COP9 signalosome subunit 7 from Arabidopsis interacts with and regulates the small subunit of ribonucleotide reductase (RNR2).

The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.

Metagenomic retrieval of a ribosomal DNA repeat array from an uncultured marine alveolate.

The aim of this study was to explore the use of large-scale sequencing to better describe the genome content of naturally occurring, uncultured protists. We constructed a metagenomic fosmid library from a picoplanktonic assemblage (0.2-3 mum size cells) collected at the Blanes Bay Microbial Observatory (Western Mediterranean). Seven clones contained a small-subunit ribosomal RNA gene (SSU rDNA) affiliating with prasinophytes and uncultured alveolates. One clone (FBB25; 35 kb in size) was completely sequenced and found to be a tandem repeat array (5.5 times) of the rDNA operon, including three rRNA genes (SSU, large-subunit and 5.8S rDNAs) and three spacer regions (internal transcribed spacers 1, 2 and intergenic spacer). The SSU rDNA of FBB25 affiliated with the marine alveolates group I, cluster 1, and was almost identical to sequences retrieved only in marine surveys from a wide geographic and ecological range. Phylogenetic trees using the different rRNA genes showed FBB25 as an independent branch among the main alveolate groups, but their closest affiliation varied between the SSU tree (dinoflagellates) and the large-subunit and 5.8S trees (perkinsids). The spacer regions of FBB25 were particularly short when compared with other eukaryotes, indicating a possible genome streamlining in this picoeukaryote. Finally, not a single polymorphism was found in the rDNA repeat array, suggesting that the high SSU rDNA variability typically found in molecular surveys derives from organismal and not intragenomic diversity. This first report on the rDNA genomic structure of an uncultured marine alveolate improves their phylogenetic position and helps interpreting data generated during picoeukaryotic molecular surveys.

Phylogenetic analysis of the phytochrome superfamily reveals distinct microbial subfamilies of photoreceptors.

Phys (phytochromes) are a superfamily of photochromic photoreceptors that employ a bilin-type chromophore to sense red and far-red light. Although originally thought to be restricted to plants, accumulating genetic and genomic analyses now indicate that they are also prevalent among micro-organisms. By a combination of phylogenetic and biochemical studies, we have expanded the Phy superfamily and organized its members into distinct functional clades which include the phys (plant Phys), BphPs (bacteriophytochromes), Cphs (cyanobacterial Phys), Fphs (fungal Phys) and a collection of Phy-like sequences. All contain a signature GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain, which houses the bilin lyase activity. A PHY domain (uppercase letters are used to denote the PHY domain specifically), which helps stabilize the Pfr form (far-red-light-absorbing form of Phy), is downstream of the GAF region in all but the Phy-like sequences. The phy, Cph, BphP and Fph families also include a PLD [N-terminal PAS (Per/Arnt/Sim)-like domain] upstream of the GAF domain. Site-directed mutagenesis of conserved residues within the GAF and PLD motifs supports their importance in chromophore binding and/or spectral activity. In agreement with Lamparter, Carrascal, Michael, Martinez, Rottwinkel and Abian [(2004) Biochemistry 43, 3659-3669], a conserved cysteine within the PLD of several BphPs was found to be necessary for binding the chromophore via the C-3 vinyl side chain on the bilin A ring. Phy-type sequences were also discovered in the actinobacterium Kineococcus radiotolerans and collections of microorganisms obtained from marine and extremely acidic environments, thus expanding further the range of these photoreceptors. Based on their organization and distribution, the evolution of the Phy superfamily into distinct photoreceptor types is proposed.

The HWE histidine kinases, a new family of bacterial two-component sensor kinases with potentially diverse roles in environmental signaling.

Two-component signal transduction pathways play a major role in the response of bacteria to external cues. These pathways are initiated by large collection of histidine kinases (HKs) containing a sensor domain that perceives the environmental signal followed by an HK domain that triggers a histidine-aspartate phosphorelay. Previous phylogenetic analyses identified 11 major families of two-component HKs by comparing signature motifs within the HK domain. Here we describe a new family with homology to Agrobacterium tumefaciens BphP2, an HK first discovered by the presence of a phytochrome sensor domain involved in light perception. Members of this sensor HK family differ from most others by the absence of a recognizable F box and the presence of several uniquely conserved residues, including a histidine in the N box and a tryptophan-X-glutamic acid sequence in the G1 box, which we have used to define the family (HWE). At least 81 members were identified in a variety of alpha- and gamma-proteobacteria, with a significant enrichment in the Rhizobiaceae family. Several representatives were shown to have HK activity in vitro, supporting their proposed participation in phosphorelays. One or more domains related to signal transduction were evident N-terminal to the HK domain, including chemotactic methyltransferase domains, suggesting that this family has multiple roles in environmental signaling. The discovery of the HWE family further extends the diversity within the HK superfamily and expands the importance of two-component signaling in bacteria.

The pair of bacteriophytochromes from Agrobacterium tumefaciens are histidine kinases with opposing photobiological properties.

Bacteriophytochrome photoreceptors (BphPs) are a family of phytochrome-like sensor kinases that help a wide variety of bacteria respond to their light environment. In Agrobacterium tumefaciens, a unique pair of BphPs with potentially opposing roles in light sensing are present. Both AtBphPs contain an N-terminal chromophore-binding domain that covalently attaches a biliverdin chromophore. Whereas AtBphP1 assumes a Pr ground state, AtBphP2 is unusual in that it assumes a Pfr ground state that is produced nonphotochemically after biliverdin binding through a transient Pr-like intermediate. Photoconversion of AtBphP2 with far-red light then generates Pr but this Pr is also unstable and rapidly reverts nonphotochemically to Pfr. AtBphP1 contains a typical two-component histidine kinase domain at its C terminus whose activity is repressed after photoconversion to Pfr. AtBphP2 also functions as a histidine kinase but instead uses a distinct two-component kinase motif that is repressed after photoconversion to Pr. We identified sequences related to this domain in numerous predicted sensing proteins in A. tumefaciens and other bacteria, indicating that AtBphP2 might represent the founding member of a family of histidine phosphorelay proteins that is widely used in environmental signaling. By using these mutually opposing BphPs, A. tumefaciens presumably has the capacity to simultaneously sense red light-rich and far-red light-rich environments through deactivation of their associated kinase cascades.