ABSTRACT
Amoebae of the Naegleria genus carry all ribosome-encoding DNA on closed circular extrachromosomal elements (CERE). We report the sequence of the CERE of Naegleria jadini (strain Willaert and Ray).
ANNOUNCEMENT
Ribosomal DNA (rDNA) of the Naegleria genus is encoded on a closed circular extrachromosomal element (CERE) (1) in the nucleolus (2–6). Each trophozoite has approximately 4,000 copies of the CERE (2, 3), which are postulated to replicate independently (2). A single origin of DNA replication (7) was mapped in the Naegleria gruberi CERE nonribosomal sequences (NRS) (6). The NRS are poorly conserved between species (1). N. jadini was isolated in Antwerp, Belgium, in 1971 (8) and is taxonomically related to N. fowleri (9, 10) but antigenically distinct (11). N. jadini was obtained from the ATCC.
N. jadini trophozoites were cultured in modified PYNFH medium at 25°C. Confluent flasks were chilled for 5 min, and then detached trophozoites were harvested. N. jadini CERE were isolated using a plasmid minikit (Qiagen). Supercoiled CERE DNA was isolated from 0.8% agarose gels and purified using the Monarch DNA gel extraction kit (New England Biolabs); as previously determined Naegleria CERE sequences have numerous repeat regions, shearing of the DNA was not desired. CERE DNA was digested with BspEI (New England Biolabs), yielding a linear product of approximately 11 to 12 kbp as determined by agarose gel electrophoresis. Further processing and sequencing were performed at the Roy J. Carver Biotechnology Center at the University of Illinois—Urbana Champaign. DNA quality and quantification were determined using the Agilent Femto Pulse system (Agilent). The library was prepared using the PacBio SMRTbell express template prep kit 3.0 (Pacific Biosciences), and sequencing was performed on a PacBio Sequel IIe using the circular consensus sequencing (CCS) sequencing mode and 30-h movies.
As mitochondrial DNA restriction fragments were coisolated in this procedure, a BLAST alignment identified mitochondrial sequences from the resultant sequence files. Sequences with ≥95% mitochondrial identity to the mitochondrial genome of N. gruberi (GenBank accession number NC_002573.1) were removed from the analysis (E value, 1e-50; remaining settings “default”), filtering out 891/72,572 (1.2%) reads. Due to the homology of the rDNA between Naegleria species, a BLAST alignment was performed on the remaining 71,681 reads to identify homology with N. gruberi rDNA (GenBank accession number AB298288). The BLAST parameters were ≥95% homology to the rDNA sequence, E value of 1e−50, and the remaining parameters default. A total of 51,695/71,681 (72.0%) read sequences were identified as having rDNA identity. The remaining 19,986/71,681 (28.0%) were removed from analysis. Reads were assembled through SPADES Assembler 3.15.5 into contigs and a consensus. Options were “--isolate” and “—plasmid—only-assembler.” The remaining parameters were default. The total number of reads over 10 kbp was 5,848. Characteristics of the CERE are provided in Table 1.
TABLE 1.
CERE characteristics of N. jadini Willaert and Ray
| Characteristic | Value |
|---|---|
| Genome size (bp) | 11,825 |
| GC content (%) | 42.1 |
| No. of contigs | 51 |
| BLAST coverage to N. gruberi rDNA (%) | 50 |
| Avg read length (bp)a | 3,635.9 |
| Total no. of readsa | 51,695 |
| N 50 | 10,186 |
| GenBank accession no. | OP997288 |
| SRA accession no. (PacBio) | SRR22270901 |
| BioProject no. | PRJNA887419 |
| BioSample no. | SAMN31166777 |
| SRA accession no. (Plasmidsaurus) | SRR23509813 |
Values represent figures after BLAST alignment against mitochondrial and N. gruberi rDNA as discussed in the text. When sequences were further filtered to identify sequences between 10 and 13 kbp, the average read length was 11,665 bp and total reads were 5,848.
To test that a complete assembly consensus was obtained, uncut CERE DNA was sequenced with Oxford Nanopore technology (https://www.plasmidsaurus.com/), using the V10 chemistry library prep kit with the R9.4.1 flow cell. The base call software was Guppy. A Clustal alignment of the Plasmidsaurus sequences and the assembled consensus was performed to verify that there was no insertion at the BspEI restriction site in the 18S rDNA sequenceBandage, using two sequencing platforms, and long reads (10 to 13 kbp) were used to resolve repeat regions and ambiguities to complete the assembly consensus.
Data availability.
The consensus sequence has been deposited in GenBank under accession number OP997288. The first nucleotide of the 18S rDNA subunit has been designated position 1 in the sequence. The PacBio raw reads have been deposited in the NCBI Sequence Read Archive (SRA) and can be found under accession number SRR22270901. The BioProject and BioSample are deposited under accession numbers PRJNA887419 and SAMN31166777, respectively. The Plasmidsaurus raw reads are deposited under accession number SRR23509813.
Contributor Information
Kristen M. Drescher, Email: kristendrescher@creighton.edu.
Jason E. Stajich, University of California, Riverside
REFERENCES
- 1.Nguyen BT, Chapman NM, Tracy S, Drescher KM. 2021. The extrachromosomal elements of the Naegleria amoebae: how little we know. Plasmid 115:102567. doi: 10.1016/j.plasmid.2021.102567. [DOI] [PubMed] [Google Scholar]
- 2.Clark CG, Cross GAM. 1987. rRNA genes of Naegleria gruberi are carried exclusively on a 14-kilobase-pair plasmid. Mol Cell Biol 7:3027–3031. doi: 10.1128/mcb.7.9.3027-3031.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Clark CG, Cross GAM. 1988. Circular ribosomal RNA genes are a general feature of schizopyrenid amoebae. J Protozool 35:326–329. doi: 10.1111/j.1550-7408.1988.tb04352.x. [DOI] [PubMed] [Google Scholar]
- 4.Fritz-Laylin LK, Cande WZ. 2010. Ancestral centriole and flagella proteins identified by analysis of Naegleria differentiation. J Cell Sci 123:4024–4031. doi: 10.1242/jcs.077453. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Mullican JC, Chapman NM, Tracy S. 2018. Complete genome sequence of the circular extrachromosomal element of Naegleria gruberi strain EGB ribosomal DNA. Genome Announc 6:e00020-18. doi: 10.1128/genomeA.00020-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Mullican JC, Drescher KM, Chapman NM, Tracy S. 2020. Complete genome sequence of the Naegleria fowleri (strain LEE) closed circular extrachromosomal ribosomal DNA element. Microbiol Resour Announc 9:e01055-20. doi: 10.1128/MRA.01055-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Mullican JC, Chapman NM, Tracy S. 2019. Mapping the single origin of replication in the Naegleria gruberi extrachromosal DNA element. Protist 170:141–152. doi: 10.1016/j.protis.2019.02.001. [DOI] [PubMed] [Google Scholar]
- 8.Jadin JB, Hermanne J, Robyn G, Willaert E, Van Maercke Y, Stevens W. 1971. Trois cas de meningo-encephalite amibienne primitive observés à Anvers (Belgique). Ann Soc Belges Med Trop Parasitol Mycol 51:255–266. [PubMed] [Google Scholar]
- 9.Clark GC, Cross GAM, De Jonckheere JF. 1989. Evaluation of evolutionary divergence in the genus Naegleria by analysis of ribosomal DNA plasmid restriction patterns. Mol Biochem Parasitol 34:281–296. doi: 10.1016/0166-6851(89)90057-1. [DOI] [PubMed] [Google Scholar]
- 10.De Jonckheere JF. 2014. What do we know by now about the genus Naegleria? Exp Parasitol 145(Suppl):S2–S9. doi: 10.1016/j.exppara.2014.07.011. [DOI] [PubMed] [Google Scholar]
- 11.Willaert E, Le Ray D. 1973. Caracteres morphologiques, biologiques et immunochimiques de Naegleria jadini sp. nov. (Amoebida, Vahlkampfiidae). Protistologica IX:417–426. [Google Scholar]
Associated Data
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Data Availability Statement
The consensus sequence has been deposited in GenBank under accession number OP997288. The first nucleotide of the 18S rDNA subunit has been designated position 1 in the sequence. The PacBio raw reads have been deposited in the NCBI Sequence Read Archive (SRA) and can be found under accession number SRR22270901. The BioProject and BioSample are deposited under accession numbers PRJNA887419 and SAMN31166777, respectively. The Plasmidsaurus raw reads are deposited under accession number SRR23509813.