Evidence for methionine-sulfoxide-reductase gene transfer from Alphaproteobacteria to the transcriptionally active (macro)nucleus of the ciliate, Euplotes raikovi
© Dobri et al.; licensee BioMed Central Ltd. 2014
Received: 7 January 2014
Accepted: 7 November 2014
Published: 25 November 2014
Deleterious phenomena of protein oxidation affect every aerobic organism and methionine residues are their elective targets. The reduction of methionine sulfoxides back to methionines is catalyzed by methionine-sulfoxide reductases (Msrs), enzymes which are particularly active in microorganisms because of their unique nature of individual cells directly exposed to environmental oxidation.
From the transcriptionally active somatic genome of a common free-living marine protist ciliate, Euplotes raikovi, we cloned multiple gene isoforms encoding Msr of type A (MsrA) committed to repair methionine-S-sulfoxides. One of these isoforms, in addition to including a MsrA-specific nucleotide sequence, included also a sequence specific for a Msr of type B (MsrB) committed to repair methionine-R-sulfoxides. Analyzed for its structural relationships with MsrA and MsrB coding sequences of other organisms, the coding region of this gene (named msrAB) showed much more significant relationships with Msr gene coding sequences of Rhodobacterales and Rhizobiales (Alphaproteobacteria), than of other eukaryotic organisms.
Based on the fact that the msrAB gene is delimited by Euplotes-specific regulatory 5’ and 3’ regions and telomeric C4A4/G4T4 repeats, it was concluded that E. raikovi inherited the coding region of this gene through a phenomenon of horizontal gene transfer from species of Alphaproteobacteria with which it coexists in nature and on which it likely feeds.
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 ,. Aerobic organisms thus urgently need to repair their methionine-oxidized proteins and their minimal gene sets systematically include Msr genes ,.
Increasing concentrations of water-borne signaling proteins (pheromones), which Euplotes raikovi uses to promote its vegetative (mitotic) growth and the sexual phenomenon of conjugation , were observed to undergo oxidation in cause-effect relationships with cell ageing . 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 , as is the case in other cell systems . 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 .
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 . 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.
Results and discussion
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 . 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.
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 ,, this regulatory protein is known to be determined by genes carried by DNA regions destined to be transferred from one to another bacterial genome .
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.
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 -. 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 , and the fact that Rhizobiales include numerous symbiotic species  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,, 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 . 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 -. Furthermore, they contribute to the Mediterranean subsurface microbial community of which E. raikovi is a common member .
Euplotes raikovi cultures used in this study derive from the wild-type strain #13 deposited at the ATCC Center (catalog, #PRA-327), and collected (June 1979) from a sandy coastal site (Porto Recanati, 43 26’N, 13 43’E) of the Adriatic cost of Italy . They were fed on green algae, Dunaliella tertiolecta, grown in pasteurized natural seawater enriched with Walne medium.
DNA purification and amplification
Total DNA preparations were obtained, according to a published procedure , from cultures deprived of food for 34 days and concentrated by centrifugation (2500 x g, for 5min). Degenerate primers were designed with the CODEHOP (Consensus-Degenerate Hybrid Oligonucleotide Primers) method  on the basis of the following two MsrA conserved sequence stretches: Leu-Ala-Gly-Gly-Cys-Phe-Trp and His-Asp-Pro-Thr-Thr-Leu-Asn-Arg-Gln-Gly. All the PCR amplifications were run in an Eppendorf Mastercycler (Eppendorf, AG, Hamburg, Germany), using 0.5-μg DNA aliquots as template in 50 μl-reaction mixtures containing 0.25 μM of each primer, 0.3mM dNTP, 1x buffer, and 1U of Perfect-Taq DNA Polymerase (Eppendorf). After an initial DNA denaturation step at 95C for 4min, 35cycles of 95C for 30sec, 58C for 40sec, and 72C for 1min were run. A final incubation step, at 72C for 7min, was added to the last cycle. Gel-purified PCR products were ligated into pGEM-T Easy Vector (Promega, WI) and transformed into TOPO 10 cells (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA). Colonies were selected for PCR amplification to screen the presence of inserts using standard M13 primers and the products were sequenced at the BMR Genomics Center of the University of Padua.
RNA extraction and cDNA synthesis
RNA was extracted from cells incubated with H2O2 (300 μM), for 30min, harvested by centrifugation, and lysed in Trizol reagent (Ambion, Life Technologies Corporation, Carlsbad, CA, USA). It was then purified with the PureLink RNA mini kit (Ambion) following the procedure described by the manufacturer, and digested with RNAse-free DNAse I to remove contaminating DNA. Single-stranded cDNA was synthesized following the 3’ RACE protocol of the FirstChoice RLM-RACE kit (Ambion) and 50ng-aliquots were next used in PCR analysis.
Sequence analysis and accession number
BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST) and ClustalW (http://www.genome.jp/tools/clustalw) were used to search for the nearest relative sequences and perform multiple sequence alignments, respectively. The msrAB sequence has been deposited to GenBank under the accession number KM197136.
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.
This work was financially supported by the Ministero dellIstruzione, Universit e Ricerca (PRIN research projects). Expert advice of Dr. Martha Dunbar for the language revision of the text is gratefully acknowledged.
- Friguet B: Oxidized protein degradation and repair in ageing and oxidative stress. FEBS Lett. 2006, 580: 2910-2916. 10.1016/j.febslet.2006.03.028.View ArticlePubMedGoogle Scholar
- Stadtman ER, Moskovitz J, Levine RL: Oxidation of methionine residues of proteins: biological consequences. Antioxi Redox Sign. 2003, 5: 577-582. 10.1089/152308603770310239.View ArticleGoogle Scholar
- Delaye L, Becerra A, Orgel L, Lazcano A: Molecular evolution of peptide methionine sulfoxide reductases (MsrA and MsrB): on the early development of a mechanism that protects against oxidative damage. J Mol Evol. 2007, 64: 15-32. 10.1007/s00239-005-0281-2.View ArticlePubMedGoogle Scholar
- Zhang XH, Weissbach H: Origin and evolution of the protein-repairing enzymes methionine sulphoxide reductases. Biol Rev. 2008, 83: 249-257. 10.1111/j.1469-185X.2008.00042.x.View ArticlePubMedGoogle Scholar
- Vallesi A, Giuli G, Bradshaw RA, Luporini P: Autocrine mitogenic activity of pheromones produced by the protozoan ciliate Euplotes raikovi. Nature. 1995, 376: 522-524. 10.1038/376522a0.View ArticlePubMedGoogle Scholar
- Alimenti C, Vallesi A, Luporini P, Buonanno F, Ortenzi C: Cell aging-induced methionine oxidation causes an autocrine to paracrine shift of the pheromone activity in the protozoan ciliate, Euplotes raikovi. Exp Cell Res. 2012, 318: 144-151. 10.1016/j.yexcr.2011.10.011.View ArticlePubMedGoogle Scholar
- Jahn CL, Klobutcher LA: Genome remodeling in ciliated protozoa. Annu Rev Microbiol. 2002, 56: 489-520. 10.1146/annurev.micro.56.012302.160916.View ArticlePubMedGoogle Scholar
- Dobri N, Oumarou EE, Alimenti C, Ortenzi C, Luporini P, Vallesi A: Methionine sulfoxide reduction in ciliates: characterization of the ready-to-use methionine sulfoxide-R-reductase genes in Euplotes. Gene. 2013, 515: 110-116. 10.1016/j.gene.2012.11.019.View ArticlePubMedGoogle Scholar
- Kim YO, Park S, Nam BH, Kang SJ, Hur YB, Kim DG, Oh TK, Yoon JH: Description of Litoreibacter meonggei sp. nov., isolated from the sea squirt Halocynthia roretzi, reclassification of Thalassobacter arenae as Litoreibacter arenae comb. nov. and emended description of the genus Litoreibacter Romanenko et al. 2011. Int J Syst Evol Microbiol. 2012, 62: 1825-1831. 10.1099/ijs.0.035113-0.View ArticlePubMedGoogle Scholar
- Turanov AA, Lobanov AV, Formenko DE, Morrison HG, Sogin ML, Klobutcher LA, Hatfield DL, Gladyshev VN: Genetic code supports targeted insertion of two amino acids by one codon. Science. 2009, 323: 259-261. 10.1126/science.1164748.PubMed CentralView ArticlePubMedGoogle Scholar
- Prez-Rueda E, Collado-Vides J: Common history at the origin of the position-function correlation in transcriptional regulators in archaea and bacteria. J Mol Evol. 2001, 53: 172-179. 10.1007/s002390010207.View ArticleGoogle Scholar
- Maddocks SE, Oyston PCF: Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology. 2008, 154: 3609-3623. 10.1099/mic.0.2008/022772-0.View ArticlePubMedGoogle Scholar
- Riedel T, Fiebig A, Petersen J, Gronow S, Kyrpides NC, Gker M, Klenk HP: Genome sequence of the Litoreibacter arenae type strain (DSM 19593T), a member of the Roseobacter clade isolated from sea sand. Stand Genomic Sci. 2013, 9: 117-127. 10.4056/sigs.4258318.PubMed CentralView ArticlePubMedGoogle Scholar
- Galardini M, Mengoni A, Brilli M, Pini F, Fioravanti A, Lucas S, Lapidus A, Cheng JF, Goodwin L, Pitluk S, Land M, Hauser L, Woyke T, Mikhailova N, Ivanova N, Daligault H, Bruce D, Detter C, Tapia R, Han C, Teshima H, Mocali S, Bazzicalupo M, Biondi EG: Exploring the symbiotic pangenome of the nitrogen-fixing bacterium Sinorhizobium meliloti. BMC Genomics. 2011, 12: 235-10.1186/1471-2164-12-235.PubMed CentralView ArticlePubMedGoogle Scholar
- Barnett MJ, Fisher RF, Jones T, Komp C, Abola AP, Barloy-Hubler F, Bowser L, Capela D, Galibert F, Gouzy J, Gurjal M, Hong A, Huizar L, Hyman RW, Kahn D, Kahn ML, Kalman S, Keating DH, Palm C, Peck MC, Surzycki R, Wells DH, Yeh KC, Davis RW, Federspiel NA, Long SR: Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid. Proc Natl Acad Sci U S A. 2001, 98: 9883-9888. 10.1073/pnas.161294798.PubMed CentralView ArticlePubMedGoogle Scholar
- Galardini M, Pini F, Bazzicalupo M, Biondi EG, Mengoni A: Replicon-dependent bacterial genome evolution: the case of Sinorhizobium meliloti. Genome Biol Evol. 2013, 5: 542-558. 10.1093/gbe/evt027.PubMed CentralView ArticlePubMedGoogle Scholar
- Martnez-Abarca F, Martnez-Rodrguez L, Lpez-Contreras JA, Jimnez-Zurdo JI, Toro N: Complete genome sequence of the alfalfa symbiont Sinorhizobium/Ensifer meliloti strain GR4. Genome Announc. 2013, 1: e00174-12. 10.1128/genomeA.00174-12.Google Scholar
- Lauro FM, McDougald D, Thomas T, Williams TJ, Egan S, Rice S, DeMaere MZ, Ting L, Ertan H, Johnson J, Ferriera S, Lapidus A, Anderson I, Kyrpides N, Munk AC, Detter C, Hang CS, Brown MV, Robb FT, Kjelleberg S, Cavicchioli R: The genomic basis of trophic strategy in marine bacteria. Proc Natl Acad Sci U S A. 2009, 106: 15527-15533. 10.1073/pnas.0903507106.PubMed CentralView ArticlePubMedGoogle Scholar
- Rocha EPC: With a little help from Prokaryotes. Science. 2013, 339: 1154-1155. 10.1126/science.1234938.View ArticlePubMedGoogle Scholar
- Keeling PJ, Palmer JD: Horizontal gene transfer in eukaryotic evolution. Nat Rev Genet. 2008, 9: 605-618. 10.1038/nrg2386.View ArticlePubMedGoogle Scholar
- Keeling PJ: Functional and ecological impacts of horizontal gene transfer in eukaryotes. Curr Opin Genet Dev. 2009, 19: 613-619. 10.1016/j.gde.2009.10.001.View ArticlePubMedGoogle Scholar
- Grtz HD: Intracellular bacteria in ciliates. Int Microbiol. 2001, 4: 143-150.Google Scholar
- Engelhardt T, Sahlberg M, Cypionka H, Engelen B: Biogeography of Rhizobium radiobacter and distribution of associated temperate phages in deep subseafloor sediments. ISME J. 2013, 7: 199-209. 10.1038/ismej.2012.92.PubMed CentralView ArticlePubMedGoogle Scholar
- Schrallhammer M, Schweikert M, Vallesi A, Verni F, Petroni G: Detection of a novel subspecies of Francisella noatunensis as endosymbiont of the ciliate Euplotes raikovi. Microb Ecol. 2011, 61: 455-464. 10.1007/s00248-010-9772-9.View ArticlePubMedGoogle Scholar
- Sjdin A, hrman C, Bckman S, Lrkeryd A, Granberg M, Lundmark E, Karlsson E, Nilsson E, Vallesi A, Tellgren-Roth C, Stenberg P, Thelaus J: Complete genome sequence of Francisella endociliophora strain FSC1006, isolated from a laboratory culture of the marine ciliate Euplotes raikovi. Genome Announc 2014, 2:e0122714.,Google Scholar
- Doolittle WF: You are what you eat: a gene transfer ratchet could account for bacterial genes in eukaryotic nuclear genomes. Trends Genet. 1998, 14: 307-311. 10.1016/S0168-9525(98)01494-2.View ArticlePubMedGoogle Scholar
- Gonzlez JM, Moran MA: Numerical dominance of a group of marine bacteria in the α-subclass of the class Proteobacteria in coastal seawater. Appl Environ Microbiol. 1997, 63: 4237-4242.Google Scholar
- Macin MC, Arahal DR, Garay E, Ludwig W, Schleifer KH, Pujalte MJ:Thalassobacter stenotrophicus gen. nov., sp. nov., a novel marine α-proteobacterium isolated from Mediterranean sea water. Int J Syst Evol Microbiol. 2005, 55: 105-110. 10.1099/ijs.0.63275-0.View ArticleGoogle Scholar
- Dang H, Li T, Chen M, Huang G: Cross-ocean distribution of Rhodobacterales bacteria as primary surface colonizers in temperate coastal marine waters. Appl Environ Microbiol. 2008, 74: 52-60. 10.1128/AEM.01400-07.PubMed CentralView ArticlePubMedGoogle Scholar
- DHondt S, Jrgensen BB, Miller DJ, Batzke A, Blake R, Cragg BA, Cypionka H, Dickens GR, Ferdelman T, Hinrichs KU, Holm NG, Mitterer R, Spivack A, Wang G, Bekins B, Engelen B, Ford K, Gettemy G, Rutherford SD, Sass H, Skilbeck CG, Aiello IW, Gurin G, House CH, Inagaki F, Meister P, Naehr T, Niitsuma S, Parkes RJ, Schippers A: Distributions of microbial activities in deep subseafloor sediments. Science. 2004, 306: 2216-2221. 10.1126/science.1101155.View ArticleGoogle Scholar
- Sss J, Schubert K, Sass H, Cypionka H, Overmann J, Engelen B: Widespread distribution and high abundance of Rhizobium radiobacter within Mediterranean subsurface sediments. Environ Microbiol. 2006, 8: 1753-1763. 10.1111/j.1462-2920.2006.01058.x.View ArticleGoogle Scholar
- Miceli C, Luporini P, Bracchi P: Morphological description, breeding system, and nuclear changes during conjugation of Euplotes raikovi Agamaliev from Mediterranean Sea. Acta Protozool. 1981, 20: 215-224.Google Scholar
- Vallesi A, Di Pretoro B, Ballarini P, Apone F, Luporini P: A novel protein kinase from the ciliate Euplotes raikovi with close structural identity to the mammalian intestinal and male-germ cell kinases: characterization and functional implications in the autocrine pheromone signaling loop. Protist. 2010, 161: 250-263. 10.1016/j.protis.2009.12.002.View ArticlePubMedGoogle Scholar
- Rose TM, Henikoff JG, Henikoff S: CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Res. 2003, 31: 3763-3766. 10.1093/nar/gkg524.PubMed CentralView ArticlePubMedGoogle Scholar
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