Mechanisms and Applications of RecA-independent recombination in Legionella pneumophila.

Abstract

Recombination is fundamental to genome maintenance and contributes to diversity in all Domains of life by generating novel sequences from the breaking and joining of DNA. RecA is central to one of the best studied mechanisms of recombination, but RecA-independent pathways also have implications for genetic engineering, evolution, and DNA replication. Here, I harness two forms of RecA-independent recombination for engineering of Legionella pneumophila, while also probing replication and repair machinery. Specifically, I make unmarked deletions in L. pneumophila using the Flp site-specific recombinase while also examining single-stranded nucleases and mismatch repair. I also describe a distinct form of recombination that does not require expression of recombinase genes. Oligonucleotides (oligos) generate mutations on the L. pneumophila chromosome by a mechanism that requires homologous DNA, but not RecA, RadA or any known phage recombinase. Instead, DNA replication likely contributes, since oligo-induced mutagenesis required ≥ 21 nucleotides of homology, was strand-dependent, and was most efficient in exponential phase. Mutagenesis appeared to be distinct from previously described mechanisms of oligo recombination, as it did not require canonical 5’ phosphate or 3’ hydroxyl groups, but did require the primosomal protein PriA and DNA Pol I. After electroporation, oligos stimulated excision of 2.1 kb of chromosomal DNA or insertion of 18 bp, and can be exploited to generate chromosomal deletions and to insert an epitope into a chromosomal coding sequence. The frequency of mutagenesis also increased substantially when either its RecJ and ExoVII nucleases were inactivated or the oligos were modified by nuclease-resistant bases. L. pneumophila is a diverse species and has a surprising number of Eukaryotic-like genes. As the organism is naturally transformable, it is thought that the species acquired some of these genes by horizontal gene transfer. Since divergent Eukaryotic sequences may be inefficiently incorporated into the genome by RecA-mediated recombination due to the extensive homology required, oligo-induced mutagenesis may have evolutionary implications as a mechanism to incorporate divergent DNA sequences with only short regions of homology. Since oligo mutagenesis appears to be conserved, it may play a role in remodeling the genomes of L. pneumophila and species across the Bacterial Domain.