Insertional mutagenesis of the Haemophilus influenzae chromosome was accomplished by a novel method employing a 2.2-kbp element, TSTE. This element, consisting of the neo gene of TnS flanked by Haemophilusspecific uptake sequences, was ligated to circularized chromosomal fragments before transformation into the homologous strain. Eight mutants defective in the production of haemocin were detected. This strategy provides an efficient mechanism for the insertional mutagenesis of H. influenzae.Generation of mutants by insertion of a transposable antibiotic marker is a useful approach in the characterization of bacterial genes in a wide range of bacterial species (9,13,17,25,33,35 (14). Although this approach is useful, the large size of Tn9O6 limits its utility as a marker for cloning the flanking DNA, which is important for further characterization of the mutagenized gene. Furthermore, the use of transposons is limited, since certain transposons are inherently unstable (4, 8) and nonrandom insertions due to hot spots are common (15,19,26,32,36).Insertional mutagenesis without transposons has been applied to Streptococcus pneumoniae and other species that are naturally transformed. This method employs transformation of bacteria with genomic DNA fragments ligated to an antibiotic resistance determinant (16,31). In this study, we report the use of a novel mutagenesis element that is ligated at high frequency into random positions of the H. influenzae chromosome.The bacterial strains and plasmids used are listed in

During growth in high concentrations of iron nitrate, H. influenzae produces compounds reactive in biochemical assays for hydroxamates. Mixing experiments established that nitrate was responsible for inducing these compounds. Analysis by 1H and 13C NMR and high resolution mass spectrometry identified the active species as 2,2-bis(3'-indolyl)indoxyl. Bacterial production of the latter compound has been previously observed only in Pseudomonas aureofaciens. A mutant defective in the production of 2,2-bis(3'-indolyl)indoxyl was constructed by marker insertion. The formation of indole and 2,2-bis (3'-indolyl)indoxyl was quantitated by reverse-phase high pressure liquid chromatography during growth in high concentrations of nitrate. The mutant produced high concentrations of indole, but only minimal amounts of 2,2-bis(3'-indolyl)indoxyl, and also proved to be defective in nitrate reduction. These data suggest that indole may function as an electron donor for nitrate reductase in H. influenzae.

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