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Protistology 16 (2): 135-142 (2022) | doi:10.21685/1680-0826-2022-16-2-7 Pl'OtiStOlO&y
Original article
Mitochondrial genome of Thecamoeba quadrilineata - the first mt genome among the representatives of the order Thecamoebida (Amoebozoa, Discosea)
Natalya I. Bondarenko1*, Elena S. Nassonova2, Yelisei S. Mesentsev1, and Alexey V. Smirnov1
1 Department of Invertebrate 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
| Submitted October 5, 2021 | Accepted December 8, 2021 |
Summary
We have sequenced and described the mitochondrial genome of Thecamoeba quadrilineata (Amoebozoa, Discosea, Thecamoebida). This is the first sequenced mitochondrial genome for amoebae of Thecamoebida lineage. The circular mitochondrial DNA of this species has 50942 bp in length and contains 23 protein-coding genes, 2 ribosomal RNAs, 18 transfer RNAs, and 26 open reading frames. In contrast with the shorter mitochondrial genomes of vannellid amoebae, it shows no evidence of RNA editing. This finding supports the hypothesis on the multiple origins of editing in different phylogenetic lineages of Amoebozoa.
Key words: Amoebozoa, Thecamoebida, mitochondrion, mitochondrial genome
Abbreviations: mt — mitochondria; cox1-3 — cytochrome oxidase subunit I, II, and III genes; tRNA — transfer RNA genes; rrnL, rrnS — ribosomal RNA genes; ORFs — open reading frames; PCGs — protein-coding genes; rps — small ribosomal subunit protein genes; rpl — large ribosomal subunit protein genes
Introduction
Mitochondrial genomes (mt genomes) ofAmoe-bozoa are sequenced for a limited number of phylogenetic lineages. The majority ofknown mt genomes belong to Centramoebida, Eumycetozoa and Van-nellida lineages; there are only a few genomes belonging to the representatives of other Amoebozoa clades. Until now, far not all phylogenetic lineages have been sampled (reviewed by Bondarenko et al.,
https://doi.org/10.21685/1680-0826-2022-16-2-7
© 2022 The Author(s)
Protistology © 2022 Protozoological Society Affiliated with RAS
2019a). The mitochondrial genome of Amoebozoa shows significant differences in size and gene composition. While closely related amoebozoan species show almost identical mt genomes (Bondarenko et al., 2019b; Karlyshev, 2019), the level ofmt genome synteny drops dramatically as the phylogenetic distance increases (Bondarenko et al., 2018a, 2018b, 2018c). Many of the sequenced mt genomes show extensive RNA editing, and among them the representative of Myxogastria — namely, Physarum
Corresponding author: Natalya I. Bondarenko. Department oflnverte-brate Zoology, Faculty ofBiology, St. Petersburg State University, Univer-sitetskaya Emb. 7/9, 199034 St. Petersburg, Russia; n.bondarenko@spbu.ru
Fig. 1. Light microscopy of Thecamoeba quadrilineata strain CCAP 1583/10. A-C — Locomotive forms; D and E — higher magnification of the cell showing the granuloplasm and the nucleus. Abbreviations: cv — contractile vacuole, f — dorsal fold, n — nucleus, nu — nucleolus. Scale bars: A-C — 10 ^m, D and E — 5 ^m.
polycephalum — demonstrates one of the most complex and extensive patterns of RNA editing among eukaryotes (Takano et al., 2001; Houtz et al., 2018). However, lineages showing extensive editing are often located in the crown of the tree, while representatives of basal clades may show little or no editing (Bondarenko et al., 2019c). These data vote for the independent origin of editing in different lineages of Amoebozoa. It makes the study of the distribution, patterns and mechanisms of RNA editing among Amoebozoa especially fascinating and important and may lead to new findings in this field. However, these studies are seriously limited by the absence of a comprehensive picture of the distribution of RNA editing phenomena across the Amoebozoa tree.
The present paper reports data on the mitochondrial genome of Thecamoeba quadrilineata (Carter, 1856) Lep§i, 1960. This species shows no RNA editing. Ifwe assume the current phylogeny of Discosea clade (Kang et al., 2017; Melton et al., 2018), our finding further evidences for the independent origin of editing in different Amoebozoa lineages.
Material and methods
The studied strain Thecamoeba quadrilineata CCAP 1583/10 was obtained from Dr. RolfMichel. He maintained it as a host for Nucleophaga amoebae strain KTq2 (Michel, 2008; Michel et al., 2009). The culture was maintained on NN agar (Panreac agar-agar, American Type QB, Spain) as described by Page (1988) made on PJ medium (Prescott and James, 1955) on accompanying bacteria. Live cells
were photographed on object slides (wet mounts in PJ medium) using a Leica DM2500 microscope (Fig. 1). The SSU sequence of this strain was obtained by Claudia Wylezich and deposited in GenBank under the number DQ122381 (see Walochnik et al., 2003; Michel et al., 2006; Kamyshatskaya et al., 2018).
To obtain a DNA sample, individual amoebae cells were collected from culture dishes using a tapered-tip Pasteur pipette, washed three times in Millipore-sterilized (0.2 ^m pore) PJ medium, and placed with 1—2 ^l of the medium in 200-^l PCR tubes. DNA was extracted using the Arcturus PicoPure DNA Extraction Kit (Thermo Fischer Scientific, USA). The extraction mixture was prepared according to the manufacturer's instructions; 10 ^l of the mixture was added to the tube containing the single cell. Further, we performed whole genome amplification using the REPLI-g Single Cell DNA Amplification Kit (Qiagen, Hilden, Germany). Multiple Displacement Amplification (MDA) was performed according to the manufacturer's instructions. Approximately 127 million reads with a length of 150 bp were obtained using HiSeq 2500 sequencing system (Illumina). Quality control checking 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 the 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
Table 1. Nucleotide composition characteristics of Thecamoeba quadrilineata
mitochondrial genome.
Species GC% A% T% G% C% AT-skew GC-skew
Thecamoeba quadrilineata 23.92 41.06 35.02 13.82 10.10 0.079 0.155
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 of the mt genome was generated by our original script written in Python.
Results and discussion
The mitochondrial genome of Thecamoeba quadrilineata is a double-stranded circular DNA molecule with a length of 50942 bp (Fig. 2). It is the first sequenced mt genome among amoebae belonging to Thecamoebida lineage (order Thecamoebi-da). It shows GC content of23.92% (Table 1), which is a rather low level. The prevalence of adenine over thymine and guanine over cytosine in the majority strand provides both positive AT-skew and GC-skew. This picture ofAT-skew resembles the one observed in many other organisms (e.g., Bernt et al., 2013b; Bondarenko et al., 2019b). Therefore, the nucleotide composition of Thecamoeba quadrilineata mt genome is significantly biased toward A and T bases, which unavoidably leads to the predominance of certain codons and amino acids in proteins.
Thecamoeba quadrilineata mt genome contains the set of 23 PCGs (atpl, 6, 9, cob, cox1-3, nadl-5,7,9,11, rpl and rps genes), 18 tRNA, two rRNA genes (rrnL and rrnS) and 28 open reading frames (ORFs) (Table 2). All genes and ORFs are located on H-strand.
The set of PCG genes differs from other known Amoebozoa mt genomes by the absence of nad4l, nad5, nad6and cob genes. BLAST search for nad4l provides a set of low-significant matches in the region cor-responding to orf9 (Table 2, Fig. 2). The set of rpl and rps genes in the mt genome ofthis strain also differs from that in other known Amoebozoa mt genomes (Burger et al., 1995; Ogawa et al., 2000; Greninger et al., 2015; Tanifuji et al., 2017; Bondarenko et al., 2018a, 2018b, 2018c). The total
length of all PCGs in Thecamoeba quadrilineata mt genome, excluding termination codons, is 22022 bp, which constitutes 43.2% of the total genome length. It is due to the fact that over 40% of the mt genome is occupied by ORFs (total length is 20460 bp) and the remaining 6.5% are tRNA, rRNA and intergenic regions. This mt genome of Thecamoeba quadrilineata differs from other known Amoebozoa mt genomes by high density in genes and ORFs. The mt genome of this species has a significant number of gene overlaps and only two non-coding regions longer than 100 bp (Table 2). The largest overlap is 44 bp; it is located between orf7 and orf8. The non-coding regions constitute 1398 bp in total and 2.74% of the total mt genome size (Table 2). The largest non-coding region is 454 bp long; it is located between tRNAHis and orf5. Also, the mt genome contains two rather long ORFs with lengths 2163 and 3216 bp (orf3 and of 7, respectively). All ORFs are unique to this mt genome and have no homologs among other Amoebozoa.
There are two alternative start codons in Thecamoeba quadrilineata mt genome. Most PCGs and ORFs use ATG as a start codon, while orf8 uses ATA, and orf15 uses ATT as a start codon. There are only two stop codons in Thecamoeba quadrilineata mt genome (TAA and TAG); TGA stop codon was not found in this mt genome. In contrast to most of sequenced mt genomes of Amoebozoa, mt genome in Thecamoeba quadrilineata uses the genetic code 1 (the standard code).
The large and the small ribosomal RNA genes (rrnL and rrnS) in Thecamoeba quadrilineata mt genome are located close to each other between tRNAMetand tRNAAla (Table 2, Fig. 2). The length of rrnL and rrnS is 2874 bp and 1911 bp, respectively. tRNA genes have a total length of1191 bp, and most of them are located between rrnS and of5 genes. All tRNAs have the typical cloverleaf secondary structure. This mt genome contains additional arginine, serine, and three methionine tRNA genes. These features are not unique. Three methionine tRNA genes are also known in other Amoebozoa mt genomes (Pombert et al., 2013; Bondarenko et al., 2018c, 2021). We observed significant differences in the nucleotide composition between tRNAMet and the
Fig. 2. Mitochondrial genome map of Thecamoeba quadrilineata. The tRNA genes are labeled based on the IUPACIUB single-letter amino acid codes.
other two duplicates of this gene. These differences and location of obtained duplications suggest the ancient nature of tRNAMet duplication compared with tRNAArgg and tRNASer, which show much smaller differences in the nucleotide composition between the duplicates of these genes.
If we accept the current phylogeny of Discosea clade as revealed by Kang with coauthors (2017) and Melton with coauthors (2018), our finding further evidences for the independent origin of editing in different Amoebozoa lineages. Among the lineages constituting the Flabellinia clade, up
to now editing is known in Vannellida. Available data on Dactylopodida clade (sister clade to Vannellida) show no evidence of RNA editing in sequenced mitochondrial genomes (Bondarenko et al., 2019c). Yet, several sequences of the Cox I gene of Korotnevella spp. deposited with the GenBank suggest the presence of frameshift in poly-T areas. Also, it might be a sequencing problem (Zlatogurski et al. 2016). Another case of RNA editing is known in Acanthopodida, which belongs to Centramoebia clade (Burger et al., 1995; Fucikova and Lahr, 2016). The present finding is the first evidence
Table 2. Thecamoeba quadrilineata mitochondrial genome organization.
Gene Strain Position (start-stop) Length (bp) Intergenic space (bp) Start codon Stop codon
orf1 + 110-799 690 209 ATG TAG
rpl2 + 803-1807 1005 -3 ATG TAA
orf2 + 1788-2153 366 -20 ATG TAA
rps3 + 2159-4249 2091 -5 ATG TAA
rpl16 + 4242-4709 468 -8 ATG TAA
orf3 + 4710-6872 2163 0 ATG TAA
orf4 + 6875-7678 804 2 ATG TAA
trnE + 8771-8841 71 -8
trnM1 + 8860-8931 72 18
rrnL + 9077-11950 2874 45
rrnS + 11978-13890 1911 27
trnA + 13890-13961 72 -1
trnQ + 13970-14041 72 8
trnP + 14048-14119 72 6
trnK + 14125-14197 73 5
trnW + 14214-14284 71 16
trnS1 + 14291-14375 85 6
trnD + 14381-14453 72 5
trnS2 + 14463-14547 85 9
trnY + 14555-14637 83 7
trnC + 14644-14714 71 6
trnM2 + 14719-14790 72 4
trnF + 14795-14867 73 4
trnR1 + 14870-14944 75 2
trnR2 + 14944-15015 72 -1
trnM3 + 15027-15098 72 11
trnH + 15119-15189 71 20
orf5 + 15653-16015 363 454
orf6 + 16140-17657 1518 124 ATG TAA
nad1 + 17662-18675 1014 4 ATG TAA
orf7 + 18770-21961 3192 -6 ATG TAA
orf8 + 21918-22748 831 -44 ATA TAA
orf9 + 22773-23111 339 23 ATG TAA
cox3 + 23160-24005 846 48 ATG TAA
orf10 + 24025-24597 573 19 ATG TAA
orf11 + 24664-25818 1155 66 ATG TAA
nad3 + 25824-26237 414 5 ATG TAA
orf12 + 26221-26967 747 -17 ATG TAA
atp9 + 27042-27278 237 74 ATG TAG
rps4 + 27302-28039 738 23 ATG TAA
orf13 + 28052-28885 834 12 ATG TAA
orf14 + 28892-29713 822 6 ATG TAA
atp1 + 29790-31397 1608 76 ATG TAG
orf15 + 31402-32187 786 4 ATG TAA
cox1 + 32224-34326 2123 36 ATG TAA
orf16 + 34289-34744 456 -38 ATT TAA
Table 2. (Continuation).
orf17 + 34752-36962 2211 7 ATG TAG
nad4 + 36998-38449 1452 35 ATG TAA
orf18 + 38453-39187 735 3 ATG TAG
nad2 + 39194-40735 1542 6 ATG TAA
atp6 + 40790-41830 1041 54 ATG TAA
rpl14 + 41834-42262 429 3 ATG TAA
orf19 + 42282-42509 228 19 ATG TAA
orf20 + 42499-43095 597 -11 ATG TAA
orf21 + 43088-43936 849 -8 ATG TAA
orf22 + 43942-44199 258 5 ATG TAA
orf23 + 44206-44550 345 6 ATG TAA
orf24 + 44555-45169 615 4 ATG TAA
orf25 + 45172-45471 300 2 ATG TAA
rps8 + 45480-45854 375 8 ATG TAA
orf26 + 45859-46434 576 4 ATG TAA
rps13 + 46436-46918 483 1 ATG TAA
nad9 + 46930-47559 630 11 ATG TAA
nad7 + 47546-48727 1182 -14 ATG TAA
rpl11 + 48733-49386 654 5 ATG TAA
orf27 + 49379-50011 633 -8 ATG TAG
rps12 + 49972-50358 387 60 ATG TAG
orf28 + 50330-50842 513 -29 ATG TAA
for the absence of editing in Thecamoebida. The Thecamoebida clade belongs to Flabellinia, but branches earlier than Dactylopodida/Vannellida clade (Kang et al., 2017; Melton et al., 2018). So, if the absence of editing in Thecamoebida is further confirmed with sequences or a larger number of mt genomes, it would prove that the point of origin of RNA editing in Vannellida, belonging to Flabellinia clade, is isolated in the phylogenetic tree from that in Acanthopodida, which belongs to Centramoebia clade.
Acknowledgments
The research was supported by the RSF project 20-14-00195. This study utilized equipment of the Core Facilities Centers "Centre for Culture Collection of Microorganisms," "Centre for Molecular and Cell Technologies," "Biobank," and "Computing Centre" of the Research Park of St Petersburg State University. This paper is dedicated to the Year of Zoology 2022 in St. Petersburg State University.
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