Exploring optimization parameters to increase ssDNA recombineering inLactococcus lactisandLactobacillus reuteri

Abstract

Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution.

The Lactococcal Phages Tuc2009 and TP901-1 Incorporate Two Alternate Forms of Their Tail Fiber into Their Virions for Infection Specialization*

“…Recombineering was performed as described previously (32). However, specific modifications optimized for L. lactis such as phosphorothioate linkages and transforming 500 g of oligonucleotides were also employed (33). Recombineering oligonucleotides were ordered from Integrated DNA Technologies (Leuven, Belgium), whereas all other oligonucleotides were ordered from Eurofins MWG (Ebersberg, Germany).…”

“…(4, 48 -50), mutagenesis was instead performed on the Tal 901-1 genome. However, rather than using the rather laborious pGhost mutagenesis method (51), we decided to employ recombineering, a mutagenesis system that has recently been adopted for L. lactis (32,33).…”

Lactobacillus reuteri-Specific Immunoregulatory Gene rsiR Modulates Histamine Production and Immunomodulation by Lactobacillus reuteri

“…The rsiR gene was inactivated by singlestranded DNA (ssDNA) recombineering as previously described (23,24). Briefly, L. reuteri 6475/pJP042 carrying recombinase recT was cultured in MRS medium with 10 g/ml erythromycin to mid-exponential phase (OD 600 , 0.6) at 37°C.…”

Novel two-component regulatory systems play a role in biofilm formation of Lactobacillus reuteri rodent isolate 100-23

“…With these methods, a plasmid-borne antibiotic-resistance gene cassette remains in the chromosome of the mutant strain and limits the subsequent inactivation of other genes of interest. Singlestranded DNA recombineering (Van Pijkeren et al, 2012) or site-specific homologous recombination (Su et al, 2011) are alternative approaches that were recently employed for genetic modification of L. reuteri. The multiple-deletion method employed in this study allowed the generation of double-gene knockout mutants.…”