Gonococcal genes encoding transferrin-binding proteins A and B are arranged in a bicistronic operon but are subject to differential expression.
Neisseria gonorrhoeae is 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 order tbpB-tbpA but are separated by an inverted repeat of unknown function. In this study, we sought to understand the transcriptional organization and regulation of the tbp genes, using a combination of lacZ transcriptional fusion analysis and reverse transcriptase PCR (RT-PCR). First, we demonstrated that tbpB and tbpA are cotranscribed and coregulated from the common upstream promoter that precedes tbpB. Using beta-galactosidase activity as a surrogate for tbp-specific transcription, we found that tbpB-specific transcripts were more prevalent than tbpA-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 that tbpB-specific transcripts were approximately twofold more prevalent than tbpA-specific transcripts at steady state. In iron-stressed cultures, the ratio of tbpB- to tbpA-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 on tbp mRNA levels.
Ronpirin
C
C
Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.
Jerse
A E
AE
Cornelissen
C N
CN
D019677:Artificial Gene Fusion; D002352:Carrier Proteins / Q000235:genetics*; D003470:Culture Media; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007501:Iron; D033862:Iron-Binding Proteins; D007763:Lac Operon; D008024:Ligands; D009344:Neisseria gonorrhoeae / Q000235:genetics* / Q000254:growth & development; D009876:Operon; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic; D033863:Transferrin-Binding Proteins
D002352:Carrier Proteins; D003470:Culture Media; D033862:Iron-Binding Proteins; D008024:Ligands; D033863:Transferrin-Binding Proteins; D007501:Iron