A 69.9-kb long inverted repeat increases genome instability in a strain of <i>Lactobacillus crispatus</i>

Long inverted repeats (LIRs) of DNA sequences longer than 30 kb are rare in prokaryotes. Here, we identified two 69.9-kb LIRs in the genome of <i>Lactobacillus crispatus</i> M247_Siena, a derivative of strain M247. Complete genome sequence of M247_Siena was determined using Nanopore and Illumina technologies, while genome structure was analyzed using ultra-long Nanopore read mapping and polymerase chain reaction (PCR). In the parental M247 genome, there was only one copy of the 69.9-kb segment, while a 15.4-kb DNA segment was present instead of the second 69.9-kb segment copy. Both segments were delimited by the same insertion sequences (IS<i>1201</i> and IS<i>Lcr2</i>), and PCR analysis of the M247 population revealed low rates (∼1.28 per 10⁴ chromosomes) of chromosomal rearrangements involving these regions. In contrast, the 69.9-kb LIRs in M247_Siena increased genomic instability, as evidenced by two alternative chromosomal structures detected at frequencies of 23.3% and 76.7% (∼1 out of 5 chromosomes). Comparative analysis of <i>L. crispatus</i> genomes revealed no LIRs similar to those of M247_Siena. However, long repeats of other DNA segments and chromosomal rearrangements, mostly associated with insertion sequences, were detected in 8 and 9 out of 25 <i>L. crispatus</i> genomes, respectively, highlighting genomic instability as a trait of the species.