Genome alterations associated with improved transformation efficiency in Lactobacillus reuteri

Abstract

BackgroundLactic acid bacteria (LAB) are one of the microorganisms of choice for the development of protein delivery systems for therapeutic purposes. Although there are numerous tools to facilitate genome engineering of lactobacilli; transformation efficiency still limits the ability to engineer their genomes. While genetically manipulating Lactobacillus reuteri ATCC PTA 6475 (LR 6475), we noticed that after an initial transformation, several LR 6475 strains significantly improved their ability to take up plasmid DNA via electroporation. Our goal was to understand the molecular basis for how these strains acquired the ability to increase transformation efficiency.ResultsStrains generated after transformation of plasmids pJP067 and pJP042 increased their ability to transform plasmid DNA about one million fold for pJP067, 100-fold for pSIP411 and tenfold for pNZ8048. Upon sequencing of the whole genome from these strains, we identified several genomic mutations and rearrangements, with all strains containing mutations in the transformation related gene A (trgA). To evaluate the role of trgA in transformation of DNA, we generated a trgA null that improved the transformation efficiency of LR 6475 to transform pSIP411 and pJP067 by at least 100-fold, demonstrating that trgA significantly impairs the ability of LR 6475 to take-up plasmid DNA. We also identified genomic rearrangements located in and around two prophages inserted in the LR 6475 genome that included deletions, insertions and an inversion of 336 Kb. A second group of rearrangements was observed in a Type I restriction modification system, in which the specificity subunits underwent several rearrangements in the target recognition domain. Despite the magnitude of these rearrangements in the prophage genomes and restriction modification systems, none of these genomic changes impacted transformation efficiency to the level induced by trgA.ConclusionsOur findings demonstrate how genetic manipulation of LR 6475 with plasmid DNA leads to genomic changes that improve their ability to transform plasmid DNA; highlighting trgA as the primary driver of this phenotype. Additionally, this study also underlines the importance of characterizing genetic changes that take place after genome engineering of strains for therapeutic purposes.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0986-8) contains supplementary material, which is available to authorized users.

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“…In fact, methylation and endonuclease systems have been studied in probiotic bacteria, but usually for genetic engineering purposes. SMRT sequencing was used to detect multiple R-M systems in strains of Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus reuteri [35,36]. Similar epigenomic studies analyzed the genetic accessibility of Bifidobacterium breve Bifidobacterium longum subsp.…”

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“…Lowering the induction temperature slows down cell proliferation processes including the rate of transcription and translation, which results in lower proportions of mis-folded protein and minimizes protein aggregation [ 41 ]. A higher protein expression at 30 ℃ was also reported for L. reuteri DSM 20016 with a pTRKH3- erm GFP expression plasmid [ 35 ].…”

“…In contrast, high transformation efficiency was seen with the constructs based on pSIP411, which harbors an SH71rep origin of replication. In contrast to 256rep, SH71rep is a high copy number replicon from L. lactis [ 35 ] that is reported as a broad-host-range replicon and is also functional in L. reuteri DSM20016 and L. reuteri ATCC PTA 6475 [ 22 ] as well as L. reuteri KUB-AC5.…”

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“…[ 119 ] A recent report suggests increased plasmid transformation efficiency with the mutation of genomic transformation‐related gene A ( trgA ), assumed to affect the genetic acceptability of the plasmid from the initial transformation. [ 120 ]…”