Overlapping embedded genes, such as htgA/yaaW, are assumed to be rare in prokaryotes. In Escherichia coli O157:H7, gfp fusions of both promoter regions revealed activity and transcription start sites could be determined for both genes. Both htgA and yaaW were inactivated strand specifically by introducing a stop codon. Both mutants exhibited differential phenotypes in biofilm formation and metabolite levels in a nontargeted analysis, suggesting that both are functional despite YaaW but not HtgA could be expressed. While yaaW is distributed all over the Gammaproteobacteria, an overlapping htgA-like sequence is restricted to the Escherichia-Klebsiella clade. Full-length htgA is only present in Escherichia and Shigella, and htgA showed evidence for purifying selection. Thus, htgA is an interesting case of a lineage-specific, nonessential and young orphan gene.