Characterization and transcriptional analysis of hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase from a unicellular cyanobacterium.
The structural genes (hupSL) encoding an uptake hydrogenase in the unicellular cyanobacterium Gloeothece sp. ATCC 27152, a strain capable of aerobic N(2) fixation, were identified and sequenced. 3'-RACE experiments uncovered the presence of an additional ORF 184 bp downstream of hupL, showing a high degree of sequence identity with a gene encoding an uptake-hydrogenase-specific endopeptidase (hupW) in other cyanobacteria. In addition, the transcription start point was identified 238 bp upstream of the hupS translational start. RT-PCR experiments revealed that hupW is co-transcribed with the uptake hydrogenase structural genes in Gloeothece sp. ATCC 27152. In addition, Northern hybridizations clearly showed that hupSLW are transcribed under nitrogen fixing conditions, but not in the presence of combined nitrogen. A putative NtcA binding site was identified in the promoter region upstream of hupS, centred at -41.5 bp with respect to the transcription start point. Electrophoretic retardation of a labelled DNA fragment (harbouring the putative NtcA-binding motif) was significantly affected by an Escherichia coli cell-free extract containing overexpressed NtcA, suggesting that NtcA is involved in the transcriptional regulation of hupSLW.
Oliveira
Paulo
P
Department of Botany, Faculty of Sciences, University of Porto, Rua do Campo Alegre 1191, 4150-181 Porto, Portugal.
Leitão
Elsa
E
Institute for Molecular and Cell Biology - Cellular and Applied Microbiology Unit, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
Tamagnini
Paula
P
Institute for Molecular and Cell Biology - Cellular and Applied Microbiology Unit, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.|Department of Botany, Faculty of Sciences, University of Porto, Rua do Campo Alegre 1191, 4150-181 Porto, Portugal.
Moradas-Ferreira
Pedro
P
Instituto de Ciências Biomédicas Abel Salazar, University of Porto, Largo Abel Salazar 2, 4099-003 Porto, Portugal.|Institute for Molecular and Cell Biology - Cellular and Applied Microbiology Unit, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
Oxelfelt
Fredrik
F
Institute for Molecular and Cell Biology - Cellular and Applied Microbiology Unit, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D015152:Blotting, Northern; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D023061:Gene Order; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D009586:Nitrogen Fixation; D010088:Oxidoreductases / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D024363:Transcription Initiation Site
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D010088:Oxidoreductases; C029746:uptake hydrogenase; D009584:Nitrogen