Whole-genome shotgun and metagenomic sequencing projects have provided a new and powerful tool in the study of community organization and metabolism in natural microbial communities (35–37
). Recently, such methods have been extended to analyze symbiotic relationships. One project involved an analysis of microbes from a marine oligochaete O. algarvensis
, which lacks a mouth, gut, anus or nephridial excretory system, and contains several bacterial endosymbionts that are located just below the worm cuticle (26
). These endosymbionts include two sulfur-oxidizing gammaproteobacteria (γ1 and γ3) and two sulfate-reducing deltaproteobacteria (δ1 and δ4). Identification of selenoprotein genes in such an unusual symbiotic system may help understand the role of selenium and other micronutrients in the intricate interactions that form such a complex, adaptive consortium.
In the present study, we employed a procedure that analyzes Sec/Cys pairs in homologous sequences to characterize the selenoproteomes of symbiotic microorganisms in the gutless worm. A total of 82 genes that belonged to 24 previously described prokaryotic selenoprotein families and 17 sequences that belonged to six new selenoprotein families were identified. Most selenoproteins were found to occur in δ1 symbiont, which contained 44 known selenoproteins (21 families) and 13 new selenoproteins (6 families). Although the genome size of δ1 symbiont is ~13.5
Mb, which is larger than most other deltaproteobacteria, its reconstruction revealed a single species (26
). If this is the case, then our study identified an organism, which has the largest selenoproteome reported to date (57 selenoproteins) of any organism, including eukaryotes and archaea.
Most detected selenoproteins were homologs of thiol-based redox enzymes and contained conserved redox motifs. In contrast, such known redox motifs were largely absent in new selenoproteins identified in the metagenomic dataset. In addition, analysis of secondary structures revealed that these new selenoproteins did not contain thioredoxin-like fold, which is a dominant fold in selenoproteins identified in several marine environmental sequencing projects (23
). Perhaps, additional redox reactions that are carried out by new selenoproteins occur in these symbionts.
Besides the unusually high number of selenoproteins, 10 Pyl-containing proteins were identified in the metagenomic dataset. δ1 contained eight of these sequences that belonged to MtbB and MttB families. Thus, the δ1 symbiont is also the organism, which has the largest number of Pyl-containing proteins in bacteria. Previously, only one bacterial protein, from D. hafniense, was known to possess Pyl. Therefore, identifying so many pyrroproteins in the same bacterium is truly remarkable.
We previously proposed that UAG may be an ambiguous codon in some archaea, wherein it could serve as either Pyl codon or a stop signal. However, in D. hafniense, UAG is frequently used as a stop signal, suggesting an unknown mechanism that allows ribosomes to recognize function of specific UAG codons. By analogy to Sec, which is inserted with the help of SECIS elements, PYLIS elements may be present in bacterial pyrroprotein genes. However, our analysis of genes coding for Pyl-containing proteins revealed no common RNA structures. Additional RNA structure searches should be carried out in the future. The current set of Pyl-containing proteins provides an excellent dataset for further interrogation.
Given that most symbiotic and host-associated bacteria have lost the ability to utilize Sec or only possess a limited number of selenoproteins, the dramatic abundance of selenoproteins in the two endosymbiotic deltaproteobacteria, especially δ1 that also contains many Pyl-containing proteins, is remarkable, raising a series of questions regarding evolution and function of these proteins, as well as their roles in symbiosis. It has been suggested that most selenoproteins evolved from their Cys-containing homologs and anaerobic environments could support the use of Sec (27
). Compared to most other symbionts and host-associated organisms, which seem to live under aerobic or microaerobic conditions, the obligate anaerobic environment of the two symbionts may be one reason for evolution of new selenoproteins. In addition, compared to the environments where other hosts live, seawater could provide a constant supply of selenium for Sec biosynthesis in these symbionts. An alternative hypothesis is that the host worm needs more efficient metabolism and waste management, which are provided by its symbionts because of the lack of digestive and excretory systems. These special needs might have led to selective advantage of harboring multiple symbionts that utilize amino acids that provide catalytic advantages to various metabolic systems, such as Sec in many redox proteins and Pyl in methylamine methyltransferases.
Symbiotic deltaproteobacteria in the gutless worm evolved as organisms that support the broadest use of the genetic code, utilizing 63 of 64 codons to code for 22 amino acids. It would be interesting to examine if this and other symbiotic systems provide selective advantage to further expand the genetic code, either utilizing a third stop signal, UAA, or using some codons to insert multiple non-canonical or common amino acids.