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La forma y el tamaño celular son caracteres codificados en el genoma bacteriano y, por tanto, hereditarios. En cada especie bacteriana, estos rasgos son constantes, aunque pueden sufrir modificaciones a lo largo del ciclo celular o bajo ciertas condiciones ambientales. En bacterias, la forma celular es crucial para la interacción con el medio ambiente y, como tal, un carácter seleccionado evolutivamente. La pared celular de peptidoglicano (PG) que rodea a las células bacterianas es la estructura que determina su morfología. En bacterias con morfología bacilar, la síntesis de PG se lleva a cabo por dos complejos enzimáticos conocidos como el elongasoma y el divisoma, que permiten respectivamente el crecimiento y la división celular. Estos procesos se han caracterizado, sobre todo, en bacterias unicelulares. Sin embargo, en bacterias multicelulares la información disponible acerca de la síntesis de PG es escasa. En este trabajo se ha estudiado el efecto de algunas condiciones ambientales en la morfología y el tamaño celular, así como en la longitud de los filamentos de Anabaena sp. PCC 7120, un modelo paradigmático de multicelularidad bacteriana. Esta cianobacteria crece como filamentos uniseriados en los cuales cada célula se comunica con las células adyacentes mediante unas estructuras proteicas llamadas septal junctions. Las características peculiares de su envuelta celular, incluyendo la presencia de una membrana externa que alberga a todas las células del filamento y un periplasma continuo, que alberga un sáculo de PG también continuo, hacen de Anabaena un modelo atractivo para el estudio de la síntesis del PG. Además, en ausencia de compuestos nitrogenados, algunas células del filamento de Anabaena pueden diferenciarse en células especializadas en la fijación del nitrógeno atmosférico, llamadas heterocistos, que presentan una morfología distintiva y unas características peculiares en su pared celular. Los resultados de este trabajo han mostrado que la fuente de

As compared to eukaryotes, bacteria have a reduced tRNA gene set encoding between 30 and 220 tRNAs. Although in most bacterial phyla tRNA genes are dispersed in the genome, many species from distinct phyla also show genes forming arrays. Here, we show that two types of arrays with distinct evolutionary origins exist. This work focuses on long tRNA gene arrays (L-arrays) that encompass up to 43 genes, which disseminate by horizontal gene transfer and contribute supernumerary tRNA genes to the host. Although in the few cases previously studied these arrays were reported to be poorly transcribed, here we show that the L-array of the model cyanobacterium Anabaena sp. PCC 7120, encoding 23 functional tRNAs, is largely induced upon impairment of the translation machinery. The cellular response to this challenge involves a global reprogramming of the transcriptome in two phases. tRNAs encoded in the array are induced in the second phase of the response, directly contributing to cell survival. Results presented here show that in some bacteria the tRNA gene set may be partitioned between a housekeeping subset, which constantly sustains translation, and an inducible subset that is generally silent but can provide functionality under particular conditions.

As compared to eukaryotes, bacteria have a reduced tRNA gene set encoding between 30 and 220 tR-NAs. Although in most bacterial phyla tRNA genes are dispersed in the genome, many species from distinct phyla also show genes forming arrays. Here, we show that two types of arrays with distinct evolutionary origins exist. This work focuses on long tRNA gene arrays (L-arrays) that encompass up to 43 genes, which disseminate by horizontal gene transfer and contribute supernumerary tRNA genes to the host. Although in the few cases previously studied these arrays were reported to be poorly transcribed, here we show that the L-array of the model cyanobacterium Anabaena sp. PCC 7120, encoding 23 functional tRNAs, is largely induced upon impairment of the translation machinery. The cellular response to this challenge involves a global reprogramming of the transcriptome in two phases. tRNAs encoded in the array are induced in the second phase of the response, directly contributing to cell survival. Results presented here show that in some bacteria the tRNA gene set may be partitioned between a housekeeping subset, which constantly sustains translation, and an inducible subset that is generally silent but can provide functionality under particular conditions.

Filamentous cyanobacteria grow by intercalary cell division, which should involve distinct steps compared to those producing separate daughter cells. The N-terminal region of FtsZ is highly conserved in the clade of filamentous cyanobacteria capable of cell differentiation. A derivative of the model strain Anabaena sp. PCC 7120 expressing only an FtsZ lacking the amino acids 2-51 of the N-terminal peptide (1N-FtsZ) could not be segregated. Strain CSL110 expresses both 1N-FtsZ, from the endogenous ftsZ gene promoter, and the native FtsZ from a synthetic regulated promoter. Under conditions of 1N-FtsZ predominance, cells of strain CSL110 progressively enlarge, reflecting reduced cell division, and show instances of asymmetric cell division and aberrant Z-structures notably differing from the Z-ring formed by FtsZ in the wild type. In bacterial 2-hybrid assays FtsZ interacted with 1N-FtsZ. However, 1N-FtsZ-GFP appeared impaired for incorporation into Z-rings when expressed together with FtsZ. FtsZ, but not 1N-FtsZ, interacted with the essential protein SepF. Both FtsZ and 1N-FtsZ polymerize in vitro exhibiting comparable GTPase activities. However, filaments of FtsZ show a distinct curling forming toroids, whereas 1N-FtsZ form thick bundles of straight filaments. Thus, the N-terminal FtsZ sequence appears to contribute to a distinct FtsZ polymerization mode that is essential for cell division and division plane location in Anabaena.

tRNAs are charged with cognate amino acids by aminoacyl-tRNA synthetases (aaRSs) and subsequently delivered to the ribosome to be used as substrates for gene translation. Whether aminoacyl-tRNAs are channeled to the ribosome by transit within translational complexes that avoid their diffusion in the cytoplasm is a matter of intense investigation in organisms of the three domains of life. In the cyanobacterium Anabaena sp. PCC 7120, the valyl-tRNA synthetase (ValRS) is anchored to thylakoid membranes by means of the CAAD domain. We have investigated whether in this organism ValRS could act as a hub for the nucleation of a translational complex by attracting other aaRSs to the membranes. Out of the 20 aaRSs, only ValRS was found to localize in thylakoid membranes whereas the other enzymes occupied the soluble portion of the cytoplasm. To investigate the basis for this asymmetric distribution of aaRSs, a global search for proteins interacting with the 20 aaRSs was conducted. The interaction between ValRS and the FoF1 ATP synthase complex here reported is of utmost interest and suggests a functional link between elements of the gene translation and energy production machineries

Las aminoacil tRNA sintetasas (aaRSs) son las enzimas que catalizan la carga del aminoácido en el tRNA y son las responsables de mantener la fidelidad en la traducción del código genético. Las aaRSs son componentes esenciales de la síntesis proteica y son ubicuas en todos los dominios de la vida (Ibba y Sol, 2000; Perona y Hadd, 2012). La cianobacteria filamentosa Anabaena sp.PCC 7120 contiene dos genes de treonil tRNA sintetasa, alr0335(thrS1) y all4723 (thrS2), originados por un evento de duplicación génica ocurrido en el phylum (Luque et al., 2008). Si bien ambos genes cifran ThrRSs bioquímicamente activas, su patrón de regulación es muy diferente: mientras que thrS1 se expresa constitutivamente en condiciones estándar de cultivo, thrS2 solo se induce tras la adición de TPEN –un quelante de iones divalentes-al cultivo (Napolitano et al., 2012). En esta tesis se ha identificado el regulador que reprime la expresión del gen thrS2, el regulador Zur, que permite la expresión en condiciones de deficiencia de zinc. Se han analizado el conjunto de genes que se encuentra bajo la acción de este regulador (regulón), entre los que se encuentras transportadores de la membrana externa e interna y homólogos que utilizan cofactores alternativos al zinc, lo que ha permitido delinear la estrategia de adaptación de Anabaena a la carencia de dicho metal. Se ha caracterizado asimismo el sistema compuesto por las dos treonil-tRNA sintetasas, T1 y T2. La enzima T1 es la encargada de aminoacilar el tRNA en condiciones estándar, mientras que esta actividad es llevaba a cabo por T2 en condiciones de bajo zinc. Sorprentemente, ambas proteínas utilizan exclusivamente zinc como cofactor, por el que poseen una afinidad similar. Aunque ambas son proteínas diméricas, la pérdida del cofactor metálico induce la monomerización de T1, pero no de T2. La enzima T2 es defectiva en la edición y produce tRNA cargado con el aminoácido ilegítimo serina a una elevada tasa. Ambas enzimas pueden formar hete

tRNAs are charged with cognate amino acids by aminoacyl-tRNA synthetases (aaRSs) and subsequently delivered to the ribosome to be used as substrates for gene translation. Whether aminoacyl-tRNAs are channeled to the ribosome by transit within translational complexes that avoid their diffusion in the cytoplasm is a matter of intense investigation in organisms of the three domains of life. In the cyanobacterium Anabaena sp. PCC 7120, the valyl-tRNA synthetase (ValRS) is anchored to thylakoid membranes by means of the CAAD domain. We have investigated whether in this organism ValRS could act as a hub for the nucleation of a translational complex by attracting other aaRSs to the membranes. Out of the 20 aaRSs, only ValRS was found to localize in thylakoid membranes whereas the other enzymes occupied the soluble portion of the cytoplasm. To investigate the basis for this asymmetric distribution of aaRSs, a global search for proteins interacting with the 20 aaRSs was conducted. The interaction between ValRS and the FoF1 ATP synthase complex here reported is of utmost interest and suggests a functional link between elements of the gene translation and energy production machineries.

Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNAThr synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNAThr. Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs.

Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNAThr synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNAThr. Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs.

In the cyanobacterium Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120), a zinc-responsive operon (all4725-all4721) has been described, which contains 4 distinct promoters. The two most upstream ones bind Zur with high affinity, whereas the other two do not or do so with a very low affinity. In this paper, a detailed characterization of the four promoters is presented, showing that all four were induced by metal depletion, and they were constitutively derepressed in a zur mutant, despite the two downstream promoters not being direct targets for this regulator. Crucially, induction by metal depletion of the two downstream promoters was abrogated when transcription initiated at the upstream promoters was interrupted by a polar insertion midway in the operon. In contrast, insertion of a nitrogen-responsive promoter at a roughly similar position provoked the two downstream promoters to adopt a regulatory pattern mimicking that of the inserted promoter. Thus, regulation of the two downstream promoters is apparently influenced by transcription from promoters upstream. Evidence is presented indicating that the activity of the two downstream promoters is kept basal in Anabaena by repression. A regulatory model compatible with these results is proposed, where promoters controlled by repression in bacterial operons may be subjected to a hierarchical regulation depending on their position in the operon. According to this model, internal promoters may respond to stimuli governing the activity of promoters upstream by an indirect regulation and to specific stimuli by a direct regulation.

Prochlorococcus is responsible for a significant part of CO2 fixation in the ocean. Although it was long considered an autotrophic cyanobacterium, the uptake of organic compounds has been reported, assuming they were sources of limited biogenic elements. We have shown in laboratory experiments that Prochlorococcus can take up glucose. However, the mechanisms of glucose uptake and its occurrence in the ocean have not been shown. Here, we report that the gene Pro1404 confers capability for glucose uptake in Prochlorococcus marinus SS120. We used a cyanobacterium unable to take up glucose to engineer strains that express the Pro1404 gene. These recombinant strains were capable of specific glucose uptake over a wide range of glucose concentrations, showing multiphasic transport kinetics. The Ks constant of the high affinity phase was in the nanomolar range, consistent with the average concentration of glucose in the ocean. Furthermore, we were able to observe glucose uptake by Prochlorococcus in the central Atlantic Ocean, where glucose concentrations were 0.5-2.7 nM. Our results suggest that Prochlorococcus are primary producers capable of tuning their metabolism to energetically benefit from environmental conditions, taking up not only organic compounds with key limiting elements in the ocean, but also molecules devoid of such elements, like glucose.

Multiple reaction monitoring (MRM) is emerging as a promising technique for the detection and quantification of protein biomarkers in complex biological samples. Compared to Western blotting or enzyme assays, its high sensitivity, specificity, accuracy, assay speed, and sample throughput represent a clear advantage for being the approach of choice for the analysis of proteins. MRM assays are capable of detecting and quantifying proteolytic peptides differing in mass unique to particular proteins, that is, proteotypic peptides, through which different protein isoforms can be distinguished. We have focused on polyphenol oxidase (PPO), a plant conspicuous enzyme encoded by a multigenic family in loquat (Eriobotrya japonica Lindl.) and other related species. PPO is responsible for both the protection of plants from biotic stress as a feeding deterrent for herbivore insects and the enzymatic browning of fruits and vegetables. The latter makes fruit more attractive to seed dispersal agents but is also a major cause of important economic losses in agriculture and food industry. An adequate management of PPO at plant breeding level would maximize the benefits and minimize the disadvantages of this enzyme, but it would require a precise knowledge of the biological role played by each isoform in the plant. Thus, for the functional study of the PPOs, we have cloned and overexpressed fragments of three PPO isoforms from loquat to develop MRM-based methods for the quantification of each isoform. The method was developed using an ion trap instrument and validated in a QQQ instrument. It resulted in the selection of at least two peptides for each isoform that can be monitored by at least three transitions. A combination of SDS-PAGE and MRM lead to detect two out of three monitored isoforms in different gel bands corresponding to different processing stages of PPO. The method was applied to determine the amount of the PPO2 isoform in protein extracts from fruit samples using extern

Zur regulators control zinc homeostasis by repressing target genes under zinc-sufficient conditions in a wide variety of bacteria. This paper describes how part of a survey of duplicated genes led to the identification of the open reading frame all2473 as the gene encoding the Zur regulator of the cyanobacterium Anabaena sp. strain PCC 7120. All2473 binds to DNA in a zinc-dependent manner, and its DNA-binding sequence was characterized, which allowed us to determine the relative contribution of particular nucleotides to Zur binding. A zur mutant was found to be impaired in the regulation of zinc homeostasis, showing sensitivity to elevated concentrations of zinc but not other metals. In an effort to characterize the Zur regulon in Anabaena, 23 genes containing upstream putative Zur-binding sequences were identified and found to be regulated by Zur. These genes are organized in six single transcriptional units and six operons, some of them containing multiple Zur-regulated promoters. The identities of genes of the Zur regulon indicate that Anabaena adapts to conditions of zinc deficiency by replacing zinc metalloproteins with paralogues that fulfill the same function but presumably with a lower zinc demand, and with inducing putative metallochaperones and membrane transport systems likely being involved in the scavenging of extracellular zinc, including plasma membrane ABC transport systems and outer membrane TonB-dependent receptors. Among the Zur-regulated genes, the ones showing the highest induction level encode proteins of the outer membrane, suggesting a primary role for components of this cell compartment in the capture of zinc cations from the extracellular medium.

The PipX factor is a regulatory protein that seems to occur only in cyanobacteria. In the filamentous, heterocyst-forming Anabaena sp. strain PCC 7120, open reading frame (ORF) asr0485, identified as the pipX gene, is expressed mainly under conditions of combined-nitrogen deprivation dependent on the global N regulator NtcA and the heterocyst-specific regulator HetR. Primer extension and 5′ rapid amplification of cDNA ends (RACE) analyses detected three transcription start points corresponding to a canonical NtcA-activated promoter (to which direct binding of NtcA was observed), an NtcA- and HetR-dependent promoter, and a consensus-type promoter, the last with putative -35 and -10 determinants. Activation of pipX took place in cells differentiating into heterocysts at intermediate to late stages of the process. Accordingly, disruption of pipX led to impaired diazotrophic growth, reduced nitrogenase activity, and impaired activation of the nitrogenase structural genes. The nitrogenase activity of the mutant was low under oxic conditions, likely resulting from inefficient protection against oxygen. In line with this, the activation of the coxB2A2C2 and coxB3A3C3 operons, encoding heterocyst-specific terminal respiratory oxidases responsible for internal oxygen removal, was deficient in the pipX mutant. Therefore, the Anabaena PipX factor shows a spatiotemporal specificity contributing to normal heterocyst function, including full activation of the nitrogenase structural genes and genes of the nitrogenase-protective features of the heterocyst.

PII, one of the most conserved signal transduction proteins, is believed to be a key player in the coordination of nitrogen assimilation and carbon metabolism in bacteria, archaea, and plants. However, the identity of PII receptors remains elusive, particularly in photosynthetic organisms. Here we used yeast two-hybrid approaches to identify new PII receptors and to explore the extent of conservation of PII signaling mechanisms between eubacteria and photosynthetic eukaryotes. Screening of Synechococcus sp. strain PCC 7942 libraries with PII as bait resulted in identification of N-acetyl glutamate kinase (NAGK), a key enzyme in the biosynthesis of arginine. The integrity of Ser49, a residue conserved in PII proteins from organisms that perform oxygenic photosynthesis, appears to be essential for NAGK binding. The effect of glnB mutations on NAGK activity is consistent with positive regulation of NAGK by PII. Phylogenetic and yeast two-hybrid analyses strongly suggest that there was conservation of the NAGK-PII regulatory interaction in the evolution of cyanobacteria and chloroplasts, providing insight into the function of eukaryotic PII-like proteins.

Los objetivos de este trabajo han sido profundizar en el estudio de la estructura genética de los genes implicados en la asimilación de nitrato en Synechococcus sp. PCC 7942, y contribuir al esclarecimiento del mecanismo de regulación de dichos genes mediante la proteína NtcA.