Complete sequence of the closed circular extrachromosomal element (CERE) of Naegleria australiensis De Jonckheere (strain PP 397)

ABSTRACT Ribosomal RNA is not encoded in chromosomal DNA in amoebae of the Naegleria genus but the rRNA genes are located on closed circular extrachromosomal ribosomal DNA (rDNA)-containing elements (CERE). In this report, we describe the sequence of the CERE of Naegleria australiensis De Jonckheere (strain PP397).

A moebas of the Naegleria genus have no ribosomal DNA (rDNA) in the nuclear DNA but rather in the nucleolus (1-4) on closed circular extrachromosomal elements [CERE (5) ].One trophozoite contains ~4,000 CERE (1,2).CERE also contain a large non-ribosomal sequence (NRS) with a single origin of replication (6).Unlike highly conserved rDNA cistrons, the NRS show limited conservation between Naegleria species (5).Naegleria australiensis De Jonckheere (PP397) was isolated from drainage water in Australia in 1973 (7) and is not pathogenic for humans but in mice induces brain pathology like Naeglaria fowleri (8) as well as pneumonitis (9).
N. australiensis was obtained from ATCC (Manassas, VA) and trophozoites cultured in modified PYNFH (peptone/yeast extract/nucleic acid/folic acid/heme with 10% fetal calf serum) medium at 25°C and split every 48 h.CERE DNA was isolated using the Plasmid Mini Kit (Qiagen), electrophoresed on 0.8% agarose gels, and unnicked supercoiled DNA purified using the Monarch Gel Extraction Kit (NEB).As known, Naegleria CERE sequences have numerous repeat regions (5); CERE were digested with PstI (NEB) in lieu of shearing the DNA, resulting in a product of approximately 12-13 kbp.All remaining process ing/sequencing was performed at the Roy J. Carver Biotechnology Center (University of Illinois-Urbana-Champaign).The Agilent Femto Pulse System was used to determine the DNA quality.The PacBio SMRTbell Express Template Prep Kit 3.0 (Pacific Biosciences) was used to prepare the library, and the PacBio Sequel IIe (SMRT Link v11.0) was used for sequencing in CCS mode and 30 h movies.
The 130,147 raw reads were filtered against the mitochondrial sequence of Naegleria gruberi (GenBank accession NC_002573.1),and sequences with at least 70% identity (33,735/25.92%)were removed from analysis (E-value 1e-50).Due to the close iden tity of rDNA between Naegleria species, a BLASTn alignment was executed on the outstanding 96,412 reads against the N. gruberi rDNA (GenBank accession AB298288; BLASTn parameters: >75% identity to the rDNA sequence, E-value 1e-50).A total of 51,009/96,412 (52.90%) sequences with rDNA identity were retained for analysis.These 51,009 reads were separated into FASTA files with read between 9 and 15 kbp (5,894/51,009; 11.5% of reads), based on the predicted size of the CERE.Reads were assembled using the SPADES Assembler 3.15.5 into contigs and a consensus (options: "-isolate, " "-only-assembler"; K-mer-125).Characteristics of the CERE are presented in Table 1.
To verify the predicted consensus, uncut CERE DNA was reisolated and sequenced using Oxford Nanopore technology (plasmidsaurus.com),using the V10 Chemistry Library Prep Kit, with the R9.4.1 flow cell.Guppy (v.6.3.8;SUP) was used to call bases.A 360 bp addition in a repeat region was identified in the Plasmidsaurus sequence at position 8,036, compared to the PacBio consensus.To resolve this discrepancy, PCR was performed using primers (Table 1) spanning the region.Fig. 1 indicates that three repeats are in the majority of CERE.

FIG 1
FIG 1 Resolution of repeat region in the N. australiensis sequence.To resolve the difference in repeat number between the PacBio and Oxford Nanopore consensus sequences, PCR was performed on the 7,041 bp CERE fragment obtained by digestion of the CERE with PacI and XcmI.Primers used are listed in

TABLE 1
CERE characteristics of Naegleria australiensis De Jonckheere (strain PP 397) Values indicated 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 9 and 15 kbp, the average read length was 11,276.8bp and total reads were 5,894. a