Wide diversity in structure and expression profiles among members of the Caenorhabditis elegans globin protein family

<p>Abstract</p> <p>Background</p> <p>The emergence of high throughput genome sequencing facilities and powerful high performance bioinformatic tools has highlighted hitherto unexpected wide occurrence of globins in the three kingdoms of life. <it>In silico </it>analysis of the genome of <it>C. elegans </it>identified 33 putative globin genes. It remains a mystery why this tiny animal might need so many globins. As an inroad to understanding this complexity we initiated a structural and functional analysis of the globin family in <it>C. elegans</it>.</p> <p>Results</p> <p>All 33 <it>C. elegans </it>putative globin genes are transcribed. The translated sequences have the essential signatures of single domain <it>bona fide </it>globins, or they contain a distinct globin domain that is part of a larger protein. All globin domains can be aligned so as to fit the globin fold, but internal interhelical and N- and C-terminal extensions and a variety of amino acid substitutions generate much structural diversity among the globins of <it>C. elegans</it>. Likewise, the encoding genes lack a conserved pattern of intron insertion positioning. We analyze the expression profiles of the globins during the progression of the life cycle, and we find that distinct subsets of globins are induced, or repressed, in wild-type dauers and in <it>daf-2(e1370)</it>/insulin-receptor mutant adults, although these animals share several physiological features including resistance to elevated temperature, oxidative stress and hypoxic death. Several globin genes are upregulated following oxygen deprivation and we find that HIF-1 and DAF-2 each are required for this response. Our data indicate that the DAF-2 regulated transcription factor DAF-16/FOXO positively modulates <it>hif-1 </it>transcription under anoxia but opposes expression of the HIF-1 responsive globin genes itself. In contrast, the canonical globin of <it>C. elegans</it>, ZK637.13, is not responsive to anoxia. Reduced DAF-2 signaling leads to enhanced transcription of this globin and DAF-16 is required for this effect.</p> <p>Conclusion</p> <p>We found that all 33 putative globins are expressed, albeit at low or very low levels, perhaps indicating cell-specific expression. They show wide diversity in gene structure and amino acid sequence, suggesting a long evolutionary history. Ten globins are responsive to oxygen deprivation in an interacting HIF-1 and DAF-16 dependent manner. Globin ZK637.13 is not responsive to oxygen deprivation and regulated by the Ins/IGF pathway only suggesting that this globin may contribute to the life maintenance program.</p

A Phylogenetic Analysis of the Globins in Fungi

BACKGROUND: ALL GLOBINS BELONG TO ONE OF THREE FAMILIES: the F (flavohemoglobin) and S (sensor) families that exhibit the canonical 3/3 α-helical fold, and the T (truncated 3/3 fold) globins characterized by a shortened 2/2 α-helical fold. All eukaryote 3/3 hemoglobins are related to the bacterial single domain F globins. It is known that Fungi contain flavohemoglobins and single domain S globins. Our aims are to provide a census of fungal globins and to examine their relationships to bacterial globins. RESULTS: Examination of 165 genomes revealed that globins are present in >90% of Ascomycota and ∼60% of Basidiomycota genomes. The S globins occur in Blastocladiomycota and Chytridiomycota in addition to the phyla that have FHbs. Unexpectedly, group 1 T globins were found in one Blastocladiomycota and one Chytridiomycota genome. Phylogenetic analyses were carried out on the fungal globins, alone and aligned with representative bacterial globins. The Saccharomycetes and Sordariomycetes with two FHbs form two widely divergent clusters separated by the remaining fungal sequences. One of the Saccharomycete groups represents a new subfamily of FHbs, comprising a previously unknown N-terminal and a FHb missing the C-terminal moiety of its reductase domain. The two Saccharomycete groups also form two clusters in the presence of bacterial FHbs; the surrounding bacterial sequences are dominated by Proteobacteria and Bacilli (Firmicutes). The remaining fungal FHbs cluster with Proteobacteria and Actinobacteria. The Sgbs cluster separately from their bacterial counterparts, except for the intercalation of two Planctomycetes and a Proteobacterium between the Fungi incertae sedis and the Blastocladiomycota and Chytridiomycota. CONCLUSION: Our results are compatible with a model of globin evolution put forward earlier, which proposed that eukaryote F, S and T globins originated via horizontal gene transfer of their bacterial counterparts to the eukaryote ancestor, resulting from the endosymbiotic events responsible for the origin of mitochondria and chloroplasts

Genome-wide analysis of the globin gene family of C. elegans

The aim of our study was to annotate sequences for 35 putative globins from the nematode Caenorhabditis elegans. All these proteins are expressed, but seven of these differ from the gene predictions in Wormbase. The entire polypeptide sequences for 31 genes and the core globin domain of four proteins were confirmed or corrected. All core globin domains were aligned manually following a procedure that was designed to fit the putative sequences to the crystal structure based alignment of 56 known globin crystal structures. Neighbor-joining analysis of the resulting alignment showed that the majority of these globins are very divergent from each other, possibly suggesting a long evolutionary divergence. The surprisingly high number and low sequence conservation of putative globins in this small organism urges a detailed functional analysis

Bacterial phyla whose globins may share ancestry with fungal globins.

<p>Bacterial phyla whose globins may share ancestry with fungal globins.</p

Bayesian phylogenetic tree of fungal and bacterial FHbs.

<p>Bayesian tree based on a T-COFFEE 9.01 alignment of the globin domains of 55 representative fungal FHbs and 54 representative bacterial FHbs. Support values at branches represent Bayesian posterior probabilities (>0.5). All the bacterial FHbs are in the blue boxes. The sequences are identified by the first three letters of the binary species name, the number of residues, and the full phylum and family names (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031856#pone.0031856.s011" target="_blank">Table S1</a>). Sac – Saccharomycetes.</p

Bayesian phylogenetic tree of fungal FHbs.

<p>Bayesian tree based on a MAFFT v.6.850 alignment of 62 fungal FHb globin domains using two plant nonsymbiotic Hbs as outgroup. Support values at branches represent Bayesian posterior probabilities (>0.5). The sequences are identified by the first three letters of the binary species name, the number of residues, and the full phylum and family names (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031856#pone.0031856.s011" target="_blank">Table S1</a>). Sac – Saccharomycetes.</p

Bayesian phylogenetic tree of fungal and bacterial T1 globins.

<p>Bayesian tree based on a T-COFFEE 9.01 alignment of 2 fungal (red and black arrows), 70 bacterial (blue), 4 euryarchaeote (purple) and 10 chlorophyte (green) T1 globins, using 2 <i>Physcomitrella</i> nsHbs as outgroup. Support values at branches represent Bayesian posterior probabilities (>0.5). All the bacterial FHbs are in the blue boxes. The sequences are identified by the first three letters of the binary species name, the number of residues, and the full phylum and family names (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031856#pone.0031856.s011" target="_blank">Table S1</a>).</p

Diagrammatic representation of fungal phylogeny based on ref. 18 and of their putative globins.

<p>Estimates of the numbers of species in parentheses are from the <i>Dictionary of the Fungi </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031856#pone.0031856-Kirk1" target="_blank">[23]</a>. Red bar indicates multicellularity with differentiated tissues; FHb* - unknown N-terminal domain linked to a FHb with an incomplete reductase domain, T1gb - T globin group1. Ratios refer to the number of genomes containing globins versus the total number of genomes analyzed.</p