Evidence for methionine-sulfoxide-reductase gene transfer from Alphaproteobacteria to the transcriptionally active (macro)nucleus of the ciliate, Euplotes raikovi

Methionine residues of polypeptide chains are common targets of oxidation phenomena which alter conformation, sub-cellular localization, and aggregation state of proteins causing detrimental effects on vital cell functions and activities [1],[2]. Aerobic organisms thus urgently need to repair their methionine-oxidized proteins and their minimal gene sets systematically include Msr genes [3],[4].

Increasing concentrations of water-borne signaling proteins (pheromones), which Euplotes raikovi uses to promote its vegetative (mitotic) growth and the sexual phenomenon of conjugation [5], were observed to undergo oxidation in cause-effect relationships with cell ageing [6]. This oxidation hits the methionine residues that are more exposed on the surface of the pheromone molecular structure and was shown to cause remarkable modifications of protein activity [6], as is the case in other cell systems [2]. To shed light on the molecular mechanism evolved by E. raikovi to repair its methionine-oxidized pheromones, attention was focused on the Msr genes that are transcribed in the cell somatic nucleus (macronucleus) characterized by an eccentric sub-chromosomic organization in which individual, gene-size DNA molecules are replicated in thousands of copies fully autonomous for both replication and transcription [7].

Differently from the MsrB gene showing a single form, the gene specifying MsrA was found to be present in the E. raikovi macronucleus in multiple isoforms [8]. One isoform, designated as msrAB gene, is described here for its unique nucleotide sequence containing information for the synthesis of MsrA and MsrB proteins characterized by unequivocal structural relationships with MsrA and MsrB of Alphaproteobacteria.

The msrAB gene cloning involved two PCR steps. A 231-bp MsrA-specific DNA fragment was first generated through amplification of total DNA preparations run with a combination of degenerate oligonucleotides (labeled #1 and 2 in Additional file 1: Table S1) specific to amino acid sequence stretches conserved in MsrA proteins of various organisms. In a second step, two nested PCR amplifications were run using primers (from #3 to #6 in Additional file 1: Table S1) specific to this DNA fragment in combination with a primer (#7 in Additional file 1: Table S1) specific to the C₄A₄/G₄T₄ repeats that are distinctive of the telomeric ends of every Euplotes macronuclear gene-size molecule [7]. Among four structurally distinct gene isoforms that were obtained, we reconstructed the full-length sequence of the longest isoform (1595bp) by overlapping the individual sequences. The reconstructed sequence was then confirmed by sequencing the amplification product of a PCR run with primers (#8 and 9 in Additional file 1: Table S1) specific to regions located close to its telomeric ends.

Instead of including a single open reading frame (ORF) like the other three gene isoforms (obtained incomplete at their 3’ regions), the 1595-bp isoform exceptionally included three potential ORFs (Figure1).

The first ORF (ORF-1), spanning from ATG at position 763 to TAA at position 1269, matched the ORF of the three other gene sequences (Additional file 2: Figure S1). It encodes a 168-amino acid MsrA protein showing a much closer structural identity (79^_90%) to MsrAs of Rhodobacterales, such as Thalassobacter (re-classified as Litoreibacter[9]) and Oceanicola, and Rhizobiales such as Sinorhizobium, than to any eukaryotic MsrA those of ciliates such as Tetrahymena and Paramecium included (Figure2).

The second ORF (ORF-2), spanning from ATG at position 305 to TAG at position 1150 and partially overlapping with ORF-1, includes (at the beginning of the overlapping region) an in-frame TGA codon which, however, is most likely not committed to stop translation. At least in principle, it should code for cysteine, or selenocysteine, so as TGA usually does in Euplotes[10]. The 152-amino acid N-terminal region of the 281-amino acid sequence encoded by this ORF shows significant relationships not with other MsrA proteins, but with bacterial MsrBs lacking Cys-Xxx-Xxx-Cys Zn-ion binding motifs [3],[4]. Its alignment is much closer (72^_78% of structural identity) to MsrBs of Rhodobacterales such as Roseovarius, Roseobacter, Thalassobacter and Oceanicola, and Rhizobiales such as Sinorhizobium and Rhizobium, than to any eukaryotic MsrB including the MsrB of E. raikovi itself (Figure3).

The third ORF (ORF-3), spanning from ATG at position 78 to TAA at position 305 and containing another in-frame TGA_, encodes a 75-amino acid protein not related to Msr proteins. Its 40-amino acid N-terminal segment is 55^_60% identical to the C-terminal sequence of the LysR-type transcription regulator of Rhizobium, Sinorhizobium, and Sphingopyxis. In addition to being strongly conserved among Rhizobiales and Sphingomonadales [11],[12], this regulatory protein is known to be determined by genes carried by DNA regions destined to be transferred from one to another bacterial genome [11].

To obtain evidence that the msrAB gene is a functional and effectively expressed gene, cDNA preparations were obtained from cells previously induced to increase their anti-oxidative enzyme synthesis by a mild oxidative stress (generated by a 30-min suspension with 300-μM H₂O₂ concentration), and subjected to PCR amplification with primer combinations specific to each ORF (Additional file 3: Figure S2). Two MsrA-specific 368-bp and 660-bp products were obtained, indicating that ORF-1 is either the only one to be expressed, or is expressed to a much higher extent than the other two ORFs.

The bacterial origin of the three ORFs of the E. raikovi macronuclear msrAB gene is well explained by a comparative analysis with the organization of the MsrA, MsrB, and transcription-regulator gene sequences in Thalassobacter arenae, Sinorhizobium meliloti and Sphingopyxis alaskensis genomes [13]-[18]. In all these Alphaproteobacteria, the MsrB and MsrA coding genes lie adjacent to one another and the TGA stop codon of the MsrB coding region partially overlaps (T. arenae), or is separated by none (S. meliloti), or only two nucleotides (S. alaskensis) from the ATG start codon of the MsrA coding region (Figure4 and Additional file 4: Figures S3-S5). In addition, in T. arenae and S. alaskensis the transcription-activator gene is located apart from the Msr coding genes [13],[18]. In S. meliloti, instead, the distance is only 114-bp from the ATG of the MsrB coding region [14] and the MsrA/MsrB/transcription-activator gene cluster does not lie in the chromosome, but in one of the two symbiotic mega-plasmids (or chromids) [14]-[17].

Genome analysis from a large variety of pro- and eukaryotes indicates that gene transfer among the three domains of life is a recurrent phenomenon in biological evolution. It also suggests that eukaryotic genomes preferentially retain those prokaryotic genes which encode enzymes capable of conferring adaptive and evolutionary advantages [19]-[21]. The finding that E. raikovi uses Msrs from Alphaproteobacteria to repair methionine-oxidized proteins supports these concepts, and implies that ciliates in general expand their genetic resources from the acquisition of bacterial gene sequences.

The pervasive tendency of Euplotes species to host endosymbiotic bacteria in their cytoplasm [22], and the fact that Rhizobiales include numerous symbiotic species [23] would suggest that the origin of the msrAB coding sequence lies in some Sinorhizobium species living as endosymbionts in E. raikovi. However, present-day stable cytoplasmic hosts of E. raikovi appear to be Gammaproteobacteria, in primis Francisella endociliophora[24],[25], which have Msr genes with sequences markedly different from those of the E. raikovi msrAB coding sequences (personal communication from Dr. Andreas Sjdin, CBRN Defence and Security Department, Swedish Defence Research Agency, Ume).

An alternative hypothesis accounting for the origin of the msrAB gene is suggested by the Doolittles aphorism you are what you eat [26]. It considers that the origin of the msrAB gene resides in some Rhodobacterales or Rhizobiales species that are usually ingested as food by E. raikovi. Molecular investigations and cultivation-based studies have consistently revealed that both Rhodobacterales of the so-called marine alpha group and Rhizobiales of the genus Rhizobium are cosmopolitan and dominant members of microbial communities in marine sediments [27]-[31]. Furthermore, they contribute to the Mediterranean subsurface microbial community of which E. raikovi is a common member [32].

ND and AV conceived the study. PL prepared the biological material for the experiments. ND, AC and FR carried out the experiments. AV analyzed the data. AV and PL wrote the manuscript. All the authors have read the article and approved the final manuscript.