Staphylococcus aureus haem biosynthesis and acquisition pathways are linked through haem monooxygenase IsdG.

Haem is an essential cofactor in central metabolic pathways in the vast majority of living systems. Prokaryotes acquire haem via haem biosynthesis pathways, and some also utilize haem uptake systems, yet it remains unclear how they balance haem requirements with the paradox that free haem is toxic. Here, using the model pathogen Staphylococcus aureus, we report that IsdG, one of two haem oxygenase enzymes in the haem uptake system, inhibits the formation of haem via the internal haem biosynthesis route. More specifically, we show that IsdG decreases the activity of ferrochelatase and that the two proteins interact both in vitro and in vivo. Further, a bioinformatics analysis reveals that a significant number of haem biosynthesis pathway containing organisms possess an IsdG-homologue and that those with both biosynthesis and uptake systems have at least two haem oxygenases. We conclude that IsdG-like proteins control intracellular haem levels by coupling the two pathways. IsdG is thus a target for the treatment of S. aureusinfections.

Desulfovibrio vulgaris CbiKP cobaltochelatase: evolution of a haem binding protein orchestrated by the incorporation of two histidine residues.

The sulfate-reducing bacteria of the Desulfovibrio genus make three distinct modified tetrapyrroles, haem, sirohaem and adenosylcobamide, where sirohydrochlorin acts as the last common biosynthetic intermediate along the branched tetrapyrrole pathway. Intriguingly, D. vulgaris encodes two sirohydrochlorin chelatases, CbiKP and CbiKC , that insert cobalt/iron into the tetrapyrrole macrocycle but are thought to be distinctly located in the periplasm and cytoplasm respectively. Fusing GFP onto the C-terminus of CbiKP confirmed that the protein is transported to the periplasm. The structure-function relationship of CbiKP was studied by constructing eleven site-directed mutants and determining their chelatase activities, oligomeric status and haem binding abilities. Residues His154 and His216 were identified as essential for metal-chelation of sirohydrochlorin. The tetrameric form of the protein is stabilized by Arg54 and Glu76, which form hydrogen bonds between two subunits. His96 is responsible for the binding of two haem groups within the main central cavity of the tetramer. Unexpectedly, CbiKP is shown to bind two additional haem groups through interaction with His103. Thus, although still retaining cobaltochelatase activity, the presence of His96 and His103 in CbiKP , which are absent from all other known bacterial cobaltochelatases, has evolved CbiKP a new function as a haem binding protein permitting it to act as a potential haem chaperone or transporter.

Staphylococcus aureus haem biosynthesis: characterisation of the enzymes involved in final steps of the pathway.

Haem is a life supporting molecule that is ubiquitous in all major kingdoms. In Staphylococcus aureus, the importance of haem is highlighted by the presence of systems both for the exogenous acquisition and endogenous synthesis of this prosthetic group. In this work, we show that in S. aureus the formation of haem involves the conversion of coproporphyrinogen III into coproporphyrin III by coproporphyrin synthase HemY, insertion of iron into coproporphyrin III via ferrochelatase HemH, and oxidative decarboxylation of Fe-coproporphyrin III into protohaem IX by Fe-coproporphyrin oxidase/dehydrogenase HemQ. Together, this route represents a transitional pathway between the classic pathway and the more recently acknowledged alternative biosynthesis machinery. The role of the haem biosynthetic pathway in the survival of the bacterium was investigated by testing for inhibitors of HemY. Analogues of acifluorfen are shown to inhibit the flavin-containing HemY, highlighting this protein as a suitable target for the development of drugs against S. aureus. Moreover, the presence of a transitional pathway for haem biosynthesis within many Gram positive pathogenic bacteria suggests that this route has the potential not only for the design of antimicrobials but also for the selective discrimination between bacteria operating different routes to the biosynthesis of haem.

The bc:caa3 supercomplexes from the Gram positive bacterium Bacillus subtilis respiratory chain: a megacomplex organization?

The respiratory chain of some prokaryotes was shown to be organized in supercomplexes. This association has been proposed to improve enzyme stability and the overall efficiency of the oxidative phosphorylation process. Here, we have revisited recent data on the supercomplexes of Bacillus subtilis respiratory chain, by means of 1D and 2D-BN-PAGE, sucrose gradient fractionation of solubilized membranes, and mass spectrometry analysis of BN-PAGE bands detected in gel for succinate and cytochrome c oxidoreductase activities. The cytochrome bc:caa3 oxygen oxidoreductase supercomplex was observed in different stoichiometries, (bc)4:(caa3)2, (bc)2:(caa3)4 and 2[(bc)2:(caa3)4], suggesting for the first time the string association model of supercomplexes in a Gram positive bacterium. In addition, the presence of a succinate:quinone oxidoreductase:nitrate reductase supercomplex was confirmed by the co-localized succinate:nitroblue tetrazolium and methylviologen:nitrate oxidoreductase activities detected in gel and corroborated by LC-MS/MS analysis.

The novel NhaE-type Na(+)/H (+) antiporter of the pathogenic bacterium Neisseria meningitidis.

Neisseria meningitidis is a pathogenic bacterium responsible for meningitis. The mechanisms underlying the control of Na(+) transmembrane movement, presumably important to pathogenicity, have been barely addressed. To elucidate the function of the components of the Na(+) transport system in N. meningitidis, an open reading frame from the genome of this bacterium displaying similarity with the NhaE type of Na(+)/H(+) antiporters was expressed in Escherichia coli and characterized for sodium transport ability. The N. meningitidis antiporter (NmNhaE) was able to complement an E. coli strain devoid of Na(+)/H(+) antiporters (KNabc) respecting the ability to grow in the presence of NaCl and LiCl. Ion transport assays in everted vesicles prepared from KNabc expressing NmNhaE from a plasmid confirmed its ability to translocate Na(+) and Li(+). Here is presented the characterization of the first NhaE from a pathogen, an important contribution to the comprehension of sodium ion metabolism in this kind of microorganisms.

In Campylobacter jejuni, a new type of chaperone receives heme from ferrochelatase.

Intracellular heme formation and trafficking are fundamental processes in living organisms. Bacteria and archaea utilize three biogenesis pathways to produce iron protoporphyrin IX (heme b) that diverge after the formation of the common intermediate uroporphyrinogen III (uro'gen III). In this study, we identify and provide a detailed characterization of the enzymes involved in the transformation of uro'gen III into heme in Campylobacter jejuni, demonstrating that this bacterium utilizes the protoporphyrin-dependent (PPD) pathway. In general, limited knowledge exists regarding the mechanisms by which heme b reaches its target proteins after this final step. Specifically, the chaperones necessary for trafficking heme to prevent the cytotoxic effects associated with free heme remain largely unidentified. In C. jejuni, we identified a protein named CgdH2 that binds heme with a dissociation constant of 4.9 ± 1.0 µM, and this binding is impaired upon mutation of residues histidine 45 and 133. We demonstrate that C. jejuni CgdH2 establishes protein-protein interactions with ferrochelatase, suggesting its role in facilitating heme transfer from ferrochelatase to CgdH2. Furthermore, phylogenetic analysis reveals that C. jejuni CgdH2 is evolutionarily distinct from the currently known chaperones. Therefore, CgdH2 is the first protein identified as an acceptor of intracellularly formed heme, expanding our knowledge of the mechanisms underlying heme trafficking within bacterial cells.

The aerobic respiratory chain of Escherichia coli: from genes to supercomplexes.

In spite of the large number of reports on the aerobic respiratory chain of Escherichia coli, from gene transcription regulation to enzyme kinetics and structural studies, an integrative perspective of this pathway is yet to be produced. Here, a multi-level analysis of the aerobic respiratory chain of E. coli was performed to find correlations between gene transcription, enzyme activity, growth dynamics, and supercomplex formation and composition. The transcription level of all genes encoding the aerobic respiratory chain of E. coli varied significantly in response to bacterial growth. Coordinated expression patterns were observed between the genes encoding NADH : quinone oxidoreductase and complex I (NDH-1), alternative NADH : quinone oxidoreductase (NDH-2) and cytochrome bdI, and also between sdhA and appC, encoding succinate dehydrogenase and cytochrome bdII, respectively. In general, the rates of the respiratory chain activities increased from mid-exponential to late-stationary phase, with no significant further variation occurring until the mid-stationary phase. Multi-level correlations between gene transcription, enzyme activity and growth dynamics were also found in this study. The previously reported NADH dehydrogenase and formate : oxygen oxidoreductase supercomplexes of E. coli were already assembled at mid-exponential phase and remained throughout growth. A new succinate oxidase supercomplex composed of succinate dehydrogenase and cytochrome bdII was identified, in agreement with the suggestion provided by the coordinated transcription of sdhA and appC.

Defenses of multidrug resistant pathogens against reactive nitrogen species produced in infected hosts.

Bacterial pathogens have sophisticated systems that allow them to survive in hosts in which innate immunity is the frontline of defense. One of the substances produced by infected hosts is nitric oxide (NO) that together with its derived species leads to the so-called nitrosative stress, which has antimicrobial properties. In this review, we summarize the current knowledge on targets and protective systems that bacteria have to survive host-generated nitrosative stress. We focus on bacterial pathogens that pose serious health concerns due to the growing increase in resistance to currently available antimicrobials. We describe the role of nitrosative stress as a weapon for pathogen eradication, the detoxification enzymes, protein/DNA repair systems and metabolic strategies that contribute to limiting NO damage and ultimately allow survival of the pathogen in the host. Additionally, this systematization highlights the lack of available data for some of the most important human pathogens, a gap that urgently needs to be addressed.

Identification of the sirohaem biosynthesis pathway in Staphylococcus aureus.

Sirohaem is a modified tetrapyrrole and a key prosthetic group of several enzymes involved in nitrogen and sulfur metabolisms. This work shows that Staphylococcus aureus produces sirohaem through a pathway formed by three independent enzymes. Of the two putative sirohaem synthases encoded in the S. aureus genome and annotated as cysG, one is herein shown to be a uroporphyrinogen III methyltransferase that converts uroporphyrinogen III to precorrin-2, and was renamed as UroM. The second cysG gene encodes a precorrin-2 dehydrogenase that converts precorrin-2 to sirohydrochlorin, and was designated as P2D. The last step was found to be performed by the gene nirR that, in fact, codes for a protein with sirohydrochlorin ferrochelatase activity, labelled as ShfC. Additionally, site-directed mutagenesis studies of S. aureus ShfC revealed that residues H22 and H87, which are predicted by homology modelling to be located at the active site, control the ferrochelatase activity. Within bacteria, sirohaem synthesis may occur via one, two or three enzymes, and we propose to name the correspondent pathways as Types 1, 2 and 3, respectively. A phylogenetic analysis revealed that Type 1 is the most used pathway in Gammaproteobacteria and Streptomycetales, Type 2 predominates in Fibrobacteres and Vibrionales, and Type 3 predominates in Firmicutes of the Bacillales order. Altogether, we concluded that the current distribution of sirohaem pathways within bacteria, which changes at the genus or species level and within taxa, seems to be the result of evolutionary multiple fusion/fission events.

The formate:oxygen oxidoreductase supercomplex of Escherichia coli aerobic respiratory chain.

The Escherichia coli formate:oxygen oxidoreductase supercomplex (FdOx) was investigated with respect to function and composition. Formate oxidoreductase activity was detected in blue native polyacrylamide gel electrophoresis (BN-PAGE) resolved membranes of E. coli, which were also capable of cyanide sensitive formate:oxygen oxidoreductase activity. The latter was compromised in strains devoid of specific oxygen reductases, particularly, in those devoid of cytochrome bo3 or bdI. A principal component analysis (PCA) integrating E. coli aerobic respiratory chain gene transcription, enzyme activity and growth dynamics was performed, correlating formate:oxygen oxidoreductase activity and the transcription of the genes encoding cytochromes bo3 and bdI, and corroborating previous evidence that associated these complexes in FdOx.