Complete bacterial genomic DNA sequences have allowed application of genome-scale strategies to identify essential genes. Efficient generation of conditional loss of function mutations provides a means of characterization of this class of genes. Promoter systems conferring tight regulation are particularly applicable to generating such mutations and we sought to apply this approach for the analysis of an essential gene of Haemophilus influenzae. Therefore, we developed the use of a D-xylose-inducible promoter for verification of an essential role in growth for yraM, which encodes a putative periplasmic lipoprotein, in both H. influenzae Rd and virulent type b strains. This promoter was sufficiently tightly regulated to generate conditionally viable strains by inducible expression of YraM. A second approach was used to further characterize YraM. Natural transformation and an ordered mutant collection spanning the H. influenzae genome provide the means to target any gene of interest for mutagenesis and temperature-sensitive (TS) mutant isolation. This strategy was applied to generate a conditionally lethal allele of yraM. The resulting TS mutation was directly mapped to a single amino acid substitution within a motif conserved in all putative YraM orthologs and this mutation was shown to be sufficient to confer the TS phenotype.