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.

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.

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.

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.

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.

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.

[A hemerythrin-like protein MSMEG_3312 influences erythromycin resistance in mycobacteria].

OBJECTIVE: Reactive oxygen species are natural products of metabolism in aerobic organisms, which lead to oxidative damage, such as DNA mutation, protein inactivation and drug resistance. MSMEG_3312 was predicted as a hemerythrin-like protein, which can carry oxygen and reversibly bind to oxygen, thus it might play important roles in the process of oxygen metabolism. In this study, we explored the role of MSMEG_3312 in drug resistance. METHODS: On the basis of bioinformatics, we identified the conserved sequence of HHE domain in MSMEG_3312 and it was predicted to have typical α-helix at secondary structure. To explore potential functions of MSMEG_3312, we constructed the msmeg_3312 knockout strain and compare the susceptibility to various drugs to its parent strain, mc2155. In addition, we also measured the promoter response when treatment of erythromycin. RESULTS: Genetic results showed that MSMEG_3312 is not necessary for M. smegmatis growth at 7H9 rich medium. The msmeg_3312 knockout strain showed increased erythromycin resistance. Moreover, the drug resistance is only limited to erythromycin which its mechanism of action is by binding to the 50S subunit of the bacteria ribosomal complex and then inhibit protein synthesis. However, there were no different MICs of other antibiotics, targets for protein synthesis inhibition, but not 50S subunit, such as tetracyclines, aminoglycosides and chloramphenicol. Moreover, we also showed that the promoter of msmeg_3312 responses to erythromycin. CONCLUSIONS: Hemerythin-like protein MSMEG_3312 is involved in erythromycin resistance.

Crystal structure, exogenous ligand binding, and redox properties of an engineered diiron active site in a bacterial hemerythrin.

A nonheme diiron active site in a 13 kDa hemerythrin-like domain of the bacterial chemotaxis protein DcrH-Hr contains an oxo bridge, two bridging carboxylate groups from Glu and Asp residues, and five terminally ligated His residues. We created a unique diiron coordination sphere containing five His and three Glu/Asp residues by replacing an Ile residue with Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119 to Fe2 of the diiron site in the I119E variant was confirmed by X-ray crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible tunnel. UV-vis absorption spectra indicate that the additional coordination of E119 inhibits the binding of the exogenous ligands azide and phenol to the diiron site. The extent of azide binding to the diiron site increases at pH ≤ 6, which is ascribed to protonation of the carboxylate ligand of E119. The diferrous state (deoxy form) of the engineered diiron site with the extra Glu residue is found to react more slowly than wild type with O2 to yield the diferric state (met form). The additional coordination of E119 to the diiron site also slows the rate of reduction from the met form. All these processes were found to be pH-dependent, which can be attributed to protonation state and coordination status of the E119 carboxylate. These results demonstrate that modifications of the endogenous coordination sphere can produce significant changes in the ligand binding and redox properties in a prototypical nonheme diiron-carboxylate protein active site.

The Ferritin-like superfamily: Evolution of the biological iron storeman from a rubrerythrin-like ancestor.

BACKGROUND: The Ferritins are part of the extensive 'Ferritin-like superfamily' which have diverse functions but are linked by the presence of a common four-helical bundle domain. The role performed by Ferritins as the cellular repository of excess iron is unique. In many ways Ferritins act as tiny organelles in their ability to secrete iron away from the delicate machinery of the cell, and then to release it again in a controlled fashion avoiding toxicity. The Ferritins are ancient proteins, being common in all three domains of life. This ubiquity reflects the key contribution that Ferritins provide in achieving iron homeostasis. SCOPE OF THE REVIEW: This review compares the features of the different Ferritins and considers how they, and other members of the Ferritin-like superfamily, have evolved. It also considers relevant features of the eleven other known families within the Ferritin-like superfamily, particularly the highly diverse rubrerythrins. MAJOR CONCLUSIONS: The Ferritins have travelled a considerable evolutionary journey, being derived from far more simplistic rubrerythrin-like molecules which play roles in defence against toxic oxygen species. The forces of evolution have moulded such molecules into three distinct types of iron storing (or detoxifying) protein: the classical and universal 24-meric ferritins; the haem-containing 24-meric bacterioferritins of prokaryotes; and the prokaryotic 12-meric Dps proteins. These three Ferritin types are similar, but also possess unique properties that distinguish them and enable then to achieve their specific physiological purposes. GENERAL SIGNIFICANCE: A wide range of biological functions have evolved from a relatively simple structural unit.

The molecular determinants of the increased reduction potential of the rubredoxin domain of rubrerythrin relative to rubredoxin.

Based on the crystal structures, three possible sequence determinants have been suggested as the cause of a 285 mV increase in reduction potential of the rubredoxin domain of rubrerythrin over rubredoxin by modulating the polar environment around the redox site. Here, electrostatic calculations of crystal structures of rubredoxin and rubrerythrin and molecular dynamics simulations of rubredoxin wild-type and mutants are used to elucidate the contributions to the increased reduction potential. Asn(160) and His(179) in rubrerythrin versus valines in rubredoxins are predicted to be the major contributors, as the polar side chains contribute significantly to the electrostatic potential in the redox site region. The mutant simulations show both side chains rotating on a nanosecond timescale between two conformations with different electrostatic contributions. Reduction also causes a change in the reduction energy that is consistent with a linear response due to the interesting mechanism of shifting the relative populations of the two conformations. In addition to this, a simulation of a triple mutant indicates the side-chain rotations are approximately anticorrelated so whereas one is in the high potential conformation, the other is in the low potential conformation. However, Ala(176) in rubrerythrin versus a leucine in rubredoxin is not predicted to be a large contributor, because the solvent accessibility increases only slightly in mutant simulations and because it is buried in the interface of the rubrerythrin homodimer.

Oxidative stress protection and the repair response to hydrogen peroxide in the hyperthermophilic archaeon Pyrococcus furiosus and in related species.

Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100 degrees C. Addition of H(2)O(2) (0.5 mM) to a growing culture resulted in the cessation of growth with a 2-h lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function, while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H(2)O(2) were not more resistant to the subsequent addition of H(2)O(2) (0.5-5.0 mM). Consequently, there is little if any induced protective response to peroxide. The organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin, and alkyl hydroperoxide reductase. Related hyperthermophiles contain homologs of the proteins involved in the constitutive protective mechanism but these organisms were more sensitive to peroxide than P. furiosus and lack several of its peroxide-responsive ORFs.

Hemerythrins enhance aerobic respiration in Methylomicrobium alcaliphilum 20ZR, a methane-consuming bacterium.

Numerous hemerythrins, di-iron proteins, have been identified in prokaryote genomes, but in most cases their function remains elusive. Bacterial hemerythrin homologs (bacteriohemerythrins, Bhrs) may contribute to various cellular functions, including oxygen sensing, metal binding and antibiotic resistance. It has been proposed that methanotrophic Bhrs support methane oxidation by supplying oxygen to a core enzyme, particulate methane monooxygenase. In this study, the consequences of the overexpression or deletion of the Bhr gene (bhr) in Methylomicrobiam alcaliphillum 20ZR were investigated. We found that the bhrknockout (20ZRΔbhr) displays growth kinetics and methane consumption rates similar to wild type. However, the 20ZRΔbhr accumulates elevated concentrations of acetate at aerobic conditions, indicating slowed respiration. The methanotrophic strain overproducing Bhr shows increased oxygen consumption and reduced carbon-conversion efficiency, while its methane consumption rates remain unchanged. These results suggest that the methanotrophic Bhr proteins specifically contribute to oxygen-dependent respiration, while they have minimal, if any, input of oxygen for the methane oxidation machinery.

How the Anaerobic Enteropathogen Clostridioides difficile Tolerates Low O2 Tensions.

Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2 O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon.IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile This enteropathogen has developed efficient strategies to detoxify O2 In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

Oxygen-mediated growth enhancement of an obligate anaerobic archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1, an obligate anaerobic hyperthermophilic archaeon, showed variable oxygen (O2) sensitivity depending on the types of substrate employed as an energy source. Unexpectedly, the culture with yeast extract as a sole energy source showed enhanced growth by 2-fold in the presence of O2. Genome-wide transcriptome analysis revealed the upregulation of several antioxidant-related genes encoding thioredoxin peroxidase (TON_0862), rubrerythrin (TON_0864), rubrerythrin-related protein (TON_0873), NAD(P)H rubredoxin oxidoreductase (TON_0865), or thioredoxin reductase (TON_1603), which can couple the detoxification of reactive oxygen species with the regeneration of NAD(P)+ from NAD(P)H. We present a plausible mechanism by which O2 serves to maintain the intracellular redox balance. This study demonstrates an unusual strategy of an obligate anaerobe underlying O2-mediated growth enhancement despite not having heme-based or cytochrome-type proteins.

A phylogenomic profile of hemerythrins, the nonheme diiron binding respiratory proteins.

BACKGROUND: Hemerythrins, are the non-heme, diiron binding respiratory proteins of brachiopods, priapulids and sipunculans; they are also found in annelids and bacteria, where their functions have not been fully elucidated. RESULTS: A search for putative Hrs in the genomes of 43 archaea, 444 bacteria and 135 eukaryotes, revealed their presence in 3 archaea, 118 bacteria, several fungi, one apicomplexan, a heterolobosan, a cnidarian and several annelids. About a fourth of the Hr sequences were identified as N- or C-terminal domains of chimeric, chemotactic gene regulators. The function of the remaining single domain bacterial Hrs remains to be determined. In addition to oxygen transport, the possible functions in annelids have been proposed to include cadmium-binding, antibacterial action and immunoprotection. A Bayesian phylogenetic tree revealed a split into two clades, one encompassing archaea, bacteria and fungi, and the other comprising the remaining eukaryotes. The annelid and sipunculan Hrs share the same intron-exon structure, different from that of the cnidarian Hr. CONCLUSION: The phylogenomic profile of Hrs demonstrated a limited occurrence in bacteria and archaea and a marked absence in the vast majority of multicellular organisms. Among the metazoa, Hrs have survived in a cnidarian and in a few protostome groups; hence, it appears that in metazoans the Hr gene was lost in deuterostome ancestor(s) after the radiata/bilateria split. Signal peptide sequences in several Hirudinea Hrs suggest for the first time, the possibility of extracellular localization. Since the alpha-helical bundle is likely to have been among the earliest protein folds, Hrs represent an ancient family of iron-binding proteins, whose primary function in bacteria may have been that of an oxygen sensor, enabling aerophilic or aerophobic responses. Although Hrs evolved to function as O2 transporters in brachiopods, priapulids and sipunculans, their function in annelids remains to be elucidated. Overall Hrs exhibit a considerable lack of evolutionary success in metazoans.

Diversity and distribution of hemerythrin-like proteins in prokaryotes.

Hemerythrins are oxygen-binding proteins found in the body fluids and tissues of certain invertebrates. Oxygen is bound at a nonheme iron centre consisting of two oxo-bridged iron atoms bound to a characteristic set of conserved histidine: aspartate and glutamate residues with the motifs H-HxxxE-HxxxH-HxxxxD. It has recently been demonstrated biochemically that two bacterial proteins bearing the same motifs do in fact possess similar iron centres and bind oxygen in the same way. The recent profusion of prokaryotic genomic sequence data has shown that proteins bearing hemerythrin motifs are present in a wide variety of bacteria, and a few archaea. Some of these are short proteins as in eukaryotes; others appear to consist of a hemerythrin domain fused to another domain, generally a putative signal transduction domain such as a methyl-accepting chemotaxis protein, a histidine kinase, or a GGDEF protein (cyclic di-GMP synthase). If, as initial evidence suggests, these are in fact hemerythrin-like oxygen-binding proteins, then their diversity in prokaryotes far exceeds that seen in eukaryotes. Here, a survey is presented of prokaryotic protein sequences bearing hemerythrin-like motifs, for which the designation 'bacteriohemerythrins' is proposed, and their functions are speculated.

Gene refashioning through innovative shifting of reading frames in mosses.

Early-diverging land plants such as mosses are known for their outstanding abilities to grow in various terrestrial habitats, incorporating tremendous structural and physiological innovations, as well as many lineage-specific genes. How these genes and functional innovations evolved remains unclear. In this study, we show that a dual-coding gene YAN/AltYAN in the moss Physcomitrella patens evolved from a pre-existing hemerythrin gene. Experimental evidence indicates that YAN/AltYAN is involved in fatty acid and lipid metabolism, as well as oil body and wax formation. Strikingly, both the recently evolved dual-coding YAN/AltYAN and the pre-existing hemerythrin gene might have similar physiological effects on oil body biogenesis and dehydration resistance. These findings bear important implications in understanding the mechanisms of gene origination and the strategies of plants to fine-tune their adaptation to various habitats.

Broad Phylogenetic Occurrence of the Oxygen-Binding Hemerythrins in Bilaterians.

Animal tissues need to be properly oxygenated for carrying out catabolic respiration and, as such, natural selection has presumably favored special molecules that can reversibly bind and transport oxygen. Hemoglobins, hemocyanins, and hemerythrins (Hrs) fulfill this role, with Hrs being the least studied. Knowledge of oxygen-binding proteins is crucial for understanding animal physiology. Hr genes are present in the three domains of life, Archaea, Bacteria, and Eukaryota; however, within Animalia, Hrs has been reported only in marine species in six phyla (Annelida, Brachiopoda, Priapulida, Bryozoa, Cnidaria, and Arthropoda). Given this observed Hr distribution, whether all metazoan Hrs share a common origin is circumspect. We investigated Hr diversity and evolution in metazoans, by employing in silico approaches to survey for Hrs from of 120 metazoan transcriptomes and genomes. We found 58 candidate Hr genes actively transcribed in 36 species distributed in 11 animal phyla, with new records in Echinodermata, Hemichordata, Mollusca, Nemertea, Phoronida, and Platyhelminthes. Moreover, we found that "Hrs" reported from Cnidaria and Arthropoda were not consistent with that of other metazoan Hrs. Contrary to previous suggestions that Hr genes were absent in deuterostomes, we find Hr genes present in deuterostomes and were likely present in early bilaterians, but not in nonbilaterian animal lineages. As expected, the Hr gene tree did not mirror metazoan phylogeny, suggesting that Hrs evolutionary history was complex and besides the oxygen carrying capacity, the drivers of Hr evolution may also consist of secondary functional specializations of the proteins, like immunological functions.