Identification of regulatory elements that control expression of the tbpBA operon in Neisseria gonorrhoeae.
Iron is an essential nutrient for survival and establishment of infection by Neisseria gonorrhoeae. The neisserial transferrin binding proteins (Tbps) comprise a bipartite system for iron acquisition from human transferrin. TbpA is the TonB-dependent transporter that accomplishes iron internalization. TbpB is a surface-exposed lipoprotein that makes the iron uptake process more efficient. Previous studies have shown that the genes encoding these proteins are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA and separated from it by an inverted repeat. The operon is under the control of the ferric uptake regulator (Fur); however, promoter elements necessary for regulated expression of the genes have not been experimentally defined. In this study, putative regulatory motifs were identified and confirmed by mutagenesis. Further examination of the sequence upstream of these promoter/operator motifs led to the identification of several novel repeats. We hypothesized that these repeats are involved in additional regulation of the operon. Insertional mutagenesis of regions upstream of the characterized promoter region resulted in decreased tbpB and tbpA transcript levels but increased protein levels for both TbpA and TbpB. Using RNA sequencing (RNA-Seq) technology, we determined that a long RNA was produced from the region upstream of tbpB. We localized the 5' endpoint of this transcript to between the two upstream insertions by qualitative RT-PCR. We propose that expression of this upstream RNA leads to optimized expression of the gene products from within the tbpBA operon.
Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, Richmond, Virginia, USA.
Rosuany N
RN
Vélez Acevedo
Department of Preclinical Science, Faculty of Medicine, Thammasat University, Pathumthani, Thailand.
Chalinee
C
Ronpirin
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA.
Justin L
JL
Kandler
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA Laboratories of Microbial Pathogenesis, Department of Veterans Affairs Medical Center, Decatur, Georgia, USA.
William M
WM
Shafer
Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, Richmond, Virginia, USA cncornel@vcu.edu.
Cynthia Nau
CN
Cornelissen
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D006069:Gonorrhea / Q000382:microbiology; D055029:Inverted Repeat Sequences / Q000235:genetics; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009344:Neisseria gonorrhoeae / Q000235:genetics* / Q000378:metabolism; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics; D011993:Recombinant Fusion Proteins; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics*; D016415:Sequence Alignment; D017423:Sequence Analysis, RNA; D033881:Transferrin-Binding Protein A / Q000235:genetics* / Q000378:metabolism; D033882:Transferrin-Binding Protein B / Q000235:genetics* / Q000378:metabolism
D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D033881:Transferrin-Binding Protein A; D033882:Transferrin-Binding Protein B; D007501:Iron