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Protistology 12 (1), 47-54 (2018)
Protistology
The complete mitochondrial genome of Clydonella sawyeri (Amoebozoa, Discosea, Vannellida)
Natalya Bondarenko1, Anna Glotova1, Oksana Kamyshatskaya2, Yelisei Mesentsev1, Kirill Lotonin1, Alexey Masharsky3, Dmitry Polev4, Elena Nassonova15 and Alexey Smirnov1
1 Department oflnvertebrate Zoology, Faculty of Biology, St. Petersburg State University, 199034 St. Petersburg, Russia
2 Core Facilities Center "Culture Collection of Microorganisms", St. Petersburg State University, Stary Peterhof, 198504 St. Petersburg, Russia
3 Core Facility Centre for Molecular and Cell Technologies, St. Petersburg State University, Stary Peterhof, 198504 St. Petersburg, Russia
4 Core Facility Centre Biobank, St. Petersburg State University, Stary Peterhof, 198504 St. Petersburg, Russia
5 Laboratory of Cytology of Unicellular Organisms, Institute of Cytology RAS, 194064 St. Petersburg, Russia
| Submitted February 21, 2018 | Accepted April 10, 2018 |
Summary
We describe the complete sequence and organization of the mitochondrial genome from a brackish-water amoeba Clydonella sawyeri (Amoebozoa, Discosea, Vannellida). The circular mitochondrial DNA of this species has 31,131 bp in length and contains 17 protein-coding genes, 2 ribosomal RNAs, 21 transfer RNAs and 13 open reading frames. Length and gene content distinguish mitochondrial genome of Clydonella sawyeri from the mitochondrial genomes of other Amoebozoa species.
Key words: Amoebozoa, Vannellidae, Clydonella, mitochondrion, mitochondrial genome
Introduction
Mitochondrial genomes (mt genomes) are an important instrument for the phylogenetic studies because of their accessibility and higher evolutionary rate compared to the nuclear DNA (Castro et al., 1998; Lang et al., 1999; Gray et al. 1999). There is much data on the mt genomes in
many groups of organisms (Tan et al., 2017), while among Amoebozoa mitochondrial genomes remain relatively poorly studied. By now sequences of mt genomes for 13 amoebozoan species are available (see Bondarenko et al., 2018a, Table S1; 2018b). This dataset is dominated with the species that can be grown in axenic culture or in pure mass culture, like Acanthamoeba, Balamuthia, and Dictyostelium,
doi: 10.21685/1680-0826-2018-12-1-4 © 2018 The Author(s)
Protistology © 2018 Protozoological Society Affiliated with RAS
and it does not yet cover even all the major branches of Amoebozoa.
The evolutionary change of the gene order in the mt genomes of Amoebozoa appears to be rather rapid and the level of synteny between genomes may be rather low in different amoebozoan lineages (Heidel and Glöckner, 2008) and even among the closely related species belonging to the same phylogenetic lineage (Bondarenko et al., 2018b). The reasons for this are not yet clear. To understand this problem better we subsequently sequence mitochondrial genomes of the amoebae of the order Vannellida (Amoebozoa, Discosea), aiming to cover all major phylogenetic branches in this large and diverse group ofnaked lobose amoebae. The present paper reports data on the mitochondrial genome of the recently described species Clydonella sawyeri, the first Clydonella species, fully characterized both by the morphological and the molecular methods (Kudryavtsev and Volkova, 2018).
Material and methods
The type culture of Clydonella sawyeri isolated from the upper part ofthe littoral zone ofSeldyanaya Bay, Chupa Inlet (Kandalaksha Bay, The White Sea, North-Western Russia) was used for this study (Kudryavtsev and Volkova, 2018). Amoebae were cultured in 90 mm Petri dishes filled with Millipore-sterilized (0.2 ^m pore) artificial seawater (25%c) 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 manufacturer's instructions. Approximately 1.7 million reads with length 25-595 bp were obtained using Ion Torrent PGM system (Life Technologies). 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 MITOS web server (Bernt et al., 2013a). Artemis was used for visualization of 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. The C. sawyeri mitochondrial genome has been deposited in GenBank under the accession number MH094141.
Results and discussion
Mitochondrial genome of C. sawyeri is a double-stranded circular DNA molecule with the length of 31,131 bp (Fig. 1). Thus, it further completes the list ofthe relatively small (less than 40 kbp) mt genomes among Amoebozoa. Two other small mt genomes also belong to amoebae of the family Vannellidae (Bondarenko et al., 2018a, 2018b). Mitochondrial genome of C. sawyeri has GC content 24,5% (Table 1), which is a relatively 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). The nucleotide composition of C. sawyeri mitochondrial genome is significantly biased toward A and T bases which leads to the predominance of certain codons and amino acids in proteins (Table 2).
Clydonella sawyeri mt genome contains set of 17 PCGs (atp1, 9, cob, cox1-3, nad1-6, nad4L, rpl and rps genes), 21 tRNA, two rRNA genes (rrnL and rrnS) and 13 open reading frames (ORFs) (Table 3). The set of rpl and rps genes in C. sawyeri mt genome differs from that in Vannella simplex and Vannella croatica mt genomes (Bondarenko et al., 2018a, 2018b). The set of PCGs genes differs by absence of nad11 and presence of atp1 genes. Thirty-eight genes and thirteen ORFs are located on H-strand except for two tRNA genes on L-strand. The total length of all PCGs in C. sawyeri mt genome, excluding termination codons, is 15.065 bp. All genes in C. sawyeri mt genome contain no introns.
Table 1. Nucleotide composition characteristics of the mitochondrial genome of Clydonella sawyeri.
AT% GC% A% T% G% C% AT-skew GC-skew
75,5 24,5 31,9 43,6 14,6 9,9 -0,155 0,194
Fig. 1. Clydonella sawyeri mitochondrial genome map. The tRNA genes are labeled based on the IUPAC-IUB single letter amino acid codes.
Mitochondrial genome of C. sawyeri has five small gene overlaps and five non-coding regions longer than 100 bp. The largest overlap is 608 bp and located between ORF11 and ORF12. The non-coding regions constitute 3225 bp in total and 10,35% of the total mt genome size (Table 3). The largest non-coding region is 715 bp long and located between tRNALys and atp9.
The large ribosomal RNA (rrnL) gene in C. sawyeri is located between cox1 and cox2 genes and small ribosomal RNA (rrnS) between tRNAArg and
tRNATrp genes (Fig. 1). The length of rrnL and rrnS is 2789 bp and 1767 bp, respectively. tRNA genes have a total length of 1570 bp and most of them are located between ORF2 and rrnS genes. All tRNAs have the typical cloverleaf secondary structure. tRNA genes are better represented in mt genome of C. sawyeri as compared to other mt genomes of vannellids (Bondarenko et al., 2018a, 2018b). Its genome contains additional arginine, leucine, serine and three methionine tRNA genes (Fig. 2).
The majority of mitochondrial genomes in-
Fig. 2. Predicted secondary structure of the tRNAMet and tRNAAg genes of Clydonella sawyeri.
Table 2. Amino acid composition of genes and ORFs located in the Clydonella sawyeri
mitochondrial genome.
nad1 nad2 nad4l nad4 nad5 nad6 cob atp1 atp9 cox1 cox2 cox3 rps12 rps7 rps19 rpl12 rpl6
Ala 20 18 8 19 30 8 21 38 11 33 11 19 8 5 1 9 1
Arg 5 6 3 9 11 4 10 24 1 8 6 7 12 6 2 19 6
Asn 11 17 7 27 23 16 15 30 2 16 22 5 7 17 9 15 1
Asp 7 13 3 9 19 4 9 28 1 16 16 4 2 5 0 4 5
Cys 3 5 0 9 13 0 3 2 1 2 4 4 2 3 2 5 1
Gln 4 4 1 8 7 3 7 21 1 6 5 5 6 3 2 2 1
Glu 11 11 2 9 12 4 7 25 3 9 16 6 1 6 3 6 4
Gly 19 19 5 27 43 8 27 44 10 49 11 18 9 5 4 24 4
His 2 6 0 6 13 3 11 4 0 15 7 10 1 1 0 5 1
Ile 31 48 23 55 64 35 38 39 7 50 33 12 7 15 6 21 8
Leu 44 68 18 81 97 31 52 58 13 55 32 28 7 15 8 27 12
Lys 8 23 2 13 27 8 7 36 3 8 14 3 28 27 19 32 43
Met 10 5 4 17 20 6 8 6 3 22 8 4 1 3 2 3 1
Phe 35 89 11 82 117 30 40 24 10 51 27 29 5 7 12 20 34
Pro 13 13 0 15 14 2 24 16 1 21 12 8 6 5 2 8 0
Ser 25 48 6 39 56 19 21 44 7 30 18 18 7 6 7 30 6
Thr 8 13 1 24 23 4 16 15 4 27 11 12 6 2 2 8 3
Trp 5 3 1 9 7 1 11 1 0 12 6 8 0 2 0 2 0
Tyr 10 31 2 28 28 4 22 16 1 26 15 13 1 2 2 6 6
Val 18 25 7 25 39 17 34 44 4 29 21 15 13 5 4 15 3
Table 3. Organization of Clydonella sawyeri mitochondrial genome.
Gene Strand Location Size (bp) Anticodon Start Stop Intergenic nucleotides
atpl + 37-1584 1548 ATG TAA 81
tRNAPhe + 1617-1689 73 gaa 32
nadl + 1777-2637 861 ATG TAG 87
nad6 + 2771-3394 624 ATG TAA 133
tRNAPro + 3408-3479 72 tgg 13
tRNALeu1 + 3494-3575 82 tag 14
cox3 + 4050-4748 699 ATG TAA 474
ORF1 + 4848-5936 1089 ATG TAG 99
ORF2 + 5930-6364 435 ATG TAA -5
tRNAMet1 + 6390-6463 74 cat 25
tRNAAsn + 6470-6542 73 gtt 6
tRNAAla + 6569-6639 71 tgc 26
tRNAMet2 + 6841-6912 72 cat 1
tRNAVal + 6929-7001 73 tac 16
tRNAGlu + 7009-7080 72 ttc 7
tRNAAsP + 7094-7164 71 gtc 13
tRNA'le + 7169-7240 72 gat 4
tRNAGln + 7272-7343 72 ttg 31
tRNAArg1 + 7347-7419 73 tct 3
tRNATyr + 7540-7623 84 gta 20
tRNAMet3 + 7659-7731 73 cat 35
tRNAArg2 + 7736-7808 73 tcg 4
rrnS + 7871-9637 1767 62
tRNATrP + 9692-9763 72 cca 54
coxl + 9847-11314 1468 ATG TAA 83
rrnL + 11336-14124 2789 21
cox2 + 14207-15084 878 ATG TAA 82
nad4l + 15074-15388 315 ATG TAA -9
nad5 + 15424-17424 2001 ATG TAG 35
nad4 + 17426-18961 1536 ATG TAA 1
ORF3 + 18948-19388 441 ATG TAA -12
ORF4 + 19646-20008 363 ATG TAA 257
nad2 + 20085-21479 1395 ATG TAA 76
rpl 6 + 21562-22017 456 ATG TAA 82
ORF5 + 22044-22295 252 ATG TAA 26
ORF6 + 22344-22715 372 ATA TAA 48
ORF7 + 22642-23088 447 ATA TAA -72
tRNALeu2 - 23104-23185 82 taa 15
ORF8 + 23244-24767 1524 ATG TAA 58
tRNA»- + 24773-24847 75 gtg 5
Table 3. Continuation.
Gene Strand Location Size (bp) Anticodon Start Stop Intergenic nucleotides
tRNALys - 24869-24941 73 ttt 21
atp9 + 25657-25908 252 ATG TAA 715
tRNASer + 26001-26088 88 gct 92
cob + 26315-27466 1152 ATG TAA 226
ORF9 + 27473-27889 417 ATG TAA 6
ORF10 + 27873-28379 507 ATG TAA -15
rps12 + 28354-28761 408 ATG TAA -24
rps7 + 28799-29235 437 ATG TAA 37
rpl12 + 29240-30025 786 ATG TAA 4
rps19 + 30031-30330 300 ATG TAA 5
ORF11 + 30294-31085 792 ATG TAA -5
ORF12 + 30476-30730 255 ATG TAA -608
ORF13 + 30639-31085 447 ATA TAA 90
clude two tRNA genes for serine and leucine and only one tRNA gene for each of the other 18 amino acids (Attardi, 1985; Cantatore and Saccone, 1987; Ogawa et al., 2000). Among known Amoebozoa mt genomes Acanthamoeba castellanii, Balamuthia mandrillaris, Hartmannella vermiformis, Neoparamoeba pemaquidensis (deposited as Par-amoeba) and Vannella croatica have only two methionine tRNA genes (Burger et al., 1995; Bondarenko et al., 2018a; Greninger et al., 2015) and only Phalansterium sp. has three methionine tRNA genes (Pombert et al., 2013). The ancient nature of tRNAMet duplication in C. sawyeri mt genome is evidenced by the large difference in the nucleotide composition of these genes. In contrast, tRNAArg gene duplication occurs for the first time and has the small difference in the nucleotide composition indicating the "young" nature of this duplication. The functional significance ofthese two duplications in C. sawyeri is not clear yet.
All PCGs and ten ORFs in C. sawyeri use ATG as a start codon, three ORFs use ATA as an alternative start codon. There are two stop codons in C. sawyeri mt genome (TAA and TAG). TGA stop codon wasn't found in this mt genome. Several genes have numerous TAA stop codons within CDS. The same situation was observed in Vannella croatica and Vannella simplex mt genomes (Bondarenko et al., 2018a, 2018b). In contrast to the above mentioned mt genomes, where most ofthe genes and ORFs have stop codons, thus presuming that those are editing sites (or reading frameshifts)
(Bondarenko et al., 2018a, 2018b), in C. sawyeri mt genome the stop codons were found within five genes only, namely cox1, cox2, nad2, nad5and rps7. The mechanism ofresolving these stop codons is not clear. Like in the other vannellids, it may be either RNA editing or reading frame shifts. We translated these five genes using insertional editing. Cytosine insertion was used, as it takes place in Cox I gene of some other vannellid amoebae (Nassonova et al., 2010) and using frameshifts. Obtained amino acid sequences were aligned manually for every gene with those from all other available amoebozoan mitochondrial genomes. This analysis showed that translation with cytosine insertions leads to non-synonymous replacements in conserved regions of proteins and amino acid sequences, resulting in considerable divergence with the sequences of other amoebozoans. Translation with using reading frame shifts showed better results; the number of non-synonymous replacements is lower, and sequences could be better aligned. Certainly, the insertional editing is not limited to cytosine insertions used here, several different kinds of editing are possible within the same mitogenome (Byrne and Gott, 2004; Gott et al., 2010), hence this question requires further study.
Acknowledgments
Supported by the RSF project 17-14-01391 (culturing, treatment of cells and molecular work).
Bioinformatics supported by RFBR project 16-3460111 to Natalya Bondarenko. This study utilized equipment of the Core Facilities Centers "Culture Collection of Microorganisms", "Centre for Molecular and Cell Technologies" and "Computing Centre SPbU" ofSaint-Petersburg State University. Infrastructure supported with SPSU equipment grants 1.40.539.2017 and 1.40.509.2017. The authors thank A.A. Kudryavtsev for providing C. sawyeri culture prior to depositing it as a type strain to the collection of the Core Facilities Center "Culture Collection of Microorganisms".
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Address for correspondence: Natalya Bondarenko. Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia; e-mail: [email protected]
