The complete mitochondrial genome of Paravannella minima (Amoebozoa, Discosea, Vannellida)

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ScienceDirect European Journal of Protistology 68 (2019) 80–87

The complete mitochondrial genome of Paravannella minima (Amoebozoa, Discosea, Vannellida) Natalya Bondarenkoa,∗ , Anna Glotovaa , Elena Nassonovab , Alexey Masharskyc , Dmitry Polevd , Alexey Smirnova a

Department of Invertebrate Zoology, Faculty of Biology, St. Petersburg State University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia b Laboratory of Cytology of Unicellular Organisms, Institute of Cytology RAS, Tikhoretsky Ave. 4, 194064 St. Petersburg, Russia c Core Facility Centre for Molecular and Cell Technologies, St. Petersburg State University, Botanicheskaya ul. 17, Stary Peterhof, 198504 St. Petersburg, Russia d Core Facility Centre Biobank, St. Petersburg State University, Botanicheskaya ul. 17, Stary Peterhof, 198504 St. Petersburg, Russia Received 16 September 2018; received in revised form 24 December 2018; accepted 9 January 2019 Available online 15 January 2019

Abstract We present a complete sequence and describe the organization of the mitochondrial genome of the amoeba Paravannella minima (Amoebooza, Discosea, Vannellida). This tiny species represents a branch at the base of Vannellida tree, to the moment being its earliest-branching lineage. The circular mitochondrial DNA of this species has 53,464 bp in length and contains 30 protein-coding genes, 2 ribosomal RNAs, 23 transfer RNAs, and 15 open reading frames. This genome is significantly longer and contains more protein-coding genes than any yet sequenced mitochondrial genome of vannellid amoebae. Unlike the previously sequenced mitochondrial genomes of Vannellida, which should be translated using the “Table 4” (the mold, protozoan, and coelenterate mitochondrial code), that of P. minima can be properly translated using the universal genetic code. © 2019 Published by Elsevier GmbH. Keywords: Amoebozoa; Mitochondrion; Mitochondrial genome; Paravannella; Vannellidae

Introduction Amoebozoa remain among the groups of organisms, where basal relationships between the main phylogenetic lineages Abbreviations: mt, mitochondrial; cox1-3, cytochrome oxidase subunit I, II, and III genes; cob, cytochrome b gene; atp9, ATP synthase subunit 9 gene; nad1-6, 11, NADH dehydrogenase subunit 1–6, 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. ∗ Corresponding author. E-mail address: [email protected] (N. Bondarenko). https://doi.org/10.1016/j.ejop.2019.01.005 0932-4739/© 2019 Published by Elsevier GmbH.

(Tubulinea, Discosea, Cutosea, Variosea, Archamoebae and Eumycetozoa) are not yet resolved, and the question on the origin and evolution of Amoebozoa and on their ancestral group remains unclear (Cavalier-Smith 2013; Cavalier-Smith 2016; Fiore-Donno et al. 2010; Fiz-Palacios et al. 2013; Lahr et al. 2011; Shadwick et al. 2009; Smirnov et al. 2011; Tekle et al. 2017). It seems that the molecular phylogeny, either at the level of single gene, multiple genes or even at the level of the phylogenomic studies cannot ultimately reply to this question. Hence it is still necessary to seek for the support for molecular phylogenetic reconstructions from other approaches that can be applied for this purpose.

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One of the possible options could be the comparison of the structure, gene content and gene sequences of the mitochondrial genome. It is widely recognized as a valuable instrument for evolutionary studies in many groups of organisms (Adams et al. 2002; Bullerwell and Gray 2004; Cameron 2014; Cavalier-Smith 1987; Desmond et al. 2011; Gray et al. 1999; Gray 2012; Petersen et al. 2017 and many others). In Amoebozoa, which most probably lost sex and cell fusions in many evolutionary lineages, it is especially usable as the pattern of evolutionary changes should not be complicated with the frequent mitochondrial introgressions. However, from this point of view, Amoebozoa remain poorly studied, even despite our recent efforts. Complete mitochondrial genomes have been published only for thirteen species of this group. Eleven of them are listed in Bondarenko et al. (2018a, table S1); two more species sequenced in Bondarenko et al. (2018b,c). The comparison of available amoebozoan mitochondrial genomes under the “broad” evolutionary scale suggests a low level of synteny between phylogenetically distant taxa (Heidel and Glöckner 2008). In the same time, at the “narrow” scale, mitochondrial genomes of species belonging to the same genus may be rather similar in their organization and gene order (Bondarenko et al. 2018b; Fuˇcikova and Lahr 2016). However, it is not yet possible to trace the evolution of gene blocks and changes of the gene order in the mitochondrial genomes under the “middle” scale — between species of different genera, belonging to the same clade in the phylogenetic tree due to the absence of appropriate data. To approach this problem, we continue sequencing mitochondrial genomes of amoebae of the order Vannellida — highly supported and species-rich monophyletic clade belonging to the class Discosea. This order includes the single family Vannellidae, combining five recognized amoebae genera — Vannella, Clydonella, Lingulamoeba, Ripella and Paravannella (Kudryavtsev 2014; Nassonova et al. 2010; Page 1983, 1988; Smirnov et al. 2007). Vannellids are among the most widely distributed organisms among naked amoebae; it is hard to get an environmental sample containing no one vannellid amoebae. They readily live and multiply in culture and can feed solely on bacteria. Thus, they represent a good object for molecular studies of this sort. Studies of “core” vannellids belonging to the genus Vannella show high level of similarity between the mitochondrial genomes of two Vannella species — V. croatica and V. simplex (Bondarenko et al. 2018a,b) but also revealed considerable differences between the mitochondrial genomes of these species and that of more distant lineage of vannellids — the genus Clydonella (Bondarenko et al. 2018c). All these genomes were found to be relatively short (29–34 kbp). In the present study, we sequenced the mitochondrial genome of the most basal lineage of vannellid amoebae — Paravannella minima. The morphology and phylogeny of this organism were studied and illustrated by Kudryavtsev (2014). Its mito-

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chondrial genome is significantly larger and is more gene-rich than those of other vannellid sequenced to the moment.

Material and Methods The strain used in this study was a type strain of Paravannella minima isolated in spring, 2006, from the filter mud in a private freshwater aquarium in Berlin, Germany (Kudryavtsev 2014). Amoebae were cultured in 90 mm Petri dishes filled with Millipore-sterilized (0.2 ␮m pore) artificial seawater (25‰) 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 23.5 million reads with the length 150 bp were obtained using HiSeq 2000 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). Annotation of the mitochondrial genome sequence was performed using MITOS web server (Bernt et al. 2013a). Artemis (version 16.0; Rutherford et al. 2000) was used for the visualization of annotation files, manual correction of gene boundaries and search of the open reading frames (ORFs). All proteincoding genes (PCGs) boundaries were verified by manual comparison with the orthologs in other amoebozoans. Genes coding tRNAs were positioned using 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 using our original script written in Python. The Paravannella minima mitochondrial genome has been deposited with the GenBank under the accession number MH910097.

Results and Discussion The mitochondrial genome of Paravannella minima is a double-stranded circular DNA molecule with the length 53,464 bp (Fig. 1). Three others sequenced mt genomes of amoebae belonging to the family Vannellidae are considerably smaller, they count only 28,933–34,145 bp (Bondarenko et al. 2018a,b,c). It is also longer than the mitochondrial genomes of other sequenced Discosea — Acanthamoeba castellanii, counting 39,205–41,591 bp (Fuˇcikova and Lahr 2016), Balamuthia mandrillaris (39,896–42,823 bp) (Greninger et al. 2015) and Neoparamoeba pemaquidensis, which counts 48,522 bp (Tanifuji et al. 2017, as Paramoeba). The mitochondrial genome of P. minima in length is similar with those of the representatives of other amoebozoan lineages — e.g. Vermamoeba vermiformis (52,068 bp),

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Fig. 1. Paravannella minima mitochondrial genome map. The tRNA genes are labeled based on the IUPACIUB single letter amino acid codes.

belonging to Tubulinea, Phalansterium sp. strain PJK-2012 (53,614 bp), belonging to Variosea and Dictyostelium spp. (55,564–54,563 bp), belonging to Eumycetozoa (Fuˇcikova and Lahr 2016; Heidel and Glöckner 2008; Ogawa et al. 2000; Pombert et al. 2013). This finding shows that considerable variations in the size of the mitochondrial genome are possible even within a well-defined, robust crown clade of Amoebozoa. The mitochondrial genome of P. minima has GC content 21.6% (Table 1), which is a relatively low level. As well as in other vannellids, it demonstrates negative AT-skew and positive GC-skew (Bondarenko et al. 2018b). This picture of AT-skew is similar to that, observed in most other organisms (Bernt et al. 2013b). This leads to the predominance of certain codons and amino acids in proteins (Table 2). This is the usual pattern, shown by other studied amoebozoan mitochondrial

Table 1. Nucleotide composition characteristics of the mitochondrial genome of Paravannella minima. AT%

GC%

A%

T%

G%

C%

AT-skew

GC-skew

78,4

21,6

35,0

43,4

13,9

7,7

−0,105

0,293

genomes as well (see Bondarenko et al. 2018a; Fuˇcikova and Lahr 2016). The mitochondrial genome of Paravannella minima contains 30 protein-coding genes (atp6, 9, cob, cox1-3, nad1-7, 9, nad4L, nad11, rpl and rps genes), 23 tRNA, two rRNA genes (rrnL and rrnS) and 15 open reading frames (ORFs) of undefined function (Table 3). This mitochondrial genome in gene content is more similar with the mitochondrial genomes

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Table 2. The codon usage in Paravannella minima mitochondrial genome. Amino Acid

A

R

Y E N D C

P

T

G

Codon

Paravannella minima

GCA GCC GCG GCT CGA CGC CGG CGT AGG AGA TAT TAC GAA GAG AAT AAC GAT GAC TGT TGC CCA CCC CCG CCT ACA ACC ACG ACT GGA GGC GGG GGT

133 11 9 205 21 3 4 106 37 332 850 30 461 64 1073 63 488 26 185 12 100 4 7 217 163 4 18 241 244 17 50 353

belonging to other amoebozoan lineages rather than Vannellida, such as Centramoebida (Acanthamoeba, Balamuthia) or Eumycetozoa (Dictyostelium) (Greninger et al. 2015; Ogawa et al., 2000). If compared to other known vannellid mitochondrial genomes, the set of PCGs genes in P. minima differs by the presence of nad 7 and nad9 genes, and the absence of atp1, 4, and 8 genes (Bondarenko et al. 2018a,b,c). The set of rpl and rps genes in P. minima mitochondrial genome also differs (Table 3). Fifty-two genes and thirteen ORFs are located on H-strand except for two ORFs and one rps gene, which are located on L-strand. The total length of all PCGs, excluding termination codons, is 25,368 bp, which consists 47.45% of the total mitochondrial genome size (Table 3). All genes contain no introns. All ORFs are unique to P. minima mitochondrial genome and have not evident homologs among other species of the order Vannellida, as well as among other Amoebozoa. Paravannella minima mt genome contains two long ORFs with the length 3165 and 6773 bp, respectively. The mitochondrial genome of P. minima has five small gene overlaps and nine non-coding regions longer than 100 bp. The largest overlap is 16 bp in length and is located between

Amino Acid F

L

Q I M V

S

K H W Stop codons

Codon

Paravannella minima

TTC TTT TTA TTG CTA CTC CTG CTT CAA CAG ATC ATT ATA ATG GTA GTC GTG GTT TCA TCC TCG TCT AGT AGC AAA AAG CAT CAC TGG TGA TAG TAA

54 1718 1257 386 49 1 7 94 315 39 31 754 613 285 223 14 41 503 202 8 33 448 422 27 1201 123 215 7 142 – 6 39

atp9 and ORF3. The non-coding regions constitute 3363 bp in total, which is 6.3% of the total mitochondrial genome size (Table 3). The largest non-coding region is 252 bp long and is located between atp6 and ORF13. In general, NNA and NNT codons are the most common codon types, while NNG and NNC codons are the least used. Thus, seven most frequently used codons are TAT, AAT, TTT, TTA, ATT, ATA and AAA (Table 2). This is consistent with the high A + T content in the nucleotide composition of PCGs, and the same pattern is known in other mitochondrial genomes of Amoebozoa (see Bondarenko et al. 2018a). There are no alternative start codons in P. minima mitochondrial genome, all PCGs and ORFs use ATG as a start codon. There are two stop codons (TAA and TAG). TGA was not found in this mitochondrial genome either as a stop codon nor as a codon coding tryptophan (Table 2). In contrast to Vannella croatica, Vannella simplex and Clydonella sawyeri mitochondrial genomes, where several genes have numerous TAA codons (which are stop codons) within CDS, which presumes RNA editing (Bondarenko et al. 2018a,b), in P. minima mitochondrial genome we have not observed

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Table 3. Organization of Paravannella minima mitochondrial genome. Gene

Strand

Location

Size (bp)

ORF1 cox3 rrnL tRNAAsp tRNAHis tRNACys tRNALeu1 tRNAMet1 tRNASer1 tRNAPhe tRNAArg1 tRNATrp tRNAArg2 nad4l nad5 nad4 nad2 rps2 rps4 ORF2 rps16 atp9 ORF3 ORF4 cob ORF5 ORF6 tRNAAla tRNAMet2 rps12 rps7 rpl2 rps19 rps3 rpl16 rpl14 ORF7 rps14 rps8 rpl6 rps13 tRNAVal tRNASer2 tRNAGly ORF8 ORF9 ORF10 ORF11 ORF12 nad3 nad9 nad7 atp6 ORF13 ORF14

+ + + + + + + + + + + + + + + + + + + − − + − + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

43–981 1072–2004 2081–4777 4852–4924 4929–5001 5029–5100 5115–5198 5224–5296 5324–5407 5424–5495 5508–5581 5605–5675 5706–5778 5937–6236 6265–8367 8415–10142 10279–11784 11813–12748 12796–13998 13982–14212 14205–14459 14556–14816 14799–15503 15692–16138 16215–17432 17463–17909 17909–18472 18488–18558 18578–18650 18692–19123 19150–19653 19668–20420 20421–20747 20784–21977 22028–22447 22466–22834 22847–23368 23427–23720 23728–24126 24137–24679 24851–25228 25251–25322 25348–25434 25482–25553 25608–26024 26072–27451 27469–30633 30791–31171 31184–31744 31783–32139 32171–32761 32767–33969 34054–34863 35116–41889 42002–42268

939 933 2697 73 73 72 84 73 84 72 74 71 73 300 2103 1728 1506 936 1203 231 255 261 705 447 1218 447 564 71 73 432 504 753 327 1194 420 378 522 294 399 543 378 72 87 72 417 1380 3165 381 561 357 591 1203 810 6773 267

Anticodon

Start

Stop

Intergenic nucleotides

ATG ATG

TAG TAA

ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG

TAA TAA TAA TAA TAA TAG TAA TAA TAA TAA TAA TAA TAA TAA

ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG

TAG TAA TAA TAA TAA TAA TAA TAA TAA TAG TAA TAA

ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG ATG

TAA TAA TAA TAA TAA TAA TAA TAA TAG TAA TAA

60 90 76 74 4 27 14 25 27 13 12 23 30 158 28 47 136 28 47 −15 −6 −2 −16 188 76 30 −1 15 19 41 26 14 0 36 50 18 12 58 7 10 171 22 45 47 54 47 17 157 12 38 31 5 84 252 12

gtc gtg gca taa cat tga gaa tct cca acg

tgc cat

tac gct tcc

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Table 3 (Continued) Gene

Strand

Location

Size (bp)

cox1 nad11 nad1 cox2 nad6 tRNAGln tRNAIle tRNALys rrnS tRNATyr tRNAGlu tRNALeu2 tRNAPro tRNAAsn ORF15

+ + + + + + + + + + + + + + +

42453–44279 44320–46416 46421–47398 47520–48458 48497–49087 49111–49182 49194–49267 49309–49380 49461–51216 51223–51307 51337–51408 51419–51502 51518–51590 51613–51685 51892–53445

1827 2097 978 939 591 72 74 72 1756 85 72 84 73 73 1554

this phenomenon. The mitochondrial genome of P. minima can be properly translated using the universal genetic code. This is an interesting finding, because before this it seemed that all sequenced mitochondrial genomes of Discosea use translation Table 4, while the universal code was found in the mitochondrial genomes of organisms, belonging to other amoebozoan lineages — Eumycetozoa and Variosea (Bondarenko et al. 2018a,b,c; Heidel and Glöckner 2008; Pombert et al. 2013; Tanifuji et al. 2017). The large ribosomal RNA (rrnL) gene is located between cox3 and tRNAAsp genes and the small ribosomal RNA (rrnS) — between tRNALys and tRNATyr genes (Fig. 1). The length of rrnL and rrnS is 2697 bp and 1756 bp, respectively (Table 3). It is noticeable that rrnS follow rrnL in both sequenced mitochondrial genomes of Vannella species — V. simplex and V. croatica, while in the other phylogenetic lineage of vannellids, represented by Clydonella sawyeri and Paravannella minima they are split with several other genes. This difference seems to be a feature of the organization of this gene block in the order Vannellida. tRNA genes have a total length of 1729 bp and most of them are located between rrnL-nad4l and rrnS-nad11 genes. The average length of tRNA genes varied from 72 to 87 bp (Table 3). All tRNAs have the typical cloverleaf secondary structure. tRNA genes are better represented in the mitochondrial genome of P. minima as compared to other mitochondrial genomes of vannellids (Bondarenko et al. 2018a, b). The mitochondrial genome of P. minima contains additional arginine, leucine, serine, and methionine tRNA genes. The duplication of methionine, serine, and leucine tRNA genes is the most frequently observed event in the known amoebozoan mitochondrial genomes and is known in many species (Attardi 1985; Bondarenko et al. 2018a,b,c; Cantatore and Saccone 1987; Greninger et al. 2015; Ogawa et al. 2000). In contrast, the duplication of arginine tRNA gene occurs only in the mitochondrial genome of Clydonella

Anticodon

Start

Stop

Intergenic nucleotides

ATG ATG ATG ATG ATG

TAG TAA TAA TAA TAA

ATG

TAA

184 40 4 121 38 23 11 41 80 6 29 10 15 22 206

ttg gat ttt gta ttc caa tgg gtt

sawyeri (Bondarenko et al. 2018c). We suggest the ancient nature of tRNAMet and tRNAArg duplication in P. minima mitochondrial genome. It is evidenced by the large difference in the nucleotide composition of these genes. In contrast, tRNALeu and tRNASer gene duplications probably occur for the first time and these genes have a small difference in the nucleotide composition between duplicates, indicating the “young” age of this duplication. Overall, mitochondrial genome of Paravannella minima is unusual for amoebae of the order Vannellida both in length and in the gene content and this finding require careful attention in future. P. minima is one of the smallest vannellids known to the moment (the average length of the cell is 5.8 ␮m). We can very preliminary suggest that the tiny size of the cell, among other matters not allowing it to have numerous mitochondria in the cytoplasm, may in a certain way be related with the size and gene richness of the mitochondrial genome. We need more data on the mitochondrial genomes of tiny vannellid species to see this is a kind of a rule, or variations in genome size are more random and cannot be formalized with a simple assumption.

Acknowledgements Supported by the RSF project 17-14-01391 (culturing, treatment of cells and molecular studies). Bioinformatics supported by RFBR project 16-34-60111 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” of Saint-Petersburg State University. Infrastructure supported with SPSU equipment grants 1.40.539.2017 and 1.40.509.2017.

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