Large Plasmid Complement Resolved: Complete Genome Sequencing of Lactobacillus plantarum MF1298, a Candidate Probiotic Strain Associated with Unfavorable Effect

Abstract

Considerable attention has been given to the species Lactobacillus plantarum regarding its probiotic potential. L. plantarum strains have shown health benefits in several studies, and even nonstrain-specific claims are allowed in certain markets. L. plantarum strain MF1298 was considered a candidate probiotic, demonstrating in vitro probiotic properties and the ability to survive passage through the human intestinal tract. However, the strain showed an unfavorable effect on symptoms in subjects with irritable bowel syndrome in a clinical trial. The properties and the genome of this strain are thus of general interest. Obtaining the complete genome of strain MF1298 proved difficult due to its large plasmid complement. Here, we exploit a combination of sequencing approaches to obtain the complete chromosome and plasmid assemblies of MF1298. The Oxford Nanopore Technologies MinION long-read sequencer was particularly useful in resolving the unusually large number of plasmids in the strain, 14 in total. The complete genome sequence of 3,576,440 basepairs contains 3272 protein-encoding genes, of which 315 are located on plasmids. Few unique regions were found in comparison with other L. plantarum genomes. Notably, however, one of the plasmids contains genes related to vitamin B12 (cobalamin) turnover and genes encoding bacterial reverse transcriptases, features not previously reported for L. plantarum. The extensive plasmid information will be important for future studies with this strain.

Keywords: Illumina sequencing; Lactobacillus plantarum; Oxford Nanopore MinION sequencing; PacBio sequencing; lactic acid bacterium; plasmid assembly; probiotic.

Figure 1

Chromosomal alignment of six Lactobacillus plantarum strains. The alignment was performed using MAUVE software [27] with strain MF1298 set as the reference. Each contiguously colored region is a local collinear block (LCB), a region without rearrangement of homologous backbone sequence. LCBs below the center line of a chromosome are in the reverse complement orientation relative to the reference chromosome. Lines between genomes trace each orthologous LCB through every chromosome. The white areas inside each LCB show regions with low similarities. Regions outside LCBs represent unique regions.

Figure 2

Lactobacillus plantarum plasmid pMF1298-5 displays a mosaic structure with regions of different origins flanked by transposases/recombinases: L. plantarum rep/parA (replication and partition) and tra (conjugal transfer) region (black), L. plantarum and Pediococcus plasmids (yellow), Pediococcus parvulus retron (reverse transcriptase; RT) region (green), L. buchneri/L. brevis nrdJ/cobA region (purple), L. brevis cobalamin region (orange). Genes related to cobalamin turnover are depicted in light blue and transposase/recombinase genes in red. Genes not annotated in this illustration are shown in grey (a majority of these encode hypothetical proteins). See text for further details. Annotations of the genes are based on RAST [29] and BLAST [28] homology searches in addition to the primary PGAP annotation (GenBank no. CP013155.2).