A small basic protein from the brz-brb operon is involved in regulation of bop transcription in Halobacterium salinarum.

BACKGROUND: The halophilic archaeon Halobacterium salinarum expresses bacteriorhodopsin, a retinal-protein that allows photosynthetic growth. Transcription of the bop (bacterioopsin) gene is controlled by two transcription factors, Bat and Brz that induce bop when cells are grown anaerobically and under light. RESULTS: A new gene was identified that is transcribed together with the brz gene that encodes a small basic protein designated as Brb (bacteriorhodopsin-regulating basic protein). The translation activity of the start codon of the brb gene was confirmed by BgaH reporter assays. In vivo site-directed mutagenesis of the brb gene showed that the Brb protein cooperates with Brz in the regulation of bop expression. Using a GFP reporter assay, it was demonstrated that Brb cooperates with both Brz and Bat proteins to activate bop transcription under phototrophic growth conditions. CONCLUSIONS: The activation of the bop promoter was shown to be dependent not only on two major factors, Bat and Brz, but is also tuned by the small basic protein, Brb.

Transcriptional control by two leucine-responsive regulatory proteins in Halobacterium salinarum R1.

BACKGROUND: Archaea combine bacterial-as well as eukaryotic-like features to regulate cellular processes. Halobacterium salinarum R1 encodes eight leucine-responsive regulatory protein (Lrp)-homologues. The function of two of them, Irp (OE3923F) and lrpA1 (OE2621R), were analyzed by gene deletion and overexpression, including genome scale impacts using microarrays. RESULTS: It was shown that Lrp affects the transcription of multiple target genes, including those encoding enzymes involved in amino acid synthesis, central metabolism, transport processes and other regulators of transcription. In contrast, LrpA1 regulates transcription in a more specific manner. The aspB3 gene, coding for an aspartate transaminase, was repressed by LrpA1 in the presence of L-aspartate. Analytical DNA-affinity chromatography was adapted to high salt, and demonstrated binding of LrpA1 to its own promoter, as well as L-aspartate dependent binding to the aspB3 promoter. CONCLUSION: The gene expression profiles of two archaeal Lrp-homologues report in detail their role in H. salinarum R1. LrpA1 and Lrp show similar functions to those already described in bacteria, but in addition they play a key role in regulatory networks, such as controlling the transcription of other regulators. In a more detailed analysis ligand dependent binding of LrpA1 was demonstrated to its target gene aspB3.

Regulation of phosphate uptake via Pst transporters in Halobacterium salinarum R1.

The genome of the archaeon Halobacterium salinarum contains two copies of the pst (phosphate-specific transport) operon, the genes of which are related to well-studied bacterial homologues. Both operons (pst1 and pst2) were shown to be polycistronic and, when under P(i)-limited conditions, transcription initiated 1 bp upstream of the translational starts. Under P(i) saturation, the pst1 operon utilized an additional transcription start site 59 bp upstream of the first one. The leaderless pst1 transcript was found to be more efficiently translated than the leadered transcript. Promoter strengths differed significantly between the two operons and when P(i) levels changed. The basal pst1 promoter activity in P(i)-saturated conditions was minimal while the pst2 promoter was active. In contrast, phosphate limitation induced the pst1 operon threefold more than the pst2 operon. We identified basic and phosphate-dependent cis-acting elements in both promoters. Phosphate-uptake assays conducted with several Pst1 and Pst2 mutant strains revealed differences in the substrate affinities between the two transporters and also suggested that the P(i)-binding proteins PstS1 and PstS2 can interact with either of the two permease subunits of the transporters. The tactic behaviour of wild type and pst-deletion strains showed that the Pst1 transporter plays an important role for phosphate-directed chemotaxis.

Flavone synthase II (CYP93B16) from soybean (Glycine max L.).

Flavonoids are a very diverse group of plant secondary metabolites with a wide array of activities in plants, as well as in nutrition and health. All flavonoids are derived from a limited number of flavanone intermediates, which serve as substrates for a variety of enzyme activities, enabling the generation of diversity in flavonoid structures. Flavonoids can be characteristic metabolites, like isoflavonoids for legumes. Others, like flavones, occur in nearly all plants. Interestingly, there exist two fundamentally different enzymatic systems able to directly generate flavones from flavanones, flavone synthase (FNS) I and II. We describe an inducible flavone synthase activity from soybean (Glycine max) cell cultures, generating 7,4'-dihydroxyflavone (DHF), which we classified as FNS II. The corresponding full-length cDNA (CYP93B16) was isolated using known FNS II sequences from other plants. Functional expression in yeast allowed the detailed biochemical characterization of the catalytic activity of FNS II. A direct conversion of flavanones such as liquiritigenin, naringenin, and eriodictyol into the corresponding flavones DHF, apigenin and luteolin, respectively, was demonstrated. The enzymatic reaction of FNSII was stereoselective, favouring the (S)- over the (R)-enantiomer. Phylogenetic analyses of the subfamily of plant CYP93B enzymes indicate the evolution of a gene encoding a flavone synthase which originally catalyzed the direct conversion of flavanones into flavones, via early gene duplication into a less efficient enzyme with an altered catalytic mechanism. Ultimately, this allowed the evolution of the legume-specific isoflavonoid synthase activity.

Phosphate-dependent behavior of the archaeon Halobacterium salinarum strain R1.

Phosphate is essential for life on earth, since it is an integral part of important biomolecules. The mechanisms applied by bacteria and eukarya to combat phosphate limitation are fairly well understood. However, it is not known how archaea sense phosphate limitation or which genes are regulated upon limitation. We conducted a microarray analysis to explore the phosphate-dependent gene expression of Halobacterium salinarum strain R1. We identified a set of 17 genes whose transcript levels increased up to several hundredfold upon phosphate limitation. Analysis of deletion mutants showed that this set of genes, the PHO stimulon, is very likely independent of signaling via two-component systems. Our experiments further indicate that PHO stimulon induction might be dependent on the intracellular phosphate concentration, which turned out to be subject to substantial changes. Finally, the study revealed that H. salinarum exhibits a phosphate-directed chemotaxis, which is induced by phosphate starvation.

A small protein from the bop-brp intergenic region of Halobacterium salinarum contains a zinc finger motif and regulates bop and crtB1 transcription.

Bacteriorhodopsin, the photosynthetic protein of Halobacterium salinarum, is optimally expressed under anaerobic growth conditions. We identified Brz (OE3104F, bacteriorhodopsin-regulating zinc finger protein), a new regulator of the bop gene. It is a small protein with a zinc finger motif, encoded directly upstream of the bop gene in the same orientation. Deletion of the brz gene caused a large decrease of bop mRNA levels as shown by Northern blot and microarray analysis. A similar effect was obtained by site-directed mutagenesis of Cys and His residues in the zinc finger motif, indicating the importance of this motif for the function of the protein. In silico analysis of the genomes from H. salinarum and other archaea revealed a large family of similar small zinc finger motif proteins, some of which may also be involved in transcription regulation of their adjacent genes.