In Escherichia coli and most Gram-negative bacteria, KDO (3-deoxy-D-manno-octulosonate), a component of the lipopolysaccharide inner core, is essential for outer membrane biogenesis and cell viability. Two recently identified genes involved in KDO biosynthesis, kdsD and kdsC, belong to the yrbG-yhbG locus where four additional ORFs (yrbG, yrbK, yhbN and yhbG) with unknown function are located. We have constructed six conditional expression mutants in which the arabinose-inducible araBp promoter is respectively located upstream of each gene of the locus. Complementation analysis of these mutants indicates that the locus is organized in at least three operons and that the three distal genes (yrbK, yhbN and yhbG) are essential for E. coli viability. Surprisingly, kdsD and kdsC (encoding a D-arabinose 5-phosphate isomerase and a KDO 8-phosphate phosphatase, respectively) were shown to be non-essential, indicating genetic redundancy for these two functions. A preliminary characterization of the arabinose-dependent mutants under permissive conditions and upon depletion revealed increased sensitivity to hydrophobic toxic chemicals, suggesting that the mutants have a defective outer membrane. These genes may thus be implicated in cell envelope integrity.