THE COMPLETE MITOCHONDRIAL GENOME OF AN UNUSUAL STRAIN OF TINY VANNELLID AMOEBA (AMOEBOZOA, DISCOSEA, VANNELLIDA) ISOLATED FROM THE NIAGARA RIVER (CANADA) Текст научной статьи по специальности «Биологические науки»

Protistology 15 (4), 304-311 (2021)

Protistology

The complete mitochondrial genome of an unusual strain of tiny vannellid amoeba (Amoebozoa, Discosea, Vannellida) isolated from the Niagara River (Canada)

Natalya I. Bondarenko1, Anna A. Glotova1, Elena S. Nassonova2, Alexey E. Masharsky3, and Alexey V. Smirnov1

1 Department ofInvertebrate Zoology, Faculty of Biology, St. Petersburg State University, 199034 St. Petersburg, Russia

2 Laboratory of Cytology of Unicellular Organisms, Institute of Cytology RAS, 194064 St. Petersburg, Russia

3Core Facility Centre Biobank, St. Petersburg State University, Stary Peterhof 198504 St. Petersburg, Russia

| Submitted November 13, 2020 | Accepted December 9, 2020 |

Summary

We present a complete sequence and describe the mitochondrial genome organization of the strain of small vannellid amoeba isolated from the Niagara River (Canada) in the year 2007. The circular mitochondrial DNA of this strain has 52,924 bp in length and contains 30 protein-coding genes, two ribosomal RNAs, 25 transfer RNAs, and 13 open reading frames. It is the second in length mt genome among amoebae of the order Vannellida (Amoebozoa, Discosea). In contrast with the shorter mitochondrial genomes of crown vannellids, it shows no evidence of RNA editing. This finding supports the hypothesis on the independent origin of editing in the phylogenetic lineage corresponding to the order Vannellida (Amoebozoa, Discosea).

Key words: Amoebozoa, Vannellidae, Ripella, mitochondrion, mitochondrial genome

Abbreviations: MT — mitochondrial; cox1-3, — cytochrome oxidase subunit I, II, and III genes; cob — cytochrome b gene; atp9—ATP synthase subunit 9 gene; nad1-7, 9,11 — NADH dehydrogenase subunit 1-7, 9, 11 and 4L genes; tRNA — transfer RNA genes; rrnL, rrnS—ribosomal RNA genes; ORF — open reading frames; PCGs — protein-coding genes; rps - small ribosomal subunit protein genes; rpl — large ribosomal subunit protein genes; CDS — coding DNA sequence

doi:10.21685/1680-0826-2021-15-4-8 © 2021 The Author(s)

Protistology © 2021 Protozoological Society Affiliated with RAS

Introduction

Mitochondrial genomes (MT genomes) of Amoebozoa remain poorly studied despite significant efforts invested in this field in recent time (reviewed by Bondarenko et al., 2019b). Among amoebozoan lineages, the best taxonomic sampling in MT genome studies is achieved among the order Vannellida (Bondarenko et al., 2018a, 2018b, 2018c, 2019a). This group ofamoebae demonstrated very different sizes of the mitochondrial genomes. The genus Vannella, the crown phylogenetic lineage of this order (Smirnov et al., 2007), has the length of MT genomes 29-34 kbp and shows extensive post-translational editing (Bondarenko et al., 2018a, 2018b). The MT genome of Clydonella sawyeri, which belongs to a more basal lineage of Vannellida (Kudryavtsev and Volkova, 2018), is 31 kbp in length but shoes low level of editing in five genes only. Simultaneously, the only studied basal lineage, represented with Paravannella minima, demonstrates a much longer genome, reaching up to 53 kbp, with no editing (Bondarenko et al., 2019a). This finding suggests an independent origin of editing in this branch of amoebozoan tree; however, a limited taxonomic sample does not allow one to locate the point of the origin of editing.

The present paper reports data on the mito-chondrial genome of a strain of a small vannellid amoeba, belonging to a new, independent basal lineage among the order Vannellida and possessing a long mitochondrial genome (52 kbp), with no editing. This finding supports the idea of the independent origin of RNA editing in this amoebozoan lineage.

Material and methods

The culture of an amoeba, designated further as "Niagara strain," was isolated from the sample of the top layer of bottom sediment of theNiagara River in the area between the Niagara Falls and Ontario Lake, along the Niagara Park-way (Canada, grid reference 43.191408N, - 79.054652E). In a forthcoming paper, this strain will be described as "Simripella niagara" (should not be considered here as a taxonomic mentioning, and provided here in order to link the present data with a forthcoming publication by another author). According to the personal communication by Dr. Alexander Kudryavtsev, in the SSU phylogenetic tree, it forms an independent lineage, basal to most other vannellids. The strain is illustrated in Fig. 1.

Fig. 1. Light-microscopic images of "Niagara

strain." Phase contrast. Scale bar: 10 ^m.

Amoebae were cultured in 90 mm Petri dishes filled with Millipore-sterilized (0.2 ^m pore) artificial seawater (25%o) and one wheat grain per dish. Cells were concentrated and washed to remove bacteria as described earlier (Bondarenko et al., 2018a). Total DNA isolation was performed using NucleoSpin Tissue Kit (Macherey-Nagel, Germany) according to the manufacturer's instructions. Approximately 40,5 million reads with a length of 150 bp were obtained using HiSeq 2500 sequencing system (Illumina). Quality control check of raw sequence data was performed using FastQC (http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/), SPAdes assembler was used for de novo mitochondrial genome assembly (Bankevich et al., 2012). An annotation of mitochondrial genome sequence was performed using the MITOS web server (Bernt et al., 2013a). Artemis was used to visualize annotation files, manual correction of gene boundaries, and open reading frames (ORFs) search (version 16.0; Rutherford et al., 2000). All protein-coding genes (PCGs) boundaries were verified by manual comparison with the orthologs in other amoebozoans. Genes coding tRNAs were positioned with tRNAcan-SE Search Server v.1.21 (Lowe and Eddy, 1997). Strand asymmetry was calculated using the formulae: AT skew= [A-T]/ [A+T] and GC skew= [G-C]/ [G+C], for the H-strand (Perna and Kocher, 1995). The physical map was generated by our original script written in Python.

Results and discussion

The mitochondrial genome of"Niagara strain" is a double-stranded circular DNA molecule with a length of52,924 bp (Fig. 2). It is another sizeable MT genome (over 40 kbp) among amoebae belonging to the family Vannellidae (see Bondarenko et al.,

4llc 12k

40k Niagara strain 13|<

Fig. 2. Mitochondrial genome map of the "Niagara strain." The tRNA genes are labeled based on the IUPACIUB single-letter amino acid codes.

2019b). It shows GC content 27,1% (Table 1), which is a rather low level. The prevalence of thymine over adenine and guanine over cytosine in the majority strand provides negative AT-skew and positive GC-skew. This picture of AT-skew is similar to that observed in most other organisms (Bernt et al., 2013b; Bondarenko et al., 2018b). Therefore, the nucleotide composition of "Niagara strain" MT genome is significantly biased toward A and T bases which unavoidably leads to the predominance of certain codons and amino acids in proteins.

"Niagara strain" MT genome contains set of 30 PCGs (atpl, 4, 6, 8, 9, cob,, cox1-3, nad1-7, 9, 11, nad4L, rpland rpsgenes), 25 tRNA, two rRNA genes (rrnL and rrnS) and 13 open reading frames (ORFs) (Table 2). This MT genome in gene content is more complete than other sequenced MT genomes of vannellids (Bondarenko et al. ,2018a, 2018b, 2018c). The set ofPCGs genes differs by the presence of nad7 and nad9 genes and includes all atp genes. The set of rpl and rps genes in the MT genome of this strain also differs from that in other known Amoebozoa

Table 1. LNucleotide composition of the mitochondrial genome of Niagara strain.

AT% GC% A% T% G% C% AT-skew GC-skew

72,9 27,1 33,7 39,2 15,4 11,7 -0,076 0,135

MT genomes (Bondarenko et al. 2018a, 2018b, 2018c; Burger et al., 1995; Greninger et al., 2015; Ogawa et al., 2000; Tanifuji et al., 2017). Similar to the Clydonella sawyeri MT genome, fifty-three genes and thirteen ORFs are located on H-strand except for two tRNA genes on L-strand (Bondarenko et al., 2018c). The total length of all PCGs in the Niagara strain MT genome, excluding termination codons, is 28.135 bp which amounts to 53,16% of the total genome length. All genes in the MT genome of this strain contain no introns. All ORFs are unique to this MT genome and have no homologs among Vannellidae species as well as among other Amoebozoa. This situation is quite common, and in many other MT genomes of Amoebozoa we also met unique ORFs (Bondarenko et al., 2019b). As a remarkable character, it contains two rather long ORFs with lengths 2508 and 2739 bp (ORF 13 and 10, respectively). MT genome of Niagara strain has two small gene overlaps and eleven non-coding regions longer than 100 bp (Table 2). The largest overlap is 14 bp and is located between ORF2 and rps12. The non-coding regions constitute 3583 bp in total and 6,7% of the total MT genome size (Table 2) which is similar to the Paravannella minima MT genome (Bondarenko et al., 2019a). The largest non-coding region is 474 bp long and located between tRNA^1 and nad11.

There are two alternative start codons in the "Niagara strain" MT genome. Most of PCGs and ORFs use ATG as a start codon, and only nad11 use ATT. There are only two stop codons in the "Niagara strain" MT genome (TAA and TAG); TGA stop codon was not found in this mt genome. Similar to the Paravannella minima MT genome, the "Niagara strain" MT genome does not have TAA stop codons within CDS, which leads to reading frameshifts in the MT genomes of otherVannellidae mt genomes (Bondarenko et al., 2018a, 2018b, 2018c). Similar to most sequenced MT genomes ofAmoebozoa, the Niagara strain uses the genetic code 4.

The large ribosomal RNA (rrnL) gene in the "Niagara strain" MT genome is located between ORF13 and nad6 gene, and the small ribosomal RNA (rrnS) is situated between tRNALys and cox1 genes (Fig. 2). The length of rrnL and rrnS is 2838 bp and 1634 bp, respectively. tRNA genes have

a total length of 1895 bp, and most of them are located between ORF12 and rrnS genes. All tRNAs have the typical cloverleaf secondary structure. This MT genome contains additional arginine, serine, tyrosine, three leucine, and methionine tRNA genes. Among known Amoebozoa MT genomes, only Phalansterium sp. and Clydonella sawyeri have three methionine tRNA genes (Bondarenko et al., 2018c; Pombert et al., 2013). As for leucine, no one known amoebozoan MT genome has three tRNA genes. We observed the difference in the nucleotide composition between tRNALeu1 located in the L-strand and the other two duplications of this gene located on the H-strand. These differences in nucleotide composition and location of these duplications suggest about ancient nature of tRNALeu1 duplication. tRNA^, tRNASer, and tRNAMet duplications also have ancient nature. In contrast, tRNATyr gene duplication occurs for the first time. The copies of these duplicated genes show a small difference in the nucleotide composition indicating the "young" nature of this duplication.

The MT genome of "Niagara strain" shows no evidence of post-translational editing. The same is true for the recently sequenced species Paravannella minima (Bondarenko et al., 2019a). However, little editing was found in another lineage — Clydonella sawyeri (Bondarenko et al. 2018c) and extensive editing — in the species belonging to the genus Vannella — the crown lineage ofVannellida, namely

— Vannella croatica and V. simplex (Bondarenko et al., 2018a, 2018b). Among other amoebozoan lineages, RNA editing is known among plasmodial slime molds - Myxogastria (see Houtz et al., 2018)

— the group belonging to Evosea, which is the crown group of the entire Amoebozoa tree. Discosea is located more basally (Kang et al., 2017). At the same time, among Discosea lineage, no editing was found in the MT genomes of the genera Paramoeba and Neoparamoeba (Tanifuji et al., 2017; Bondarenko et al., 2020). Both belong to Dactylopodida clade, which in the phylogenetic tree of Discosea is more basal than the Vannellida clade. So, the present finding further confirms the suggestion on the possibility of the independent origin of editing in individual amoebozoan lineages (Bondarenko et al., 2019b).

Table 2. Organization of Niagara strain mitochondrial genome.

Gene Strand Location Size (bp) Anticodon Start Stop Intergenic nucleotides

nad2 + 22-1899 1878 ATG TAA 36

rps2 + 1903-3033 1131 ATG TAA 3

tRNALeu1 - 3036-3114 79 TAA 2

tRNAPhe - 3139-3211 73 GAA 24

rps4 + 3233-4981 1749 ATG TAA 21

ORF1 + 4985-5239 255 ATG TAA 3

tRNAHis + 5463-5535 73 GTG 223

atp9 + 5660-5914 255 ATG TAA 124

tRNASer1 + 5937-6023 87 GCT 22

tRNASer2 + 6059-6143 85 TGA 35

cob + 6409-7713 1305 ATG TAG 265

rpl11 + 7727-8149 423 ATG TAA 13

ORF2 + 8156-8677 522 ATG TAA 6

rps12 + 8664-9053 390 ATG TAA -14

ORF3 + 9113-11011 1899 ATG TAA 59

rpl2 + 11015-11733 759 ATG TAA 3

ORF4 + 11775-12044 270 ATG TAA 1

rps3 + 12054-13505 1452 ATG TAA 9

rpl16 + 13505-13924 418 ATG TAA -1

ORF5 + 13946-15529 1584 ATG TAA 41

rpl14 + 15569-15937 369 ATG TAA 39

ORF6 + 15948-16517 570 ATG TAA 10

rps14 + 16526-16825 300 ATG TAA 8

rps8 + 16836-17219 384 ATG TAG 10

ORF7 + 17222-17758 537 ATG TAA 2

rps13 + 17763-18140 378 4

ORF8 + 18162-18929 768 ATG TAA 21

tRNAPro + 18952-19024 73 TGG 22

ORF9 + 19060-19566 507 ATG TAA 35

atp8 + 19707-20090 384 ATG TAA 140

atp4 + 20092-21051 960 ATG TAA 1

tRNAAsp + 21065-21138 74 GTC 13

nad3 + 21338-21877 540 ATG TAA 199

nad9 + 21905-22648 744 ATG TAA 27

nad7 + 22650-23867 1218 ATG TAA 1

Table 2. Continuation.

atp6 + 23891-24853 963 ATG TAA 23

ORFIO + 24864-27602 2739 ATG TAA 10

atpl + 27813-29303 1491 ATG TAA 210

tRNA11 e + 29322-29394 73 CAT 18

tRNAArsl + 29432-29504 73 ACG 37

nadll + 29979-32105 2127 ATT TAA 474

nadl + 32109-33218 1110 ATG TAA 3

tRNAVal + 33299-33372 74 TAC 80

ORF11 + 33409-33750 342 ATG TAA 36

tRNAAsn + 33761-33832 72 GTT 10

tRNAArs2 + 33860-33933 74 TCT 27

tRNAJy" + 34000-34082 83 GTA 66

ORF12 + 34118-35572 1455 ATG TAA 35

tRNAGU + 35628-35699 72 TTC 55

tRNA№" + 35722-35793 72 CAT 22

tRNA№t2 + 35826-35898 73 CAT 32

tRNALeu2 + 36055-36136 82 TAG 156

tRNATrp + 36150-36222 73 CCA 13

tRNAAla + 36267-36338 72 TGC 44

tRNACys + 36351-36421 71 GCA 12

tRNALeu3 + 36438-36522 85 CAA 16

tRNAGln + 36540-36612 73 TTG 17

tRNALys + 36630-36701 72 TTT 17

rrnS + 36736-38369 1634 ATG TAA 34

coxl + 38488-40056 1569 ATG TAA 118

cox3 + 40197-41048 852 ATG TAG 140

ORFl3 + 41093-43600 2508 ATG TAA 44

rrnL + 43629-46466 2838 ATG TAA 28

nad6 + 46677-47711 1035 ATG TAG 210

cox2 + 47762-48601 840 ATG TAA 50

tRNA№t3 + 48625-48698 74 CAT 23

nad4l + 48736-49032 297 ATG TAA 37

nad5 + 49055-51172 2118 ATG TAA 22

tRNATyr2 + 51198-51280 83 GTA 25

nad4 + 51313-52908 1596 ATG TAA 32

Acknowledgments

Supported by the RSF project 20-14-00195. This study utilized equipment of the Core Facilities Centers "Culture Collection of Microorganisms," "Centre for Molecular and Cell Technologies," "Biobank," and "Computing Centre SPbU" of the Research Park ofSaint-Petersburg State University.

References

Bankevich A., Nurk S., Antipov D., Gurevich A. et al. 2012. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455-477. https://doi.org/ 10.1089/cmb.2012.0021

Bernt M., Donath A., Jühling F., Externbrink F. et al. 2013a. MITOS: improved de novo meta-zoan mitochondrial genome annotation. Mol. Phylogenet. Evol. 69, 313-319. https://doi.org/ 10.1016/j.ympev.2012.08.023

Bernt M., Bleidorn C., Braband A., Dambach J. et al. 2013b. A comprehensive analysis of bila-terian mitochondrial genomes and phylogeny. Mol. Phylogenet. Evol. 69, 352-364. https://doi.org/ 10.1016/j.ympev.2013.05.002

Bondarenko N.I., Nassonova E.S., Mijanovic O., Glotova A.A. et al. 2018a. Mitochondrial genome of Vanmlla croatica (Amoebozoa, Discosea, Van-nellida). J. Eukaryot. Microbiol. 65, 820-827. https:// doi.org/doi:10.1111/jeu.12523

Bondarenko N., Glotova A., Nassonova E., Masharsky A., Kudryavtsev A. and Smirnov A. 2018b. The complete mitochondrial genome of Vannella simplex (Amoebozoa, Discosea, Vannellida). Europ. J. Protistol. 63, 83-95. https://doi.org/ 10.1016/j.ejop.2018.01.006

Bondarenko N., Glotova A., Kamyshatskaya O., Mesentsev Y. et al. 2018c. The complete mito-chondrial genome of Clydonella sawyeri (Amo-ebozoa, Discosea, Vannellida). Protistology. 12, 47-54. https://doi.org/10.21685/1680-0826-2018-12-1-4

Bondarenko N., Glotova A., Nassonova E., Masharsky A., Polev D. and Smirnov A. 2019a. The complete mitochondrial genome of Paravannella minima (Amoebozoa, Discosea, Vannellida). Europ. J. Protistol. 68, 80-87. https://doi.org/10.1016/j. ejop.2019.01.005

Bondarenko N., Smirnov A, Nassonova E., Glotova A. and Fiore-Donno Anna-Maria. 2019b.

Mitochondrial genomes of Amoebozoa. Protistology. 13, 179-191. https://doi.org/10.21685/1680-0826-2019-13-4-1

Bondarenko N., Volkova E., Masharsky A., Kudryavtsev A. and Smirnov A. 2020. A comparative characterization of the mitochondrial genomes of Paramoeba aparasomata and Neoparamoeba pema-quidensis (Amoebozoa, Paramoebidae). J. Eukaryot. Microbiol. 67, 167-175. https://doi.org/10.1111/ jeu.12767

Burger G., Plante I., Lonergan K.M. and Gray M.W. 1995. The mitochondrial DNA of the amoeboid protozoon, Acanthamoeba castellanii: complete sequence, gene content and genome organization. J. Mol. Biol. 245, 522-537. https://doi. org/10.1006/jmbi.1994.0043

Houtz J., Cremona N. and Gott J.M. 2018. Editing of mitochondrial RNAs in Physarum poly-cephalum. In: RNA metabolism in mitochondria (Eds Cruz-Reyes J. and Gray M.W.). Springer, Cham. pp. 199-222. https://doi.org/10.1007/978-3-319-78190-7_8

Greninger A.L., Messacar K., Dunnebacke T., Naccache S.N. et al. 2015. Clinical metagenomic identification of Balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing. Genome Med. 7, 113. https://doi.org/10.1186/s13073-015-0235-2

Kang S., Tice A.K., Spiegel F.W., Silberman J.D. et al. 2017. Between a pod and a hard test: the deep evolution of amoebae. Mol. Biol. Evol. 34, 2258-2270. https://doi.org/10.1093/molbev/ msx162

Kudryavtsev A. and Volkova E. 2018. Clydonella sawyeri n. sp. (Amoebozoa, Vannellida): Morphological and molecular study and a re-definition ofthe genus Clydonella Sawyer, 1975. Europ. J. Protistol. 63, 62-71. https://doi.org/10.1016/j.ejop. 2018.01.008

Lowe T.M. and Eddy S.R. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955-964. https://doi.org/10.1093/nar/25.5.955

Ogawa S., Yoshino R., Angata K., Iwamoto M. et al. 2000. The mitochondrial DNA of Dictyostelium discoideum: complete sequence, gene content, and genome organization. Mol. Gen. Genet. 263, 514519. https://doi.org/10.1007/PL00008685

Perna N.T. and Kocher T.D. 1995. Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes. J. Mol. Evol. 41,

353-358. https://doi.org/10.1007/BF00186547

Pombert J.-F., Smirnov A., James E.R., Jan-ouskovec J., Gray M.W. and Keeling P.J. 2013. The complete mitochondrial genome from an unidentified Phalansterium species. Protist Genomics. 1, 25-32. https://doi.org/10.2478/prge-2013-0002 Rutherford K., Parkhill J., Crook J., Horsnell T. et al. 2000. Artemis: sequence visualization and annotation. Bioinformatics. 16, 944-945. https:// doi.org/10.1093/bioinformatics/16.10.944

Smirnov A.V., Nassonova E.S., Chao E. and Cavalier-Smith T. 2007. Phylogeny, evolution, and taxonomy ofvannellid amoebae. Protist. 158, 295324. https://doi.org/10.1016/j.protis.2007.04.004 Tanifuji G., Cenci U., Moog D., Dean S. et al. 2017. Genome sequencing reveals metabolic and cellular interdependence in an amoeba-kine-toplastid symbiosis. Sci. Rep. 7, 11688. https://doi. org/10.1038/s41598-017-11866-x

Address for correspondence: Natalya I. Bondarenko. Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, Universitetskaya Emb. 7/9, 199034 St. Petersburg, Russia; e-mail: [email protected]