DNA Homologous Recombination Proteins for Genetic Manipulations in Bacteria

The proteomic and functional genomic analyses of many bacterial species have been severely hindered by the lack of effective methods with which their genetic material may be accurately and efficiently modified. This is the case for the vast majority of pathogenic and commensal bacterial species commonly found within the mouth.

Objectives: To identify novel sets of DNA homologous recombination proteins and determine their potential for general bacterial chromosome engineering using a combination of in vitro and in vivo techniques.

Methods: Pairs of adjacent genes encoding (bacteriophage lambda-like) single stranded DNA (ssDNA) exonucleases and ssDNA annealing proteins (SSAPs) were identified in a beta- and a gamma-proteobacterium using a bioinformatic approach. After cloning, recombinant protein expression and purification, the biophysical activities of the two putative exonucleases were comprehensively characterized in vitro. The oligonucleotide-directed DNA repair activities of the SSAP homologues were determined in Escherichia coli, using a point-mutated kanamycin resistance reporter gene. The double-stranded DNA recombination activities of the two exonuclease/SSAP pairs were established semi-quantitatively by determining their ability to mediate the precise creation of C-terminal green fluorescent protein (GFP) fusions with genes on the E. coli chromosome.

Results: The two exonuclease homologues were both divalent metal-dependent ssDNA exonucleases, with optimal activity at pH 8-8.5. The ssDNA-directed recombination activities of the two SSAP homologues in our E. coli reporter system were comparable to those of the SSAP from bacteriophage lambda (lambda-bet). Both exonuclease/SSAP pairs could mediate the accurate creation of C-terminal GFP fusions to several genes on the E. coli chromosome with good efficiencies.

Conclusions: The novel SSAP/exonuclease systems described here can efficiently and accurately modify the chromosome of E. coli in using both oligonucleotides and dsDNA molecules to direct the desired genetic alterations. We discuss their potential for homologous recombination-based genome engineering within other bacterial species.