Revised complete genome sequences of Limosilactobacillus reuteri DSM 20016 T and ATCC PTA-6475 and confirmation of an intragenic macrosatellite in adhesin gene cmbA

Cell and mucus binding protein A (CmbA) is a cell-wall-anchored adhesin common to human isolates of Limosilactobacillus reuteri , which governs mucosal adhesion in vitro . Recent attempts to sequence cmbA in different L. reuteri strains revealed significant genomic inconsistencies with the publicly available closed genome sequences, especially with that of the L. reuteri type strain, DSM 20016 T . We report here a revised closed genome sequence for DSM 20016 T and a closed genome sequence for the closely related L. reuteri ATCC PTA-6475 (MM4-1A). Hybrid long- and short-read sequencing demonstrated that two genomic regions totaling 40 kbp, previously thought to be absent in DSM 20016 T , were in fact intact. The cmbA gene, present in one of these regions, is the longest predicted gene in both genomes and was confirmed to contain an intragenic tandem repeat region. In DSM 20016 T , the region consists of 11 identical ~290 bp tandem direct repeats totaling 3.2 kbp, while ATCC PTA-6475 has 8 repeats totaling 2.3 kbp. This macrosatellite posed a challenge to PCR-based approaches to confirm the length of the gene. Polyacrylamide gel electrophoresis of cell wall extracts from ATCC PTA-6475 showed a ~160 kDa band, which was absent from a cmbA -knockout strain, consistent with the expected size based on whole-genome sequencing and confirmed by mass spectrometry to be CmbA. Overall, we present refined publicly available genome sequences for two frequently studied L. reuteri strains and validate the length of a large gene with a conspicuously high number of identical tandem repeats. IMPORTANCE Studies comparing bacterial genomes and routine cloning work often implicitly assume that the closed genome sequences available from public databases are accurate. However, as technologies improve and we gain new data, inconsistencies can arise which prompt the resequencing of strains, sometimes with surprising results. We show here that a significant sequencing assembly artifact led to a large gap in the publicly available closed genome of the Limosilactobacillus reuteri type strain which has remained uncorrected for nearly two decades, despite a vast body of L. reuteri work over that time. Another region contained a large stretch of repetitive intragenic DNA that still posed a challenge to modern PCR techniques. Therefore, in addition to being useful to L. reuteri biologists, this work serves as an important reminder of the intrinsically experimental nature of sequencing data; it usually pays to resequence early and often.