Draft Genome Sequence of Acholeplasma laidlawii, a Common Contaminant of Cell Cultures.

Mollicutes are important cell culture contaminants which may eventually affect the results of biological assays or affect their interpretation. Acholeplasma laidlawii is one of the most frequent contaminants of cell cultures. Here, we report the complete genome sequence of A. laidlawii strain MDBK/IPV, recovered from Madin-Darby bovine kidney (MDBK) cells.

GENOME ANNOUNCEMENT

Acholeplasma species comprise bacteria from the Mollicutes class that are described as saprophyte and commensal bacteria. It has been suggested that acholeplasmas and phytoplasmas come from an Acholeplasma-like last-common ancestor (1). Unlike other mycoplasmas, members of the family Acholeplasmataceae do not require sterols for cultivation and are able to synthesize fatty acids from precursors (2). Some mycoplasmal species, among them Acholeplasma laidlawii, could persist as culture contamination on cell lineages and consequently can affect the results of biological assays or affect their interpretation. Moreover, A. laidlawii infects plants, with phytopathogenic effects analogous to Phytoplasma infection; besides, these bacteria were detected as commensals in insects (3). To our knowledge, only one complete genome sequence of A. laidlawii (strain PG-8A) has been published (4). The analysis of this A. laidlawii genome revealed a richly equipped repertoire for metabolism, SOS response, repair systems, and extensive transcriptional regulation machinery, including the two-component systems, riboswitches and T-boxes (4, 5). Here, we report the complete genome sequence of A. laidlawii strain MDBK/IPV, recovered from a Madin-Darby bovine kidney (MDBK) cell lineage.

A. laidlawii MDBK/IPV DNA was extracted from preparations of MDBK cells as follows: the supernatant of MDBK monolayers was clarified by low-speed centrifugation, filtered (0.45-µm pore size), and ultracentrifuged (6). DNA was extracted according to a phenol-chloroform protocol. Whole-genome paired-end sequencing was performed in the Illumina MiSeq platform (Illumina), with the 500-cycle kit (version 2). DNA libraries were prepared with a Nextera kit (Illumina). The original reads were imported into the Geneious software (version 8.1) and trimmed. The assembly of the A. laidlawii genome was accomplished by read mapping to the reference A. laidlawii genome (A. laidlawii PG-8A, GenBank accession no. CP000896) with the Geneious software and de novo assembly with the SPAdes assembler 3.6.0 (7). This yielded 12-fold average read depth. Gene prediction and annotation were performed automatically with the NCBI annotation tool (8). Statistics were generated by QUAST (9).

The genome of A. laidlawii MDBK/IPV consists of 29 contigs (largest contig, 212,101 bp; N50, 86,549 bp), with a total size of 1,307,942 bp. The G+C content of A. laidlawii MDBK/IPV is 31.97%. The A. laidlawii MDBK/IPV genome contains 1,250 predicted genes, two rRNA gene operons (5S-16S-23S), 33 tRNA genes, three noncoding RNAs (ncRNAs), and 25 pseudogenes. Among 1,183 potential protein-coding genes, 56% encode proteins with assigned functional roles. A high genetic repertoire was observed in A. laidlawii MDBK/IPV, which is described as necessary for adaptation to changing environmental conditions (5).

A comparative analysis between A. laidlawii MDBK/IPV and A. laidlawii PG-8A genomes shows the absence of approximately 200 kb in the MDBK/IPV genome. Interestingly, this PG-8A region is composed mainly of insertion elements (IS) and phage sequences. This genomic difference can be related to genome condensation or duplication of genetic material and integration events, which are commonly described in acholeplasmas (5).

The genome sequence of A. laidlawii MDBK/IPV will enable further investigations to better understand the molecular biology process of this bacterium that is important in culture cell contamination and in insights of minimal cellular life functions.

Accession number(s).

The draft genome of A. laidlawii strain MDBK/IPV has been deposited at DDBJ/ENA/GenBank under the accession no. MIPS00000000. The version described in this paper is version MIPS01000000.