Agustín Vioque

Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in Anabaena sp. PCC7120

The fixation of atmospheric N2 by cyanobacteria is a major source of nitrogen in the biosphere. In Nostocales, such as Anabaena, this process is spatially separated from oxygenic photosynthesis and occurs in heterocysts. Upon nitrogen step-down, these specialized cells differentiate from vegetative cells in a process controlled by two major regulators: NtcA and HetR. However, the regulon controlled by these two factors is only partially defined, and several aspects of the differentiation process have remained enigmatic. Using differential RNA-seq, we experimentally define a genome-wide map of >10,000 transcriptional start sites (TSS) of Anabaena sp. PCC7120, a model organism for the study of prokaryotic cell differentiation and N2 fixation. By analyzing the adaptation to nitrogen stress, our global TSS map provides insight into the dynamic changes that modify the transcriptional organization at a critical step of the differentiation process. We identify >900 TSS with minimum fold change in response to nitrogen deficiency of eight. From these TSS, at least 209 were under control of HetR, whereas at least 158 other TSS were potentially directly controlled by NtcA. Our analysis of the promoters activated during the switch to N2 fixation adds hundreds of protein-coding genes and noncoding transcripts to the list of potentially involved factors. These data experimentally define the NtcA regulon and the DIF+ motif, a palindrome at or close to position −35 that seems essential for heterocyst-specific expression of certain genes.

Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli☆

The genes for the protein (C5 protein) and RNA (M1 RNA) subunits of Escherichia coli RNase P have been subcloned and their products prepared in milligram quantities by rapid procedures. The interactions between the two subunits of the enzyme have been studied in vitro by a filter-binding technique. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant for the specific interactions of the subunits in the holoenzyme complex is approximately 4 x 10(-10) M. C5 protein also interacts with various RNA molecules in a non-specific manner with a dissociation constant of 2 x 10(-8) to 6 x 10(-8) M. Regions of M1 RNA required for interaction with C5 protein have been defined by deletion analysis and footprinting techniques. These interactions are localized primarily between nucleotides 82 to 96 and 170 to 270 of M1 RNA.

Unexpected diversity of RNase P, an ancient tRNA processing enzyme : Challenges and prospects

For an enzyme functioning predominantly in a seemingly housekeeping role of 5′ tRNA maturation, RNase P displays a remarkable diversity in subunit make‐up across the three domains of life. Despite the protein complexity of this ribonucleoprotein enzyme increasing dramatically from bacteria to eukarya, the catalytic function rests with the RNA subunit during evolution. However, the recent demonstration of a protein‐only human mitochondrial RNase P has added further intrigue to the compositional variability of this enzyme. In this review, we discuss some possible reasons underlying the structural diversity of the active sites, and use them as thematic bases for elaborating new directions to understand how functional variations might have contributed to the complex evolution of RNase P.

Nucleotide sequence of the phytoene desaturase gene from Synechocystis sp. PCC 6803 and characterization of a new mutation which confers resistance to the herbicide norflurazon

Carotenoids are pigments present in all photosynthetic organisms, where they act as accessory pigments and as components of the photosynthetic reaction centers [5] and also as protectors against photo-oxidative damage [5]. Phytoene desaturase is a key enzyme in carotenoid biosynthesis, responsible for the conversion of phytoene into ~-carotene [5]. Many commercially important herbicides are inhibitors of carotenogenesis [9]. Norfturazon is a specific inhibitor of phytoene desaturase. Its interaction with the enzyme has been studied in vitro and it seems to be noncompetitive [10]. The pds gene, which codes for phytoene desaturase, has been cloned and sequenced in only two green photosynthetic organisms: Synechococcus [2, 3] and soybean [1]. In Synechococcus, a point mutation has been described which confers resistance to norflurazon [3]. In this work, a spontaneous mutant (strain AV4) which is resistant to norflurazon was isolated from Synechocystis sp. PCC 6803 by plating cells directly on plates containing 70 gM of norflurazon. DNA isolated from mutant AV4 can transform wild-type sensitive cells to norflurazon resistance with a high frequency. In order to clone the gene mutated in strain AV4, total DNA from this mutant was digested with Hind III and fractionated in a low-melting agarose gel. Fifty fractions were cut from the gel and assayed individually for their ability to confer resistance to wildtype cells upon transformation by the in situ dot method of Dzelzkalns and Bogorad [4]. The fraction of approximately 3.3 kb retained the capacity to transform wild-type cells to norflurazon resistance and was used to construct a gene library in the plasmid pGEM-7zf( + ) (Promega). Clones were analyzed individually by transformation. Out of 100 clones analyzed, one was found which could confer resistance to norflurazon to wildtype cells upon transformation and thus contained the mutation. Sequence analysis of this clone (Fig. 1) identified an open reading frame which is highly homologous to the previously sequenced pds genes from Synechococcus and soybean. In order to clone the wild-type pds gene from Syn-

Enzymatic cleavage of RNA by RNA

The discovery and characterization of the catalytic RNA subunit of the enzyme ribonuclease P ofEscherichia coli is described.

A higher-plant type ζ-carotene desaturase in the cyanobacterium Synechocystis PCC6803

The genomic DNA sequence of Synechocystis was analysed for putative ζ-carotene desaturase genes. Two promising candidates slr0940 and slr0033 were found with similarities to the structurally different ζ-carotene desaturase genes from higher plants and Anabaena, respectively. Only the expression product of the analogue to the plant gene, slr0940, was able to mediate the 2-step desaturation of ζ-carotene via neurosporene to lycopene after complementation of this pathway in Escherichia coli. When enzyme reactions were carried out with this protein, activity was obtained with either ζ-carotene or neuroporene as substrates. The in vitro reaction was inhibited by the pyrimidine derivative J852 which is effective as experimental herbicide in plants. The occurrence of two different types of ζ-carotene desaturases among cyanobacteria and the phylogenetic consequences on chloroplast evolution are discussed.

Cloning and expression in Escherichia coli of the gene coding for phytoene synthase from the cyanobacterium Synechocystis sp. PCC6803

The gene coding for the carotenoid biosynthesis enzyme phytoene synthase (pys) has been cloned from the unicellular cyanobacterium Synechocystis sp. PCC6803. The gene has been functionally expressed in Escherichia coli, where it directs the biosynthesis of phytoene from geranylgeranyl pyrophosphate (GGPP). Analysis of the sequence of the Synechocystis pys protein deduced from the gene sequence shows that it is highly homologous to the tomato and Synechococcus phytoene synthases and shows conserved domains with other bacterial phytoene synthase enzymes. The pys gene starts 60 nucleotides downstream of the pds gene (which codes for phytoene desaturase). However, it seems to be transcribed mainly from its own promoters, because insertions that disrupt the pds gene do not affect significantly the expression of the pys gene.

Role of IAA-oxidase in the formation of ethylene from 1-aminocyclopropane-1-carboxylic acid

Abstract The IAA-oxidase system of olive tree ( Olea europea ) in the presence of its substrate, IAA, and cofactors, DCP and Mn 2 , forms ethylene from 1-aminocyclopropane-l-carboxylic acid (ACC) bound as a Schiffs base to pyridoxal phosphate. Similarly, olive leaf discs upon incubation with ACC liberate considerable amounts of ethylene. The results suggest that this IAA-oxidase system may be the one active in the last step in the biosynthesis of ethylene from methionine.

Transformation of Cyanobacteria

Cyanobacteria are a diverse and successful group of bacteria defined by their ability to carry out oxygenic photosynthesis. They occupy diverse ecological niches and are important primary producers in the oceans. Cyanobacteria are amenable to genetic manipulation. Some strains are naturally transformable. Many others have been transformed in the lab by conjugation or electroporation. The ability to transform cyanobacteria has been determinant in the development of the molecular biology of these organisms and has been the basis of many of their biotechnological applications. Cyanobacteria are the source of natural products and toxins of potential use and can be engineered to synthesize substances of biotechnological interest. Their high protein and vitamin content makes them useful as a dietary supplement. Because of their ability to occupy diverse ecological niches, they can be used to deliver to the medium substances of interest or as biosensors.

RNase P from bacteria. Substrate recognition and function of the protein subunit

RNase P recognizes many different precursor tRNAs as well as other substrates and cleaves all of them accurately at the expected position. RNase P recognizes the tRNA structure of the precursor tRNA by a set of interactions between the catalytic RNA subunit and the T- and acceptor-stems mainly, although residues in the 5′-leader sequence as well as the 3′-terminal CCA are important. These conclusions have been reached by several studies on mutant precursor tRNAs as well as cross-linking studies between RNase P RNA and precursor tRNAs. The protein subunit of RNase P seems also to affect the way that the substrate is recognized as well as the range of substrates that can be used by RNase P, although the protein does not seem to interact directly with the substrates. The interaction between the protein and RNA subunits of RNase P has been extensively studiedin vitro. The protein subunit sequence is not highly conserved among bacteria, however different proteins are functionally equivalent as heterologous reconstitution of the RNase P holoenzyme can be achieved in many cases.