Gonococcal Genes Encoding Transferrin-Binding Proteins A and B Are Arranged in a Bicistronic Operon but Are Subject to Differential Expression

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Abstract
Neisseria gonorrhoeaeis capable of utilizing host iron-binding proteins, such as transferrin, lactoferrin, and hemoglobin, as the sole source of iron. The receptor involved in transferrin iron acquisition is composed of two distinct transferrin-binding proteins, TbpA and TbpB. The genes that encode these proteins are linked on the chromosome in the ordertbpB-tbpAbut are separated by an inverted repeat of unknown function. In this study, we sought to understand the transcriptional organization and regulation of thetbpgenes, using a combination oflacZtranscriptional fusion analysis and reverse transcriptase PCR (RT-PCR). First, we demonstrated thattbpBandtbpAare cotranscribed and coregulated from the common upstream promoter that precedestbpB. Using β-galactosidase activity as a surrogate fortbp-specific transcription, we found thattbpB-specific transcripts were more prevalent thantbpA-specific transcripts after 2 h of growth under iron stress conditions. We confirmed the results obtained by fusion analysis by using RT-PCR applied to native RNA isolated from wild-type gonococci. Three different varieties of RT-PCR were employed: relative, competitive, and real time quantitative. The results of all analyses indicated thattbpB-specific transcripts were approximately twofold more prevalent thantbpA-specific transcripts at steady state. In iron-stressed cultures, the ratio oftbpB- totbpA-specific message was approximately 2; however, in iron-replete cultures, this ratio dropped to 1. Using these techniques, we also quantitated the effects of iron, external pH, and presence of ligand ontbpmRNA levels.