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

Abstract

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 ␤-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.Pathogenic bacteria must obtain essential nutrients in order to establish an infection; of these nutrients, iron plays a critical role (for a recent review, see reference 53).

Transcriptional organization, regulation and role of the Porphyromonas gingivalis W83 hmu haemin-uptake locus

“…Explanations for the observed differences in gene expression within the hmuYRSTUV operon include differential transcript expression or stability. There is a precedent for differential expression within bacterial operons and usually it is a consequence of secondary structures (stem-loop structures) located within mRNA (Ehrmann et al, 1998;Khun et al, 2000;Newbury et al, 1987a, b;Ronpirin et al, 2001). Sequences capable of adopting the secondary structures are usually located in the intercistronic regions between the coding regions of genes within the operon.…”

“…Differential expression within operons encoding various bacterial transport systems has been reported (Ehrmann et al, 1998;Khun et al, 2000;Ronpirin et al, 2001). This differential expression resulted in overexpression of promoter-proximal genes and was the result of either premature transcript termination or differential transcript stability.…”

The lgtABCDE Gene Cluster, Involved in Lipooligosaccharide Biosynthesis in Neisseria gonorrhoeae , Contains Multiple Promoter Sequences

“…The introduction of the ⍀ interposon in either orientation results in at least a 1,000-fold reduction in transcription, causing polar mutations (17,18). This interposon cassette has been used to determine linkage relationships and to map the location of promoters in a number of organisms, including N. gonorrhoeae (12,17,37,42). Using ⍀ interposon insertions within the lgtABCDE region, we were able to generate evidence that supports the presence of multiple promoters.…”

Identification of Regulatory Elements That Control Expression of the tbpBA Operon in Neisseria gonorrhoeae

“…The tbpA and tbpB genes are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA (17,23,24). The genes are separated by an 86-bp region that contains an inverted repeat.…”