A GENOME-BASED APPROACH FOR THE IDENTIFICATION OF ESSENTIAL BACTERIALGENES

We have used comparative genomics to identify 26 Escherichia coli open reading frames that are both of unknown function (hypothetical open r eading frames or y-genes) and conserved in the compact genome of Mycop lasma genitalium. Not surprisingly, these genes are broadly conserved in the bacterial world. We used a markerless knockout strategy to scre en for essential E. coli genes, To verify this phenotype, we construct ed conditional mutants in genes for which no null mutants could be obt ained. In total we identified six genes that are essential for E. coli (yhbZ, ygjD, ycfB, yfil, yihA, and yjeQ), The respective orthologs of the genes yhbZ, ygjD, ycfB, yjeQ, and yihA are also essential in Baci llus subtilis. This low number of essential genes was unexpected and m ight be due to a characteristic of the versatile genomes of E. coli an d B. subtilis that is comparable to the phenomenon of nonorthologous g ene displacement. The gene ygjD, encoding a sialoglycoprotease, was el iminated from a minimal genome computationally derived from a comparis on of the Haemophilus influenzas and M, genitalium genomes. We show th at ygjD and its ortholog ydiE are essential in E. coli and B. subtilis , respectively. Thus, we include this gene in a minimal genome. This s tudy systematically integrates comparative genomics and targeted gene disruptions to identify broadly conserved bacterial genes of unknown f unction required for survival on complex media.