Conserved C-Terminal Tail Is Responsible for Membrane Localization and Function of Pseudomonas aeruginosa Hemerythrin.

Many bacteria have hemerythrin (Hr) proteins that bind O2, including Pseudomonas aeruginosa, in which microoxia-induced Hr (Mhr) provide fitness advantages under microoxic conditions. Mhr has a 23 amino-acid extension at its C-terminus relative to a well-characterized Hr from Methylococcus capsulatus, and similar extensions are also found in Hrs from other bacteria. The last 11 amino acids of this extended, C-terminal tail are highly conserved in gammaproteobacteria and predicted to form a helix with positively charged and hydrophobic faces. In cellular fractionation assays, wild-type (WT) Mhr was found in both membrane and cytosolic fractions, while a MhrW143* variant lacking the last 11 residues was largely in the cytosol and did not complement Mhr function in competition assays. MhrL112Y, a variant that has a much longer-lived O2-bound form, was fully functional and had a similar localization pattern to that of WT Mhr. Both MhrW143* and MhrL112Y had secondary structures, stabilities, and O2-binding kinetics similar to those of WT Mhr. Fluorescence studies revealed that the C-terminal tail, and particularly the fragment corresponding to its last 11 residues, was sufficient and necessary for association with lipid vesicles. Molecular dynamics simulations and subsequent cellular analysis of Mhr variants have demonstrated that conserved, positively charged residues in the tail are important for Mhr interactions with negatively charged membranes and the contribution of this protein to competitive fitness. Together, these data suggest that peripheral interactions of Mhr with membranes are guided by the C-terminal tail and are independent of O2-binding.

The hemerythrin-like diiron protein from Mycobacterium kansasii is a nitric oxide peroxidase.

The hemerythrin-like protein from Mycobacterium kansasii (Mka HLP) is a member of a distinct class of oxo-bridged diiron proteins that are found only in mycobacterial species that cause respiratory disorders in humans. Because it had been shown to exhibit weak catalase activity and a change in absorbance on exposure to nitric oxide (NO), the reactivity of Mka HLP toward NO was examined under a variety of conditions. Under anaerobic conditions, we found that NO was converted to nitrite (NO2-) via an intermediate, which absorbed light at 520 nm. Under aerobic conditions NO was converted to nitrate (NO3-). In each of these two cases, the maximum amount of nitrite or nitrate formed was at best stoichiometric with the concentration of Mka HLP. When incubated with NO and H2O2, we observed NO peroxidase activity yielding nitrite and water as reaction products. Steady-state kinetic analysis of NO consumption during this reaction yielded a Km for NO of 0.44 µM and a kcat/Km of 2.3 × 105 M-1s-1. This high affinity for NO is consistent with a physiological role for Mka HLP in deterring nitrosative stress. This is the first example of a peroxidase that uses an oxo-bridged diiron center and a rare example of a peroxidase utilizing NO as an electron donor and cosubstrate. This activity provides a mechanism by which the infectious Mycobacterium may combat against the cocktail of NO and superoxide (O2•-) generated by macrophages to defend against bacteria, as well as to produce NO2- to adapt to hypoxic conditions.

Pseudomonas aeruginosa lasR mutant fitness in microoxia is supported by an Anr-regulated oxygen-binding hemerythrin.

Pseudomonas aeruginosa strains with loss-of-function mutations in the transcription factor LasR are frequently encountered in the clinic and the environment. Among the characteristics common to LasR-defective (LasR-) strains is increased activity of the transcription factor Anr, relative to their LasR+ counterparts, in low-oxygen conditions. One of the Anr-regulated genes found to be highly induced in LasR- strains was PA14_42860 (PA1673), which we named mhr for microoxic hemerythrin. Purified P. aeruginosa Mhr protein contained the predicted di-iron center and bound molecular oxygen with an apparent Kd of ∼1 µM. Both Anr and Mhr were necessary for fitness in lasR+ and lasR mutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in the lasR mutant. Among genes in the Anr regulon, mhr was most closely coregulated with the Anr-controlled high-affinity cytochrome c oxidase genes. In the absence of high-affinity cytochrome c oxidases, deletion of mhr no longer caused a fitness disadvantage, suggesting that Mhr works in concert with microoxic respiration. We demonstrate that Anr and Mhr contribute to LasR- strain fitness even in biofilms grown in normoxic conditions. Furthermore, metabolomics data indicate that, in a lasR mutant, expression of Anr-regulated mhr leads to differences in metabolism in cells grown on lysogeny broth or artificial sputum medium. We propose that increased Anr activity leads to higher levels of the oxygen-binding protein Mhr, which confers an advantage to lasR mutants in microoxic conditions.

Spectroscopic Characterization of a Diferric Mycobacterial Hemerythrin-Like Protein with Unprecedented Reactivity toward Nitric Oxide.

Hemerythrin-like proteins (HLPs) are broadly distributed across taxonomic groups and appear to play highly diverse functional roles in prokaryotes. Mycobacterial HLPs contribute to the survival of these pathogenic bacteria in mammalian macrophages, but their modes of action remain unclear. A recent crystallographic characterization of Mycobacterium kansasii HLP (Mka-HLP) revealed the unexpected presence of a tyrosine sidechain (Tyr54) near the coordination sphere of one of the two iron centers. Here, we show that Tyr54 is a true ligand to the Fe2(III) ion which, in conjunction with the presence of a µ-oxo group bridging the two iron(III), brings unique reactivity toward nitric oxide (NO). Monitoring the titration of Mka-HLP with NO by Fourier-transform infrared and electron paramagnetic resonance spectroscopies shows that both diferric and diferrous forms of Mka-HLP accumulate an uncoupled high-spin and low-spin {FeNO}7 pair. We assign the reactivity of the diferric protein to an initial radical reaction between NO and the µ-oxo bridge to form nitrite and a mixed-valent diiron center that can react further with NO. Amperometric measurements of NO consumption by Mka-HLP confirm that this reactivity can proceed at low micromolar concentrations of NO, before additional NO consumption, supporting a NO scavenging role for mycobacterial HLPs.

Repair of Iron Center Proteins-A Different Class of Hemerythrin-like Proteins.

Repair of Iron Center proteins (RIC) form a family of di-iron proteins that are widely spread in the microbial world. RICs contain a binuclear nonheme iron site in a four-helix bundle fold, two basic features of hemerythrin-like proteins. In this work, we review the data on microbial RICs including how their genes are regulated and contribute to the survival of pathogenic bacteria. We gathered the currently available biochemical, spectroscopic and structural data on RICs with a particular focus on Escherichia coli RIC (also known as YtfE), which remains the best-studied protein with extensive biochemical characterization. Additionally, we present novel structural data for Escherichia coli YtfE harboring a di-manganese site and the protein's affinity for this metal. The networking of protein interactions involving YtfE is also described and integrated into the proposed physiological role as an iron donor for reassembling of stress-damaged iron-sulfur centers.

Crystal structure of a hemerythrin-like protein from Mycobacterium kansasii and homology model of the orthologous Rv2633c protein of M. tuberculosis.

Pathogenic and opportunistic mycobacteria have a distinct class of non-heme di-iron hemerythrin-like proteins (HLPs). The first to be isolated was the Rv2633c protein, which plays a role in infection by Mycobacterium tuberculosis (Mtb), but could not be crystallized. This work presents the first crystal structure of an ortholog of Rv2633c, the mycobacterial HLP from Mycobacterium kansasii (Mka). This structure differs from those of hemerythrins and other known HLPs. It consists of five α-helices, whereas all other HLP domains have four. In contrast with other HLPs, the HLP domain is not fused to an additional protein domain. The residues ligating and surrounding the di-iron site are also unique among HLPs. Notably, a tyrosine occupies the position normally held by one of the histidine ligands in hemerythrin. This structure was used to construct a homology model of Rv2633c. The structure of five α-helices is conserved and the di-iron site ligands are identical in Rv2633c. Two residues near the ends of helices in the Mka HLP structure are replaced with prolines in the Rv2633c model. This may account for structural perturbations that decrease the solubility of Rv2633c relative to Mka HLP. Clusters of residues that differ in charge or polarity between Rv2633c and Mka HLP that point outward from the helical core could reflect a specificity for potential differential interactions with other protein partners in vivo, which are related to function. The Mka HLP exhibited weaker catalase activity than Rv2633c. Evidence was obtained for the interaction of Mka HLP irons with nitric oxide.

Recent Insights into the Diversity and Evolution of Invertebrate Hemerythrins and Extracellular Globins.

There are three broad groups of oxygen-transport proteins found in the haemolymph (blood) of invertebrates, namely the hemocyanins, the hemerythrins and the globins. Both hemerythrins and extracellular globins are iron-based proteins that are understudied when compared to the copper-containing hemocyanins. Recent evidence suggests that hemerythrins and (giant) extracellular globins (and their linker chains) are more widely distributed than previously thought and may have biological functions beyond oxygen transport and storage. Herein, we review contemporary literature of these often-neglected proteins with respect to their structural configurations on formation and ancestral states.

Identification and Characterization of a Redox Sensor Phosphodiesterase from Ferrovum sp. PN-J185 Containing Bacterial Hemerythrin and HD-GYP Domains.

The second messenger bis(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates numerous important physiological functions in bacteria. In this study, we identified and characterized the first dimeric, full-length, non-heme iron-bound phosphodiesterase (PDE) containing bacterial hemerythrin and HD-GYP domains (Bhr-HD-GYP). We found that the amino acid sequence encoded by the FV185_09380 gene from Ferrovum sp. PN-J185 contains an N-terminal bacterial hemerythrin domain and a C-terminal HD-GYP domain, which is characteristic of proteins with PDE activity toward c-di-GMP. Inductively coupled plasma optical emission spectroscopy analyses showed that Bhr-HD-GYP contains 4 equiv of iron atoms per subunit, suggesting both hemerythrin and HD-GYP domains have non-heme di-iron sites. A redox-dependent spectral change expected for oxo-bridged non-heme iron with carboxylate ligands was observed, and this redox interconversion was reversible. However, unlike marine invertebrate hemerythrin, which functions as an oxygen-binding protein, Bhr-HD-GYP did not form an oxygen adduct because of rapid autoxidation. The reduced ferrous iron complex of the protein catalyzed the hydrolysis of c-di-GMP to its linearized product, 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), whereas the oxidized ferric iron complex had no significant activity. These results suggest that Bhr-HD-GYP is a redox and oxygen sensor enzyme that regulates c-di-GMP levels in response to changes in cellular redox status or oxygen concentration. Our study may lead to an improved understanding of the physiology of iron-oxidizing bacterium Ferrovum sp. PN-J185.

Variable-Temperature ESI-IMS-MS Analysis of Myohemerythrin Reveals Ligand Losses, Unfolding, and a Non-Native Disulfide Bond.

Variable-temperature electrospray ionization combined with ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques are used to monitor structural transitions of the protein myohemerythrin from peanut worm in aqueous ammonium acetate solutions from ∼15 to 92 °C. At physiological temperatures, myohemerythrin favors a four-helix bundle motif and has a diiron oxo cofactor that binds oxygen. As the solution temperature is increased from ∼15 to 35 °C, some bound oxygen dissociates; at ∼66 °C, the cofactor dissociates to produce populations of both folded and unfolded apoprotein. At higher temperatures (∼85 °C and above), the IMS-MS spectrum indicates that the folded apoprotein dominates, and provides evidence for stabilization of the structure by formation of a non-native disulfide bond. In total, we find evidence for 18 unique forms of myohemerythrin as well as information about the structures and stabilities of these states. The high-fidelity of IMS-MS techniques provides a means of examining the stabilities of individual components of complex mixtures that are inaccessible by traditional calorimetric and spectroscopic methods.

Response of the Anaerobic Methanotroph "Candidatus Methanoperedens nitroreducens" to Oxygen Stress.

"Candidatus Methanoperedens nitroreducens" is an archaeon that couples the anaerobic oxidation of methane to nitrate reduction. In natural and man-made ecosystems, this archaeon is often found at oxic-anoxic interfaces where nitrate, the product of aerobic nitrification, cooccurs with methane produced by methanogens. As such, populations of "Ca Methanoperedens nitroreducens" could be prone to regular oxygen exposure. Here, we investigated the effect of 5% (vol/vol) oxygen exposure in batch activity assays on a "Ca Methanoperedens nitroreducens" culture, enriched from an Italian paddy field. Metagenome sequencing of the DNA extracted from the enrichment culture revealed that 83% of 16S rRNA gene reads were assigned to a novel strain, "Candidatus Methanoperedens nitroreducens Verserenetto." RNA was extracted, and metatranscriptome sequencing upon oxygen exposure revealed that the active community changed, most notably in the appearance of aerobic methanotrophs. The gene expression of "Ca Methanoperedens nitroreducens" revealed that the key genes encoding enzymes of the methane oxidation and nitrate reduction pathways were downregulated. In contrast to this, we identified upregulation of glutaredoxin, thioredoxin family/like proteins, rubrerythrins, peroxiredoxins, peroxidase, alkyl hydroperoxidase, type A flavoproteins, FeS cluster assembly protein, and cysteine desulfurases, indicating the genomic potential of "Ca Methanoperedens nitroreducens Verserenetto" to counteract the oxidative damage and adapt in environments where they might be exposed to regular oxygen intrusion.IMPORTANCE "Candidatus Methanoperedens nitroreducens" is an anaerobic archaeon which couples the reduction of nitrate to the oxidation of methane. This microorganism is present in a wide range of aquatic environments and man-made ecosystems, such as paddy fields and wastewater treatment systems. In such environments, these archaea may experience regular oxygen exposure. However, "Ca Methanoperedens nitroreducens" is able to thrive under such conditions and could be applied for the simultaneous removal of dissolved methane and nitrogenous pollutants in oxygen-limited systems. To understand what machinery "Ca Methanoperedens nitroreducens" possesses to counteract the oxidative stress and survive, we characterized the response to oxygen exposure using a multi-omics approach.

Immunological properties of oxygen-transport proteins: hemoglobin, hemocyanin and hemerythrin.

It is now well documented that peptides with enhanced or alternative functionality (termed cryptides) can be liberated from larger, and sometimes inactive, proteins. A primary example of this phenomenon is the oxygen-transport protein hemoglobin. Aside from respiration, hemoglobin and hemoglobin-derived peptides have been associated with immune modulation, hematopoiesis, signal transduction and microbicidal activities in metazoans. Likewise, the functional equivalents to hemoglobin in invertebrates, namely hemocyanin and hemerythrin, act as potent immune effectors under certain physiological conditions. The purpose of this review is to evaluate the true extent of oxygen-transport protein dynamics in innate immunity, and to impress upon the reader the multi-functionality of these ancient proteins on the basis of their structures. In this context, erythrocyte-pathogen antibiosis and the immune competences of various erythroid cells are compared across diverse taxa.

De novo design of protein-protein interactions through modification of inter-molecular helix-helix interface residues.

For de novo design of protein-protein interactions (PPIs), information on the shape and chemical complementarity of their interfaces is generally required. Recent advances in computational PPI design have allowed for de novo design of protein complexes, and several successful examples have been reported. In addition, a simple and easy-to-use approach has also been reported that arranges leucines on a solvent-accessible region of an α-helix and places charged residues around the leucine patch to induce interactions between the two helical peptides. For this study, we adopted this approach to de novo design a new PPI between the helical bundle proteins sulerythrin and LARFH. A non-polar patch was created on an α-helix of LARFH around which arginine residues were introduced to retain its solubility. The strongest interaction found was for the LARFH variant cysLARFH-IV-3L3R and the sulerythrin mutant 6L6D (KD=0.16 µM). This artificial protein complex is maintained by hydrophobic and ionic interactions formed by the inter-molecular helical bundle structure. Therefore, by the simple and easy-to-use approach to create de novo interfaces on the α-helices, we successfully generated an artificial PPI. We also created a second LARFH variant with the non-polar patch surrounded by positively charged residues at each end. Upon mixing this LARFH variant with 6L6D, mesh-like fibrous nanostructures were observed by atomic force microscopy. Our method may, therefore, also be applicable to the de novo design of protein nanostructures.

Discovery and evolution of novel hemerythrin genes in annelid worms.

BACKGROUND: Despite extensive study on hemoglobins and hemocyanins, little is known about hemerythrin (Hr) evolutionary history. Four subgroups of Hrs have been documented, including: circulating Hr (cHr), myohemerythrin (myoHr), ovohemerythrin (ovoHr), and neurohemerythrin (nHr). Annelids have the greatest diversity of oxygen carrying proteins among animals and are the only phylum in which all Hr subgroups have been documented. To examine Hr diversity in annelids and to further understand evolution of Hrs, we employed approaches to survey annelid transcriptomes in silico. RESULTS: Sequences of 214 putative Hr genes were identified from 44 annelid species in 40 different families and Bayesian inference revealed two major clades with strong statistical support. Notably, the topology of the Hr gene tree did not mirror the phylogeny of Annelida as presently understood, and we found evidence of extensive Hr gene duplication and loss in annelids. Gene tree topology supported monophyly of cHrs and a myoHr clade that included nHrs sequences, indicating these designations are functional rather than evolutionary. CONCLUSIONS: The presence of several cHrs in early branching taxa suggests that a variety of Hrs were present in the common ancestor of extant annelids. Although our analysis was limited to expressed-coding regions, our findings demonstrate a greater diversity of Hrs among annelids than previously reported.

New enzymatic pathways for the reduction of reactive oxygen species in Entamoeba histolytica.

BACKGROUND: Entamoeba histolytica, an intestinal parasite that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. A flavodiiron protein and a rubrerythrin have been characterized in this human pathogen, although their physiological reductants have not been identified. METHODS: The present work deals with biochemical studies performed to reach a better understanding of the kinetic and structural properties of rubredoxin reductase and two ferredoxins from E. histolytica. RESULTS: We complemented the characterization of two different metabolic pathways for O2 and H2O2 detoxification in E. histolytica. We characterized a novel amoebic protein with rubredoxin reductase activity that is able to catalyze the NAD(P)H-dependent reduction of heterologous rubredoxins, amoebic rubrerythrin and flavodiiron protein but not ferredoxins. In addition, the protein exhibited an NAD(P)H oxidase activity, which generates hydrogen peroxide from molecular oxygen. We describe how different ferredoxins were also efficient reducing substrates for both flavodiiron protein and rubrerythrin. CONCLUSIONS: The enzymatic systems herein characterized could contribute to the in vivo detoxification of O2 and H2O2, playing a key role for the parasite defense against reactive oxidant species. GENERAL SIGNIFICANCE: To the best of our knowledge this is the first characterization of a eukaryotic rubredoxin reductase, including a novel kinetic study on ferredoxin-dependent reduction of flavodiiron and rubrerythrin proteins.

Hemerythrin-related antimicrobial peptide, msHemerycin, purified from the body of the Lugworm, Marphysa sanguinea.

A ∼1.7 kDa antimicrobial peptide was purified from the acidified body extract of the Lugworm, Marphysa sanguinea, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase high performance liquid chromatography (HPLC). The identified peptide is composed of 14 amino acids with the N-terminal acetylation. Comparison of the identified amino acid sequences and molecular weight of this peptide with those of other known proteins or peptides revealed that this peptide had high identity to the N-terminus of hemerythrin of marine invertebrates and named the msHemerycin. The full-length hemerythrin cDNA of Lugworm was contained 1027-bp, including a 5'-untranslated region (UTR) of 60-bp, a 3'-UTR of 595-bp, and an open reading frame of 372-bp encoding 123 amino acids including the msHemerycin at the N-terminus. Tissue distribution of the msHemerycin mRNA suggests that it is constitutively expressed as a non-tissue-specific manner, however, a relatively higher expression level was observed in muscle (6.8-fold) and brain (6.3-fold), and the lowest level in digestive gland. The secondary structural prediction and homology modeling studies indicate that the msHemerycin might form an unordered structure and might act via unconventional mechanism. Our results suggest that the msHemerycin might be an innate immune component related to the host defenses in the Lugworm. This is the first report on the antimicrobial function of the peptide derived from the N-terminus of hemerythrin in the Lugworm, Marphysa sanguinea.

Hemerythrin is required for Aeromonas hydraphlia to survive in the macrophages of Anguilla japonica.

Survival in host phagocytes is an effective strategy for pathogenic microbes to spread. To understand the mechanisms of Aeromonas hydrophila survival within host macrophages, a library of mini-Tn10 transposon insertion mutants was constructed. The M85 mutant, whose survival in host macrophages was only 23.1% of that of the wild-type (WT) strain, was utilized for further study. Molecular analysis showed that a 756-bp open reading frame (ORF) (GenBank accession No. CP007576) in the M85 mutant was interrupted by mini-Tn10. This ORF encodes for a 183-amino acid protein and displays the highest sequence identity (99%) with the hemerythrin (Hr) protein of A. hydrophila subspecies hydrophila ATCC 7966. The survival of the WT, M85 mutant, and complemented M85 (Hr) strains were compared in host macrophages in vitro, and the results showed that M85 exhibited defective survival, while that of M85 (Hr) was restored. To investigate the possible mechanisms of A. hydrophila survival in host macrophages, the expression of Hr under hyperoxic and hypoxic conditions was evaluated. The results revealed that the expression of this protein was higher under hyperoxic conditions than under hypoxic conditions, which indicates that Hr protein expression is sensitive to O2 concentration. Hydrogen peroxide sensitivity tests further suggested that the M85 mutant was more sensitive to oxidative stress than the WT and M85 (Hr) strains. Taken together, these results suggest that the Hr protein may act as an O2 sensor and as a detoxifier of reactive oxygen species, and is required for A. hydrophila survival within host macrophages.

An anaerobic bacterium, Bacteroides thetaiotaomicron, uses a consortium of enzymes to scavenge hydrogen peroxide.

Obligate anaerobes are periodically exposed to oxygen, and it has been conjectured that on such occasions their low-potential biochemistry will predispose them to rapid ROS formation. We sought to identify scavenging enzymes that might protect the anaerobe Bacteroides thetaiotaomicron from the H2 O2 that would be formed. Genetic analysis of eight candidate enzymes revealed that four of these scavenge H2 O2  in vivo: rubrerythrins 1 and 2, AhpCF, and catalase E. The rubrerythrins served as key peroxidases under anoxic conditions. However, they quickly lost activity upon aeration, and AhpCF and catalase were induced to compensate. The AhpCF is an NADH peroxidase that effectively degraded low micromolar levels of H2 O2 , while the catalytic cycle of catalase enabled it to quickly degrade higher concentrations that might arise from exogenous sources. Using a non-scavenging mutant we verified that endogenous H2 O2 formation was much higher in aerated B. thetaiotaomicron than in Escherichia coli. Indeed, the OxyR stress response to H2 O2 was induced when B. thetaiotaomicron was aerated, and in that circumstance this response was necessary to forestall cell death. Thus aeration is a serious threat for this obligate anaerobe, and to cope it employs a set of defences that includes a repertoire of complementary scavenging enzymes.

Hemerythrins in the microaerophilic bacterium Campylobacter jejuni help protect key iron-sulphur cluster enzymes from oxidative damage.

Microaerophilic bacteria are adapted to low oxygen environments, but the mechanisms by which their growth in air is inhibited are not well understood. The citric acid cycle in the microaerophilic pathogen Campylobacter jejuni is potentially vulnerable, as it employs pyruvate and 2-oxoglutarate:acceptor oxidoreductases (Por and Oor), which contain labile (4Fe-4S) centres. Here, we show that both enzymes are rapidly inactivated after exposure of cells to a fully aerobic environment. We investigated the mechanisms that might protect enzyme activity and identify a role for the hemerythrin HerA (Cj0241). A herA mutant exhibits an aerobic growth defect and reduced Por and Oor activities after exposure to 21% (v/v) oxygen. Slow anaerobic recovery of these activities after oxygen damage was observed, but at similar rates in both wild-type and herA strains, suggesting the role of HerA is to prevent Fe-S cluster damage, rather than promote repair. Another hemerythrin (HerB; Cj1224) also plays a protective role. Purified HerA and HerB exhibited optical absorption, ligand binding and resonance Raman spectra typical of µ-oxo-bridged di-iron containing hemerythrins. We conclude that oxygen lability and poor repair of Por and Oor are major contributors to microaerophily in C. jejuni; hemerythrins help prevent enzyme damage microaerobically or during oxygen transients.

Oxidative protection of hemoglobin and hemerythrin by cross-linking with a nonheme iron peroxidase: potentially improved oxygen carriers for use in blood substitutes.

The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-creating powerful oxidants such as compounds I and II [formally Fe(IV)] formed in peroxidases and catalases. Rubrerythrin could, therefore, be a useful ingredient in protein-based artificial oxygen carriers. Here, we report that the oxygen-carrying proteins, hemoglobin (Hb) and hemerythrin (Hr), can each be copolymerized with rubrerythrin using glutaraldehyde yielding high molecular weight species. These copolymers show additional peroxidase activity compared to Hb-only and Hr-only polymers, respectively and also generate lower levels of free radicals in reactions that involve hydrogen peroxide. Tests on human umbilical vein endothelial cells (HUVEC) reveal slightly better performance of the Rbr copolymers compared to controls, as measured at 24 h, but not at later times.

Massive gene acquisitions in Mycobacterium indicus pranii provide a perspective on mycobacterial evolution.

Understanding the evolutionary and genomic mechanisms responsible for turning the soil-derived saprophytic mycobacteria into lethal intracellular pathogens is a critical step towards the development of strategies for the control of mycobacterial diseases. In this context, Mycobacterium indicus pranii (MIP) is of specific interest because of its unique immunological and evolutionary significance. Evolutionarily, it is the progenitor of opportunistic pathogens belonging to M. avium complex and is endowed with features that place it between saprophytic and pathogenic species. Herein, we have sequenced the complete MIP genome to understand its unique life style, basis of immunomodulation and habitat diversification in mycobacteria. As a case of massive gene acquisitions, 50.5% of MIP open reading frames (ORFs) are laterally acquired. We show, for the first time for Mycobacterium, that MIP genome has mosaic architecture. These gene acquisitions have led to the enrichment of selected gene families critical to MIP physiology. Comparative genomic analysis indicates a higher antigenic potential of MIP imparting it a unique ability for immunomodulation. Besides, it also suggests an important role of genomic fluidity in habitat diversification within mycobacteria and provides a unique view of evolutionary divergence and putative bottlenecks that might have eventually led to intracellular survival and pathogenic attributes in mycobacteria.