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.

In mixed-species communities, bacteria can deploy contact-dependent effectors to compete with other organisms, often directly injecting these proteins into neighboring cells. One current hypothesis is that the entire protein contains information specific for a single 20 species; emergence of new effectors comes from acquiring genes. Here we have characterized a family of DNA-degrading effectors that are nucleases which cause death. Like other families of chimeric nucleases, these effectors contain two domains. One is a PD-(D/E)XK-containing domain necessary for DNA cleavage. The other domain, which does not contain known DNA-binding structures, encodes species-identifying information. 25 We capitalized on the species-identifying domain to differentiate among low-abundance species, as well as to reveal domain architectures within these proteins, in human gut and oral microbiomes. Emerging are questions about how low-abundance strains use effectors for survival and how strain-identifying effectors evolve.

Dense microbial communities, like the gut and soil microbiomes, are dynamic societies. Bacteria can navigate these environments by deploying proteins (effectors) that alter foreign cells' behavior. Immunity proteins preferentially protect neighboring sibling cells, in contrast to canonical toxin-antitoxin systems. A prevailing hypothesis is that when immunity proteins are bound to specific (cognate) protein partners, it is sufficient to disrupt their function; further, there is little-to-no crosstalk with other (non-cognate) effectors. Here, we build on sporadic reports challenging these hypotheses. We show that immunity proteins from a newly defined protein family can bind and protect against non-cognate PD-(D/E)XK-containing effectors from diverse phyla. We describe the domains essential for binding and function and show that binding alone is insufficient for protective activity. Moreover, we found that these effector and immunity genes co-occur in individual human microbiomes. These results expand the growing repertoire of bacterial protection mechanisms and force us to reconsider how non-cognate interactions impact community structure within complex ecosystems.