Diversity of Globins in Myxobacteria

Globins are heme proteins that are capable of reversible oxygen binding. All globins can be classified into three families: the M (myoglobin-like), S (sensor) and T (truncated) globins. M and S globins exhibit the canonical 3/3 α-helical fold, and T globins are characterized by a 2/2 α-helical fold. Globins in the genomes of myxobacteria have not been characterized till date. Myxobacteria have very large genomes relative to other bacteria and have a unique life cycle that involves the aggregation of cells into fruiting bodies under starvation conditions. The diversity of globin like sequences in 14 sequenced genomes of myxobacteria is presented in this review. In myxobacterial globins some unusual domain architectures are identified that have not been characterized in bacteria so far; these are: i) a unique chimeric group I 2/2 HbN in the genome of Corallococcus coralloides DSM 2259; ii) M globin chimera harboring a central and a C-terminal globin domain in Sorangium cellulosum ‘so ce 56’ and Plesiocystis pacifica SIR-1 respectively; iii) two tandem globin domains on the same M globin polypeptide in the genomes of Sorangium cellulosum .


INTRODUCTION
Hemoglobins (Hbs) occur in all kingdoms of life and they have a common characteristic of reversible oxygen binding. The structures of myoglobin and Hb from vertebrates have a typical tertiary structure, the globin fold, consisting of eight α-helical segments (A-H) that are connected by short intervening loops [1]. All bacterial globins have been classified into three lineages; the M (myoglobin like) family, the S (sensor) family and the T (truncated) family [2,3]. The M family comprises of flavohemoglobin (FHb) and single domain globin (SDgb) while the S family is classified into Globin coupled sensor (GCS), Protoglobin (Pgb) and single domain sensor globins (SDSgb). Both of M and S globins display a canonical 3/3-α helical globin fold characteristic of metazoan globins [4,5]. T globins classified as trHb1, trHb2 and trHb3 for group I, II and III respectively (or N, O, and P respectively) display a 2/2 α-helical fold [6][7][8].
The chimeric globins in M family, i.e., FHb, generally comprise a N-terminal globin domain and a C-terminal ferredoxin reductase-like domain. In S globin family, chimeric globins (GCS) have a N-terminal globin domain and variable C-terminal domain that is involved either in aerotactic response or gene regulation. Only one chimeric globin of T globin family has been studied till date in bacteria [9].
Myxobacteria are Gram-negative bacteria that belong to the delta branch of proteobacteria and constitute the order Myxococcales [10]. Their genomes have high G+C content and they inhabit terrestrial and marine environments [11,12]. Myxobacteria have an unusual social behavior among bacteria; they exhibit coordinated motility, predating other members of the soil micro fauna, and they possess a communal response to starvation. Under nutrient limitation, a population of cells aggregates forms a multicellular fruiting body within which cells differentiate into myxospores [13]. These bacteria are significant for human health as they are prolific producers of bioactive secondary metabolites of pharmaceutical importance. Some of these metabolites exhibit modes of action that are rarely observed with other microbial compounds [14].
The determination of genomic sequence information of the organisms has provided a tool for the investigation of genes and predicting the corresponding protein functions. With the genome sequence available for myxobacteria, bioinformatics approaches have provided the identity of globins present in this group of social bacteria [15]. The functional information on annotated globin sequences from the genomes is also available in the Uniprot Knowlegde base. So far, among bacteria, globins have been functionally characterized largely from pathogenic bacteria and cyanobacteria where they are implicated in evasion of host defence mechanism during infection and nitrogen metabolism respectively [16][17][18]. In this review, occurrence and type of globins in myxobacteria and the conservation of important residues implicated in ligand interactions as compared to the other most studied globins are presented. The study of regulation of various globins in different stages of a complex life cycle in myxobacteria will add to the existing knowledge of the role of globins in physiology of the microbial hosts.

S globins in myxobacteria
In bacteria, GCSs are multidomain proteins that have an N-terminal myoglobin like domain appended to variable C-terminal transmitter domain. Based on their C-terminal domains, the GCSs are classified as either aerotactic or gene regulating. It is suggested that GCSs have descended from an ancient globin only progenitor, the Pgb [19]. There are two distinct types of single domain S globins, the protoglobins (Pgbs) and the SDSgbs [20].
In general, the S family members occur in ~30% of bacterial globin-containing genomes, often in combination with members of the other two families. 13/14 myxobacterial genomes analyzed were found to harbor a gene coding for S globin ( Table 1). The genomes of myxobacteria abound in GCSs where the globin domain at N-terminus is appended to histidine kinase domain at the Cterminus. Such novel heme based globincoupled oxygen sensor histidine kinase has recently been characterized from A. dehalogenans FW-109-5 [21]. The comparative analysis of the sequences of the globin domains in GCSs suggests that Tyrosine is the conserved residue at heme distal site and His is the proximal ligand (Fig. 1).
The genome of S. cellulosum So ce 56 and Cystobacter fuscus harbor two GCSs in their genomes. In S. cellulosum So ce56, the globin domains appended to STAS domain (Sulfate  Transporter  and  an anti-sigma factor antagonistic) and to a histidine kinase domain show 26% sequence identity. On the contrary, the genome of Sorangium cellulosum So0157-2 harbours one GCS where the globin domain is appended to a histidine kinase domain. The globin domains in two GCSs in the genome of C. fuscus are ~28% identical; one of these is appended to histidine kinase and the other to an MCP domain. The genomes of both the species of Sorangium harbor an SDSgb each. Sorangium cellulosum So0157-2 has a Pgb in its genome in addition to other S globins. The genome of S. cellulosum So0157-2 is 1.75 bases larger than the genome of S. cellulosum So ce56 (13.03 Mb) and it has been proposed to have acquired genes that confer flexibility for ecological adaptation [22]. The co-existence of globins from the same group indicates a diversification of their functions. It is possible that the additional globin in the former might confer the ability to adapt to a complex habitat. The largest number of S globins have recently been reported from a predatory marine bacterium Saprospira grandis the genome of which has ten copies of GCSs and each globin domain at the N-terminus in these S globins is appended to the C-terminal STAS domain [23]. The exact physiological function imparted to the cell by co-existence of GCS and Pgb/SDSgb, as detected in the genomes of S. cellulosum remains to be elucidated. It is probable that these S globins play a role in regulation of specific physiological functions, such as fruiting body formation and sporulation in myxobacteria that require optimal oxygen concentration for cell survival. Pgbs identified in the genomes of myxobacteria showed a sequence motif similar to the well conserved motif at the N-terminus of the known Pgbs, (Ile/Val)-Pro-Gly-Tyr-Xxx-(Tyr/Phe)-Gly (Xxx= any residue) [24] (Fig. 2). No such sequence correlation at N-terminus was found to be present in GCS or SDSgbs of myxobacteria.

T globins in myxobacteria
2/2 globins are short versions of one-domain Hbs that are 20-40 residues shorter than other bacterial M globins [6,25]. They are further classified into three phylogenetically distinct groups (I, II and III, or N, O, P respectively). The group I 2/2HbNs have been implicated to play a role in the alleviation of nitrosative stress and the group II 2/2HbOs are related to O 2 metabolism [26]. The role of the group III HbP characterized only from Campylobacter jejuni is not very clear [27].
In the genomes of myxobacteria sequenced till date, only trHbNs and trHbOs are detected. Analysis of occurrence of trHbNs and trHbOs indicates that 9/14 myxobacterial genomes have the genes for each of these globins (Table 1). Further, the 2/2HbN globins in myxobacteria lack the N-terminal 11 amino acid residues and the polar sequence motif constituting the pre-A helix that is implicated in optimal NO scavenging activity of HbN (Fig. 3). The deletion of pre-A helix in Mycobacterium tuberculosis has been shown to reduce the NO dioxygenase activity of HbN [28]. The putative 2/2 globins from M. xanthus DK1622 have been cloned and expressed in Escherichia coli. These globins bind heme as indicated by red colour they impart to the cells when expressed in recombinant E. coli and give the characteristic spectrum of globins (unpublished observations). The residues that are implicated in control of ligand binding in myxobacterial group I 2/2HbN are: B10Tyr-E7Gln-E11Gln; B10Tyr-E7Leu-E11Glu; B10Leu-E7Leu-E11Thr and B10Tyr-E7Ile-E11Met. Sequence analysis of trHbNs of Aneromyxobacter spp. show an unusual insertion of 15 amino acids (a non-helical structure; SWISS MODEL; [29]) between helices BC-E. Whether this insertion has some relevance in the adaptation or sustenance of these bacteria in an anaerobic niche is not known.
Two putative group I 2/2HbNs occur in the genomes of P. pacifica SIR-1 and C. coralloides DSM 2259. In P. pacifica SIR-1 genome, in addition to a 121 amino acid residue long group I 2/2HbN, a 187 amino acid T1 globin carrying an N-terminal extension of ~20 amino acids is found. The N-terminal extension conforms to the membrane lipoprotein lipid attachment site (www.ebi.ac.uk/tools; Interpro Scan) and the globin domain exhibits a high Z>20 in a FUGUE [30] search with Paramecium caudatum and Chlamydomonas eugametos group I 2/2HbNs. In the genome of C. coralloides DSM 2259, two group I 2/2HbNs -a SD (121 aas) and a chimera having globin domain at N-terminus fused to Methyl Accepting chemotaxis protein (MCP) at C-terminus were identified. Till date, the globin domains found appended to the MCP domain have been reported to have a 3-over-3 fold in bacteria. A trHbN chimera has been reported as a putative globin in fungus Allomyces macrogynus where the globin is present as the C-terminal domain and N-terminal is a ribonulease inhibitor [31]. A T2 chimeric globin has been characterized from Streptomyces avermitis where the monooxygenase domain at N-terminus is fused to globin domain (at Cterminus) [9].
The structure based sequence alignment of myxobacterial group II 2/2HbOs with the sequence of homologous trHbOs of Mycobacterium tuberculosis and Bacillus subtilis reveals that the residues of the heme distal pocket are PheB9, TyrB10, HisCD1, Thr/SerE7 and PheE11; the proximal site residues are HisF8 and TrpG8 (Fig. 4). The crystal structure of Agrobacterium tumefaciens HbO having HisCD1 has been solved and based on its structure it is suggested that it may not act as an oxygen carrier but may serve as signal for growth under low oxygen tension and the presence of His at CD1 site is indicative of the functional adaptation [32].
Myxobacterial trHbNs form a separate cluster from trHbOs and appear to form a relatively heterogeneous group, while group II 2/2HbOs are more homogeneous (Fig. 5).

M globins in myxobacteria
M globins are found restricted to 5/14 sequenced genomes in myxobacteria. Sequence alignment of the globins or globin domains of M globins with other bacterial globins indicates that structural features for adopting a 3/3 globin fold and signature sequences of typical microbial globins (B10, CD1, E7 and F8) are conserved (Fig. 6). In S. cellulosum So ce 56, two of the three M globins show unusual domain architectures: two tandem globin domains on the same polypeptide with the sequence identity of 88%; an FHb having kinase domain at N-terminus appended to a central globin domain and a hydrolase at Cterminus. An F globin chimera occurs in P. pacifica SIR-1 that has C-terminal globin domain. In all the globins reported till date from bacteria, very few globin domains have been found to be present in tandem on the same polypeptide. Some multi-globin domain proteins are known to exist in lower eukaryotes [20].
In S. cellulosum So0157-2, in addition to three SD F-globins, a chimeric F-globin having tandem globin domains on a polypeptide with the sequence identity of 83% are found. The relevance and function of the same type of globins in the genome can be assessed by studying the genetic regulation of these proteins.
--M-TIHGYTLGTGAVAKSPVSPADFEDMKKSALF    Recently, centrally located globin domains in M globins have been recognized in several fungi [31]. The study of these M globins can give further insights into the structure-function relationship of globins.

CONCLUSION
The grouping of globins in myxobacterial genomes reveals the co-occurrence of SDSgb and FHb in S. cellulosum that was thought to be exclusive to fungal genomes. It is possible that novel functions conferred on the host by the existence of combination of globins allows the host to survive in varied environmental conditions. Also, the co-existence of different globins of the same family in an organism suggests that they may be playing different functions in cellular metabolism of myxobacteria. However, the sequence information alone is not sufficient to determine the functions of globins and the function of these genes can be revealed by knocking out the genes followed by physiological studies on the null mutants. The correlation of expression of globins with the complex life cycle in myxobacteria will provide insights into the role of globins in physiology of the hosts. The study of myxobacteria have led to elucidation of many phenomena that were previously not known to exist in the prokaryotes such as coordinated social behavior, complex signal transduction networks, unique and complex motility mechanisms, and contact signaling [33]. It would be interesting to study regulation of globins in various stages of a complex life cycle in myxobacteria as this will unveil the precise role of globins in physiology of these microbes.