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Infection and Immunity, October 2001, p. 6336-6347, Vol. 69, No. 10
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.10.6336-6347.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Chalinee Ronpirin,1 Ann E. Jerse,2 and Cynthia Nau Cornelissen1,*

Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0678,1 and Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 208142

Received 27 April 2001/Returned for modification 19 June 2001/Accepted 9 July 2001

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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, P.O. Box 980678, Richmond, VA 23298-0678. Phone: (804) 827-1754. Fax: (804) 828-9946. E-mail: cncornel{at}hsc.vcu.edu.

Infection and Immunity, October 2001, p. 6336-6347, Vol. 69, No. 10
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.10.6336-6347.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.