dynamics is hard to access by experiment. Using the octahelical transmembrane architecture of the copper-transporting P1B -type ATPase from Legionella pneumophila as a model structure, we have established the site-specific labeling of internal cysteines with a polarity-sensitive fluorophore. This enabled dipolar relaxation studies in a solubilized form of the protein and in its lipid-embedded state in nanodiscs. Time-dependent fluorescence shifts revealed the site-specific hydration and dipole mobility around the conserved ion-binding motif. The spatial distribution of both features is shaped significantly and independently of each other by membrane lateral pressure.
Asunto(s)
Proteínas Bacterianas/metabolismo , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Proteínas Bacterianas/química , Dominio Catalítico , Dicroismo Circular , Cisteína/química , Colorantes Fluorescentes/química , Legionella pneumophila/enzimología , Nanoestructuras/química , Estructura Secundaria de ProteínaRESUMEN
We compared cold and hot wood extracts of 3 endemic Siberian trees-namely, Prunus padus (bird cherry), Populus tremula (aspen), and Betula sp. (birch)-on biomass production and laccase and peroxidase secretion in submerged cultures by the medicinal mushroom Lentinus edodes. Of the conditions tested, only hot Prunus extracts stimulated biomass production, whereas all extracts stimulated laccase and peroxidase secretion, albeit to different extents. A large, differential stimulation of manganese peroxidase was observed by hot Prunus extracts. The results highlight important differences between tree species in the stimulation of biomass and enzyme production by L. edodes and point to potentially interesting stimulatory factors present in hot Prunus extracts. These findings are of relevance in the use of L. edodes for medicinal or biotechnological applications.
Asunto(s)
Betula/química , Proteínas Fúngicas/metabolismo , Lentinula/enzimología , Populus/química , Prunus/química , Biomasa , Proteínas Fúngicas/genética , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Lentinula/crecimiento & desarrollo , Lentinula/metabolismo , Extractos VegetalesRESUMEN
The nineties witnessed the discovery of the copper ATPases, enzymes which transport copper across the cytoplasmic membranes of bacteria and eukaryotes. In the same decade, several other key components of copper homeostasis have also been discovered, like copper chaperones and plasma membrane copper transporters. This has finally led to a molecular understanding of two inherited human diseases related to copper: Menkes disease, manifested by systemic copper deficiency, and Wilson disease, caused by defective secretion of excess copper. A historic perspective and untold stories of the events leading up to these discoveries are presented here.
Asunto(s)
Cobre/uso terapéutico , Degeneración Hepatolenticular/historia , Síndrome del Pelo Ensortijado/historia , Proteínas de Transporte de Catión/genética , Cobre/deficiencia , Degeneración Hepatolenticular/diagnóstico , Degeneración Hepatolenticular/tratamiento farmacológico , Degeneración Hepatolenticular/genética , Historia del Siglo XX , Homeostasis , Humanos , Síndrome del Pelo Ensortijado/diagnóstico , Síndrome del Pelo Ensortijado/tratamiento farmacológico , Síndrome del Pelo Ensortijado/genética , Mutación/genéticaRESUMEN
Nanoparticles are increasingly being used for treatment and diagnostic purposes, but their effects on cells is not fully understood. Here, the interaction of fluorescent up-conversion nanoparticles (UpC-NPs) with neutrophils was investigated by imaging and measurement of membrane-cytosceletal elasticity by atomic force microscopy. It was found that UpC-NPs induce the death of neutrophils mainly by necrosis, and to a smaller extent by a novel process called 'mummification'. Necrosis occurs by gradual loss of intracellular contents and nuclei, 45-110min after exposure to UpC-NPs. Mummification is apparent as an increase in the rigidity of the neutrophils' membrane and acquisition of a characteristic bumpy shape with numerous protrusions; this structure does not change during atomic force microscopy scanning. Coating UpC-NPs with protein by incubation with serum leads to (1) formation of nanoparticle aggregates in the nm and µm size range, (2) a reduction in toxicity, (3) reduced mummification of neutrophils, and (4) no significant reduction of the elasticity of the membrane-cytoskeletal complex of neutrophils 30min after exposure to coated UpC-NPs. The study shows that serum proteins greatly curb the toxicity of nanoparticles and reveals mummification as a novel mechanism of UpC-NP-induced cell death.
Asunto(s)
Muerte Celular/efectos de los fármacos , Fluoruros/química , Nanopartículas/química , Neutrófilos/patología , Membrana Celular/efectos de los fármacos , Citoesqueleto/química , Citoesqueleto/efectos de los fármacos , Elasticidad/efectos de los fármacos , Fluorescencia , Humanos , Nanopartículas del Metal/química , Neutrófilos/efectos de los fármacos , SueroRESUMEN
UNLABELLED: Both prokaryotic and eukaryotic organisms possess mechanisms for the detoxification of heavy metals, and these mechanisms are found among distantly related species. We investigated the role of intracellular glutathione (GSH), which, in a large number of taxa, plays a role in protection against the toxicity of common heavy metals. Anaerobically grown Lactococcus lactis containing an inducible GSH synthesis pathway was used as a model organism. Its physiological condition allowed study of putative GSH-dependent uranyl detoxification mechanisms without interference from additional reactive oxygen species. By microcalorimetric measurements of metabolic heat during cultivation, it was shown that intracellular GSH attenuates the toxicity of uranium at a concentration in the range of 10 to 150 µM. In this concentration range, no effect was observed with copper, which was used as a reference for redox metal toxicity. At higher copper concentrations, GSH aggravated metal toxicity. Isothermal titration calorimetry revealed the endothermic binding of U(VI) to the carboxyl group(s) of GSH rather than to the reducing thiol group involved in copper interactions. The data indicate that the primary detoxifying mechanism is the intracellular sequestration of carboxyl-coordinated U(VI) into an insoluble complex with GSH. The opposite effects on uranyl and on copper toxicity can be related to the difference in coordination chemistry of the respective metal-GSH complexes, which cause distinct growth phase-specific effects on enzyme-metal interactions. IMPORTANCE: Understanding microbial metal resistance is of particular importance for bioremediation, where microorganisms are employed for the removal of heavy metals from the environment. This strategy is increasingly being considered for uranium. However, little is known about the molecular mechanisms of uranyl detoxification. Existing studies of different taxa show little systematics but hint at a role of glutathione (GSH). Previous work could not unequivocally demonstrate a GSH function in decreasing the presumed uranyl-induced oxidative stress, nor could a redox-independent detoxifying action of GSH be identified. Combining metabolic calorimetry with cell number-based assays and genetics analysis enables a novel and general approach to quantify toxicity and relate it to molecular mechanisms. The results show that GSH-expressing microorganisms appear advantageous for uranyl bioremediation.
Asunto(s)
Glutatión/metabolismo , Lactococcus lactis/efectos de los fármacos , Lactococcus lactis/metabolismo , Compuestos de Uranio/toxicidad , Anaerobiosis , Biotransformación , Calorimetría , Lactococcus lactis/crecimiento & desarrolloRESUMEN
Desulfosporosinus sp. OT is a Gram-positive, acidophilic sulfate-reducing firmicute isolated from copper tailings sediment in the Norilsk mining-smelting area in Siberia and represents the first Desulfosporosinus species whose genome has been sequenced. Desulfosporosinus sp. OT is exceptionally copper resistant, which made it of interest to study the resistance mechanism. It possesses a copUAZ operon which is shown here to be involved in copper resistance. The copU gene encodes a CsoR-type homotetrameric repressor. By electrophoretic mobility shift assay, it was shown that CopU binds to the operator/promoter region of the copUAZ operon in the absence of copper and is released from the DNA by Cu+ or Ag+, implying that CopU regulates the operon in a copper/silver-dependent manner. DOT_CopA is a P1B-type ATPase related to other characterized, bacterial copper ATPases. When expressed in a copper-sensitive Escherichia coli ΔcopA mutant, it restores copper resistance to WT levels. His-tagged DOT_CopA was expressed from a plasmid in E. coli and purified by Ni-NTA affinity chromatography. The purified enzyme was most active in the presence of Cu(I) and bacterial phospholipids. These findings indicate that the copUAZ operon confers copper resistance to Desulfosporosinus sp. OT, but do not per se explain the basis of the high copper resistance of this strain.
RESUMEN
Contact killing is a novel term describing the killing of bacteria when they come in contact with metallic copper or copper-containing alloys. In recent years, the mechanism of contact killing has received much attention and many mechanistic details are available. The authors here review some of these mechanistic aspects with a focus on the critical physicochemical properties of copper which make it antibacterial. Known mechanisms of contact killing are set in context to ionic, corrosive, and physical properties of copper. The analysis reveals that the oxidation behavior of copper, paired with the solubility properties of copper oxides, are the key factors which make metallic copper antibacterial. The concept advanced here explains the unique position of copper as an antibacterial metal. Based on our model, novel design criteria for metallic antibacterial materials may be derived.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Fenómenos Químicos , Cobre/química , Cobre/farmacología , Viabilidad Microbiana/efectos de los fármacos , Modelos Biológicos , Oxidación-Reducción , Fenómenos FarmacológicosRESUMEN
The well-established killing of bacteria by copper surfaces, also called contact killing, is currently believed to be a combined effect of bacterial contact with the copper surface and the dissolution of copper, resulting in lethal bacterial damage. Iron can similarly be released in ionic form from iron surfaces and would thus be expected to also exhibit contact killing, although essentially no contact killing is observed by iron surfaces. However, we show here that the exposure of bacteria to iron surfaces in the presence of copper ions results in efficient contact killing. The process involves reduction of Cu(2+) to Cu(+) by iron; Cu(+) has been shown to be considerably more toxic to cells than Cu(2+). The specific Cu(+) chelator, bicinchoninic acid, suppresses contact killing by chelating the Cu(+) ions. These findings underline the importance of Cu(+) ions in the contact killing process and infer that iron-based alloys containing copper could provide novel antimicrobial materials.
Asunto(s)
Antiinfecciosos/química , Sulfato de Cobre/farmacología , Cobre/química , Enterococcus/efectos de los fármacos , Enterococcus/fisiología , Hierro/química , Viabilidad Microbiana , Aleaciones , Antiinfecciosos/aislamiento & purificación , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Escherichia coli , Oxidación-Reducción , Quinolinas/farmacología , Propiedades de SuperficieRESUMEN
Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD-yaiAB operon of Lactococcus lactisâ IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD-yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p-benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4-hydroxymuconic semialdehyde in an oxygen-consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD-yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments.
Asunto(s)
Cobre/toxicidad , Regulación Bacteriana de la Expresión Génica , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Quinonas/metabolismo , Quinonas/toxicidad , Benzoquinonas/metabolismo , Cobre/metabolismo , Flavoproteínas/genética , Técnicas de Inactivación de Genes , Lactococcus lactis/crecimiento & desarrollo , Mutagénesis Insercional , Operón , Estrés FisiológicoRESUMEN
Fungi possess a range of light receptors to regulate metabolism and differentiation. To study the effect of light on Lentinula edodes (the shiitake mushroom), mycelial cultures were exposed to blue, green, and red fluorescent lights and light-emitting diodes, as well as green laser light. Biomass production, morphology, and pigment production were evaluated. Exposure to green light at intervals of 1 min/d at 0.4 W/m(2) stimulated biomass production by 50-100 %, depending on the light source. Light intensities in excess of 1.8 W/m(2) or illumination longer than 30 min/d did not affect biomass production. Carotenoid production and morphology remained unaltered during increased biomass production. These observations provide a cornerstone to the study of photoreception by this important fungus.
Asunto(s)
Micelio/crecimiento & desarrollo , Micelio/efectos de la radiación , Hongos Shiitake/crecimiento & desarrollo , Hongos Shiitake/efectos de la radiación , Biomasa , Color , Fluorescencia , Hifa/crecimiento & desarrollo , Hifa/efectos de la radiación , LuzRESUMEN
Copper kills bacteria rapidly by a mechanism that is not yet fully resolved. The antibacterial property of copper has raised interest in its use in hospitals, in place of plastic or stainless steel. On the latter surfaces, bacteria can survive for days or even weeks. Copper surfaces could thus provide a powerful accessory measure to curb nosocomial infections. We here investigated the effect of the copper surface structure on the efficiency of contact killing of Escherichia coli, an aspect which so far has received very little attention. It was shown that electroplated copper surfaces killed bacteria more rapidly than either polished copper or native rolled copper. The release of ionic copper was also more rapid from electroplated copper compared to the other materials. Scanning electron microscopy revealed that the bacteria nudged into the grooves between the copper grains of deposited copper. The findings suggest that, in terms of contact killing, more efficient copper surfaces can be engineered.
Asunto(s)
Cobre/toxicidad , Escherichia coli/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Cobre/química , Escherichia coli/fisiología , Microscopía Electrónica de Rastreo , Propiedades de SuperficieRESUMEN
The potential of metallic copper as an intrinsically antibacterial material is gaining increasing attention in the face of growing antibiotics resistance of bacteria. However, the mechanism of the so-called "contact killing" of bacteria by copper surfaces is poorly understood and requires further investigation. In particular, the influences of bacteria-metal interaction, media composition, and copper surface chemistry on contact killing are not fully understood. In this study, copper oxide formation on copper during standard antimicrobial testing was measured in situ by spectroscopic ellipsometry. In parallel, contact killing under these conditions was assessed with bacteria in phosphate buffered saline (PBS) or Tris-Cl. For comparison, defined Cu2O and CuO layers were thermally generated and characterized by grazing incidence X-ray diffraction. The antibacterial properties of these copper oxides were tested under the conditions used above. Finally, copper ion release was recorded for both buffer systems by inductively coupled plasma atomic absorption spectroscopy, and exposed copper samples were analyzed for topographical surface alterations. It was found that there was a fairly even growth of CuO under wet plating conditions, reaching 4-10 nm in 300 min, but no measurable Cu2O was formed during this time. CuO was found to significantly inhibit contact killing, compared to pure copper. In contrast, thermally generated Cu2O was essentially as effective in contact killing as pure copper. Copper ion release from the different surfaces roughly correlated with their antibacterial efficacy and was highest for pure copper, followed by Cu2O and CuO. Tris-Cl induced a 10-50-fold faster copper ion release compared to PBS. Since the Cu2O that primarily forms on copper under ambient conditions is as active in contact killing as pure copper, antimicrobial objects will retain their antimicrobial properties even after oxide formation.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Cobre/farmacología , Antibacterianos/química , Cobre/química , Microscopía Electrónica de RastreoRESUMEN
Lactococcus lactis possesses a pronounced extracellular Cu(2+)-reduction activity which leads to the accumulation of Cu(+) in the medium. The kinetics of this reaction were not saturable by increasing copper concentrations, suggesting a non-enzymic reaction. A copper-reductase-deficient mutant, isolated by random transposon mutagenesis, had an insertion in the menE gene, which encodes O-succinylbenzoic acid CoA ligase. This is a key enzyme in menaquinone biosynthesis. The ΔmenE mutant was deficient in short-chain menaquinones, and exogenously added menaquinone complemented the copper-reductase-deficient phenotype. Haem-induced respiration of wild-type L. lactis efficiently suppressed copper reduction, presumably by competition by the bd-type quinol oxidase for menaquinone. As expected, the ΔmenE mutant was respiration-deficient, but could be made respiration-proficient by supplementation with menaquinone. Growth of wild-type cells was more copper-sensitive than that of the ΔmenE mutant, due to the production of Cu(+) ions by the wild-type. This growth inhibition of the wild-type was strongly attenuated if Cu(+) was scavenged with the Cu(I) chelator bicinchoninic acid. These findings support a model whereby copper is non-enzymically reduced at the membrane by menaquinones. Respiration effectively competes for reduced quinones, which suppresses copper reduction. These findings highlight novel links between copper reduction, respiration and Cu(+) toxicity in L. lactis.
Asunto(s)
Cobre/metabolismo , Cobre/toxicidad , Lactococcus lactis/efectos de los fármacos , Lactococcus lactis/metabolismo , Vitamina K 2/metabolismo , Membrana Celular/metabolismo , Elementos Transponibles de ADN , Técnicas de Inactivación de Genes , Mutagénesis Insercional , Oxidación-Reducción , Succinato-CoA Ligasas/genéticaRESUMEN
Bacteria are rapidly killed on copper surfaces, and copper ions released from the surface have been proposed to play a major role in the killing process. However, it has remained unclear whether contact of the bacteria with the copper surface is also an important factor. Using laser interference lithography, we engineered copper surfaces which were covered with a grid of an inert polymer which prevented contact of the bacteria with the surface. Using Enterococcus hirae as a model organism, we showed that the release of ionic copper from these modified surfaces was not significantly reduced. In contrast, killing of bacteria was strongly attenuated. When E. hirae cells were exposed to a solid iron surface, the loss of cell viability was the same as on glass. However, exposing cells to iron in the presence of 4 mM CuSO4 led to complete killing in 100 min. These experiments suggest that contact killing proceeds by a mechanism whereby the metal-bacterial contact damages the cell envelope, which, in turn, makes the cells susceptible to further damage by copper ions.
Asunto(s)
Cationes/farmacología , Cobre/farmacología , Enterococcus/efectos de los fármacos , Hierro/farmacología , Membrana Celular/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacosRESUMEN
Enterococcus hirae ATCC 9790 is a Gram-positive lactic acid bacterium that has been used in basic research for over 4 decades. Here we report the sequence and annotation of the 2.8-Mb genome of E. hirae and its endemic 29-kb plasmid pTG9790.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/genética , Enterococcus faecalis/genética , Genoma Bacteriano , Análisis de Secuencia de ADN , Cobre/metabolismo , Metabolismo Energético , Enterococcus faecalis/fisiología , Homeostasis , Transporte Iónico , Datos de Secuencia Molecular , PlásmidosRESUMEN
Lactococcus lactis cannot synthesize haem, but when supplied with haem, expresses a cytochrome bd oxidase. Apart from the cydAB structural genes for this oxidase, L. lactis features two additional genes, hemH and hemW (hemN), with conjectured functions in haem metabolism. While it appears clear that hemH encodes a ferrochelatase, no function is known for hemW. HemW-like proteins occur in bacteria, plants and animals, and are usually annotated as CPDHs (coproporphyrinogen III dehydrogenases). However, such a function has never been demonstrated for a HemW-like protein. We here studied HemW of L. lactis and showed that it is devoid of CPDH activity in vivo and in vitro. Recombinantly produced, purified HemW contained an Fe-S (iron-sulfur) cluster and was dimeric; upon loss of the iron, the protein became monomeric. Both forms of the protein covalently bound haem b in vitro, with a stoichiometry of one haem per monomer and a KD of 8 µM. In vivo, HemW occurred as a haem-free cytosolic form, as well as a haem-containing membrane-associated form. Addition of L. lactis membranes to haem-containing HemW triggered the release of haem from HemW in vitro. On the basis of these findings, we propose a role of HemW in haem trafficking. HemW-like proteins form a distinct phylogenetic clade that has not previously been recognized.