Characterization of the Haemophilus influenzae topA locus: DNA topoisomerase I is required for genetic competence.

A gene essential for the development of genetic competence in Haemophilus influenzae (Hi) was identified as a homolog of the Escherichia coli (Ec) topA gene, which encodes DNA topoisomerase I (TopI). The Hi topA locus was initially identified by mini-Tn10kan mutagenesis. Three independent insertion events within 500 bp of each other resulted in mutant strains that shared a similar phenotype. Each was deficient in competence-induced DNA binding, showed increased sensitivity to UV irradiation, and had an increased doubling time as compared to the wild-type (wt) strain. The nucleotide sequence of a 6.6-kb fragment containing the wt allele was determined. The sequence contained an open reading frame (ORF) of 868 amino acids (aa) that was interrupted by each of the mini-Tn10kan mutations. The deduced aa sequence had a molecular mass of 98 155 Da, a pI of 8.59 and showed strong similarity to Ec TopI. Examination of the topoisomer distribution of a test plasmid in an Hi mutant carrying an insertion in this ORF showed an increase in the level of supercoiling, indicating that TopI is necessary to relax supercoiled DNA in Hi. Complementation studies and insertional inactivation of genes downstream from topA indicated that TopI and not some downstream gene product was essential for competence. Four other ORFs were identified and two of these had homology to known genes. ORF1, which was truncated at one end of the sequenced region, shared strong sequence similarity to the C-terminal end of Ec pyridine nucleotide transhydrogenase beta subunit. ORF4, which was also truncated, showed strong sequence similarity to the N-terminal end of the Ec threonyl-tRNA synthetase.