Characterization and transcriptional analysis of hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase from a unicellular cyanobacterium

Abstract

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. 39-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. INTRODUCTIONSeveral species of bacteria and cyanobacteria are capable of N 2 fixation. During the N 2 fixation process H 2 is formed as a by-product. This nitrogenase-dependent H 2 production is often compromised by the presence of an uptake hydrogenase (encoded by hupSL) that rapidly consumes the H 2 generated. In addition, a bi-directional enzyme (encoded by hoxEFUYH) may be present which, depending on the growth conditions, may display the capacity of both producing and consuming H 2 (Lambert & Smith, 1981;Houchins, 1984;Schmitz et al., 2002;Tamagnini et al., 2002). All cyanobacteria examined so far contain an uptake, a bi-directional or both the hydrogenases (Schmitz et al., 1995(Schmitz et al., , 2002Boison et al., 1996;Tamagnini et al., 2000Tamagnini et al., , 2002Sheremetieva et al., 2002; Schütz et al., 2004). Moreover, the uptake-type enzyme has been found in all nitrogen fixing cyanobacterial strains studied to date (Carrasco & Golden, 1995;Oxelfelt et al., 1998;Happe et al., 2000;Tamagnini et al., 2000Tamagnini et al., , 2002 Schütz et al., 2004).The first data on cyanobacterial hupSL transcription appeared in 1995 (Carrasco & Golden, 1995). RT-PCR experiments on Anabaena/Nostoc sp. strain PCC 7120 demonstrated that hupL transcription coincides with the formation of heterocysts. Subsequent studies, in other filamentous strains, have confirmed the induction of an hupL transcript under nitrogen-fixing conditions only (Axelsson et al., 1999;Happe et al., 2000; Hansel et al., 2001). One exception is Anabaena variabilis ATCC 29413, where a low level of hupL expression has been detected in vegetative cells grown with the addition of ammonia (Boison et al., 2000). In all the cyanobacterial strains 3Present address: Department of Physiological Botany, Evolutionary Biology Centre,...

An AbrB-Like Protein Regulates the Expression of the Bidirectional Hydrogenase in Synechocystis sp. Strain PCC 6803

“…32 P]ATP as described previously (27). For each 20-l reaction mixture, 20 fmol of each labeled DNA fragment was incubated with various amounts of Sll0359 (see below) (in a buffer described previously [26]), supplemented with 1 g of salmon sperm DNA.…”

“…NtcA, the nitrogen control regulator in cyanobacteria, has been suggested to mediate the transcription of the uptake hydrogenase in different cyanobacterial strains, Nostoc punctiforme PCC 73102 (24), Gloeothece sp. strain ATCC 27152 (27), and Lyngbya majuscula CCAP 1446/4 (23), as well as that of the hydrogenase maturation proteins in Lyngbya majuscula CCAP 1446/4 (15). A LexA-related protein, which has been proposed to not be involved in the classical regulation of DNA repair genes (11), was shown to interact in two different regions of the promoter of the hox genes in Synechocystis sp.…”

“…In addition, results obtained with two genetically modified strains of Synechocystis sp. strain PCC 6803, one with a not fully segregated inactivation mutation of the abrB-like gene and the other overexpressing the same abrB-like gene, suggest that this transcription factor functions as a regulator of hox gene expression.Two different Ni-Fe hydrogenases have been characterized physiologically, biochemically, and on the molecular level in cyanobacteria: one is named uptake hydrogenase and catalyzes the consumption of H 2 produced by the nitrogenase during N 2 fixation, and the other is termed bidirectional hydrogenase and its function is still under debate (32,37,38).Over the years, it has continuously been shown by reverse transcription-PCR and Northern blot analysis (2,3,4,15,23,27,33,38) that the transcription of cyanobacterial hydrogenases varies under different environmental conditions. Nevertheless, an understanding of which key regulators are involved is just emerging and the inherent signal transduction pathways certainly deserve better attention.…”

“…Over the years, it has continuously been shown by reverse transcription-PCR and Northern blot analysis (2,3,4,15,23,27,33,38) that the transcription of cyanobacterial hydrogenases varies under different environmental conditions. Nevertheless, an understanding of which key regulators are involved is just emerging and the inherent signal transduction pathways certainly deserve better attention.…”

Novel Insights into the Regulation of LexA in the Cyanobacterium Synechocystis sp. Strain PCC 6803

“…Even though 3Ј RACE does not exactly determine the point where a given cyanobacterial transcript is terminated (polyadenylation of transcripts in Synechocystis sp. strain PCC 6803 is known to occur [38], but associated with their degradation rather than with their stabilization, as in eukaryotes), it has been used successfully with cyanobacterial RNA to examine the 3Ј ends of different transcripts (32,38). Thus, even assuming that the real transcription stop points of the lexA and slr1735 transcripts are localized between 50 and 80 nucleotides downstream of each of the identified sites, these transcripts still would not overlap.…”