Cloning, Sequencing, and Regulation of the Glutathione Reductase Gene from the Cyanobacterium Anabaena PCC 7120

Abstract

Glutathione reductase (GR) was purified from the cyanobacterium Anabaena PCC 7120. A 3-kilobase genomic DNA fragment containing the coding sequence for the GR gene (gor) was identified and cloned by polymerase chain reaction based on sequences of selected peptides isolated from proteolyzed GR. The coding sequence encompassing 458 amino acid residues, as well as 360 base pairs of the 5-flanking region and 430 base pairs of the 3-flanking region, were determined. Genomic Southern analysis indicates that gor is a single-copy gene. A gor antisense RNA probe hybridized with a 1.4-kilobase transcript, suggesting that the gene is not part of an operon including additional genes. The deduced GR amino acid sequence shows 41 to 48% identity with those of human, Escherichia coli, Pseudomonas aeruginosa, pea, and Arabidopsis thaliana GR. The coding sequence of GR was overexpressed in a GR-deficient E. coli strain, SG5, and the recombinant protein was purified. Anabaena GR is NADPH-linked, but a Lys residue replaces an Arg residue involved in NADPH binding in GR from other species. In addition, Anabaena GR carries the GXGXXG "fingerprint" motif which otherwise characterizes NAD(H)-dependent enzymes. These differences may contribute to the lack of affinity for 2,5-ADPSepharose 4B of Anabaena GR. Three E. coli-type promoter sequences and a BifA/NtcA binding motif were found upstream of the open reading frame. The middle and the proximal promoters were shown to be active. However, the use of the middle promoter was dependent on the nitrogen source in the culture medium. Both GR activity and GR protein concentration increased in ammonium grown cultures in which both the middle and proximal promoters were used for transcriptional initiation. The BifA/NtcA-binding site overlaps the middle promoter sequence and may thus be involved in regulation of differential transcription.

Biochemical Characterization of the CDP- d -Arabinitol Biosynthetic Pathway in Streptococcus pneumoniae 17F

“…S8 in the supplemental material). The motif GXXGXXG, located near the N terminus of the proteins, is similar to the motif (GXGXXA, GXG XXG) involved in the binding of cofactor NAD(P)H (8,17). The second motif is very similar to the previously reported motif SX 24 YXXXK in a dehydrogenase from Comamonas testosteroni, in which the Ser, Tyr, and Lys residues play important roles in catalytic activity (11,14).…”

Nitrogen Control in Cyanobacteria

“…Finally, a sequence, TGTN 9 ACA, located at a position that would be compatible with an NtcA repressor site (Fig. 6) has been reported for a promoter of the Anabaena gor gene, encoding glutathione reductase, that is preferentially used in ammonium-grown cells (53). This sequence is similar to those discussed above for the NtcA-binding sites of xisA and glbN and could represent a weak binding site for NtcA (55).…”

NtcA from Microcystis aeruginosa PCC 7806 Is Autoregulatory and Binds to the Microcystin Promoter

“…Several NtcA binding sites were identified, one of which overlaps the 70 -type E. coli promoter. This organization may result in the production of multiple transcripts under cellular conditions, as needed (6). This promoter arrangement echoes that of the mcyA/D promoter, and therefore it is possible that NtcA also acts as a repressor of microcystin synthetase transcription.…”