Identification of transcription activators that regulate gonococcal adaptation from aerobic to anaerobic or oxygen-limited growth

Analysis of the Neisseria gonorrhoeae DNA sequence database revealed the pr esence of two genes, one encoding a protein predicted to be 37.5% identical (50% similar) in amino acid sequence to the Escherichia coli FNR protein a nd the other encoding a protein 41% and 42% identical (54 and 51% sequence similarity) to the E. coli NarL and NarP proteins respectively. Both genes have been cloned into E. coli and insertionally inactivated in vitro. The m utated genes have been transformed into gonococci and recombined into the c hromosome. The fnr mutation totally abolished and the narP mutation severel y diminished the ability of gonococci to: (i) grow anaerobically; (ii) adap t to oxygen-limited growth; (iii) initiate transcription from the aniA prom oter (which directs the expression of a copper-containing nitrite reductase , AniA, in response to the presence of nitrite); and (iv) reduce nitrite du ring growth in oxygen-limited media. The product of nitrite reduction was i dentified to be nitrous oxide. Immediately upstream of the narL/narP gene i s an open reading frame that, if translated, would encode a homologue of th e E. coli nitrate- and nitrite-sensing proteins NarX and NarQ. As transcrip tion from the aniA promoter was not activated during oxygen-limited growth in the presence of nitrate, the gonococcal two-component regulatory system is designated NarQ-NarP rather than NarX-NarL. As far as we are aware, this is the first well-documented example of a two-component regulatory system working in partnership with a transcription activator in pathogenic neisser ia. A 45 kDa c-type cytochrome that was synthesized during oxygen-limited, but not during oxygen sufficient, growth was identified as a homologue of c ytochrome c peroxidases (CCP) of other bacteria. The gene for this cytochro me, designated ccp, was located, and its regulatory region was cloned into the promoter probe vector pLES94. Transcription from the ccp promoter was r epressed during aerobic growth and induced during oxygen-limited growth and was totally FNR dependent, suggesting that the gonococcal FNR protein is a transcription activator of at least two genes. However, unlike AniA, synth esis of the CCP homologue was insensitive to the presence of nitrite during oxygen-limited growth.