The plo gene, encoding the Arcanobacterium pyogenes cholesterol-dependent cytolysin, pyolysin (PLO), was localized to a 2.7-kb genomic islet of reduced %G+C content and alternate codon usage frequency. This islet, conserved among isolates from diverse hosts and geographical locations, separated the housekeeping genes smc and ftsY, which are found adjacent in many prokaryotes. The ftsY and ffh genes, located downstream of the plo islet, encode components of the signal recognition particle. Mutational analysis suggested that these genes were essential for viability in A. pyogenes. The A. pyogenes ffh gene was unable to complement a conditional ffh mutant of Escherichia coli and its overexpression was toxic in E. coli. Mutagenesis of the islet-encoded orf121 did not affect plo expression, indicating that it may not be involved directly in the regulation of plo expression. Regardless, the presence of the plo gene as part of a genomic islet inserted between genes essential for normal growth may provide selective pressure for the retention of this important virulence factor.