Despite its presence in most bacteria, yqgF remains one of only 13 essential genes of unknown function in Escherichia coli. Predictions of YqgF function often derive from sequence similarity to RuvC, the canonical Holliday junction resolvase. To clarify its role, we deleted yqgF from a bacterium where it is not essential, Acinetobacter baylyi ADP1. Loss of yqgF impaired growth and increased the frequency of transformation and allelic replacement (TAR). When E. coli yqgF was inserted in place of its A. baylyi chromosomal orthologue, wild-type growth and TAR were restored. Functional similarities of yqgF in both gamma-proteobacteria were further supported by defective 16S rRNA processing by the A. baylyi mutant, an effect previously shown in E. coli for a temperature-sensitive yqgF allele. However, our data question the validity of deducing YqgF function strictly by comparison to RuvC. A. baylyi studies indicated that YqgF and RuvC can function in opposition to one another. Relative to the wild type, the ΔyqgF mutant had increased TAR frequency and increased resistance to nalidixic acid, a DNA-damaging agent. In contrast, deletion of ruvC decreased TAR frequency and lowered resistance to nalidixic acid. YqgF, but not RuvC, appears to increase bacterial susceptibility to DNA damage, including UV radiation. Nevertheless, the effects of yqgF on growth and TAR frequency were found to depend on amino acids analogous to catalytically required residues of RuvC. This new heterologous system should facilitate future yqgF investigation by exploiting the viability of A. baylyi yqgF mutants. In addition, bioinformatic analysis showed that a non-essential gene immediately upstream of yqgF in A. baylyi and E. coli (yqgE) is similarly positioned in most gamma- and beta-proteobacteria. A small overlap in the coding sequences of these adjacent genes is typical. This conserved genetic arrangement raises the possibility of a functional partnership between yqgE and yqgF.