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1.
Nucleic Acids Res ; 49(15): 8757-8776, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34379789

RESUMO

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.


Assuntos
Genes Bacterianos , Óperon , Biossíntese de Proteínas , RNA de Transferência/genética , Estresse Fisiológico/genética , Anabaena/genética , Antibacterianos/farmacologia , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Viabilidade Microbiana/genética , RNA de Transferência/metabolismo , Sequências Reguladoras de Ácido Nucleico
2.
mSphere ; 5(5)2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33115834

RESUMO

The model cyanobacterium Anabaena sp. PCC 7120 exhibits a phototrophic metabolism relying on oxygenic photosynthesis and a complex morphology. The organismic unit is a filament of communicated cells that may include cells specialized in different nutritional tasks, thus representing a paradigm of multicellular bacteria. In Anabaena, the inorganic carbon and nitrogen regime influenced not only growth, but also cell size, cell shape, and filament length, which also varied through the growth cycle. When using combined nitrogen, especially with abundant carbon, cells enlarged and elongated during active growth. When fixing N2, which imposed lower growth rates, shorter and smaller cells were maintained. In Anabaena, gene homologs to mreB, mreC, and mreD form an operon that was expressed at higher levels during the phase of fastest growth. In an ntcA mutant, mre transcript levels were higher than in the wild type and, consistently, cells were longer. Negative regulation by NtcA can explain that Anabaena cells were longer in the presence of combined nitrogen than in diazotrophic cultures, in which the levels of NtcA are higher. mreB, mreC, and mreD mutants could grow with combined nitrogen, but only the latter mutant could grow diazotrophically. Cells were always larger and shorter than wild-type cells, and their orientation in the filament was inverted. Consistent with increased peptidoglycan width and incorporation in the intercellular septa, filaments were longer in the mutants, suggesting a role for MreB, MreC, and MreD in the construction of septal peptidoglycan that could affect intercellular communication required for diazotrophic growth.IMPORTANCE Most studies on the determination of bacterial cell morphology have been conducted in heterotrophic organisms. Here, we present a study of how the availability of inorganic nitrogen and carbon sources influence cell size and morphology in the context of a phototrophic metabolism, as found in the multicellular cyanobacterium Anabaena In Anabaena, the expression of the MreB, MreC, and MreD proteins, which influence cell size and length, are regulated by NtcA, a transcription factor that globally coordinates cellular responses to the C-to-N balance of the cells. Moreover, MreB, MreC, and MreD also influence septal peptidoglycan construction, thus affecting filament length and, possibly, intercellular molecular exchange that is required for diazotrophic growth. Thus, here we identified new roles for Mre proteins in relation to the phototrophic and multicellular character of a cyanobacterium, Anabaena.

3.
Front Microbiol ; 9: 2260, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30333801

RESUMO

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 (ΔN-FtsZ) could not be segregated. Strain CSL110 expresses both ΔN-FtsZ, from the endogenous ftsZ gene promoter, and the native FtsZ from a synthetic regulated promoter. Under conditions of ΔN-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 ΔN-FtsZ. However, ΔN-FtsZ-GFP appeared impaired for incorporation into Z-rings when expressed together with FtsZ. FtsZ, but not ΔN-FtsZ, interacted with the essential protein SepF. Both FtsZ and ΔN-FtsZ polymerize in vitro exhibiting comparable GTPase activities. However, filaments of FtsZ show a distinct curling forming toroids, whereas ΔN-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.

4.
Plant Cell Physiol ; 59(9): 1860-1873, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-29878163

RESUMO

Thylakoid membranes are far from being homogeneous in composition. On the contrary, compositional heterogeneity of lipid and protein content is well known to exist in these membranes. The mechanisms for the confinement of proteins at a particular membrane domain have started to be unveiled, but we are far from a thorough understanding, and many issues remain to be elucidated. During the differentiation of heterocysts in filamentous cyanobacteria of the Anabaena and Nostoc genera, thylakoids undergo a complete reorganization, separating into two membrane domains of different appearance and subcellular localization. Evidence also indicates different functionality and protein composition for these two membrane domains. In this work, we have addressed the mechanisms that govern the specific localization of proteins at a particular membrane domain. Two classes of proteins were distinguished according to their distribution in the thylakoids. Our results indicate that the specific accumulation of proteins of the CURVATURE THYLAKOID 1 (CURT1) family and proteins containing the homologous CAAD domain at subpolar honeycomb thylakoids is mediated by multiple mechanisms including a previously unnoticed phenomenon of thylakoid membrane migration.


Assuntos
Anabaena/fisiologia , Tilacoides/fisiologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fotossíntese , Transporte Proteico
5.
Methods Mol Biol ; 1696: 147-162, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29086402

RESUMO

Targeted mass spectrometric methods such as selected/multiple reaction monitoring (SRM/MRM) have found intense application in protein detection and quantification which competes with classical immunoaffinity techniques. It provides a universal procedure to develop a fast, highly specific, sensitive, accurate, and cheap methodology for targeted detection and quantification of proteins based on the direct analysis of their surrogate peptides typically generated by tryptic digestion. This methodology can be advantageously applied in the field of plant proteomics and particularly for non-model species since immunoreagents are scarcely available. Here, we describe the issues to take into consideration in order to develop a MRM method to detect and quantify isoforms of the thylakoid-bound protein polyphenol oxidase from the non-model and database underrepresented species Eriobotrya japonica Lindl.


Assuntos
Eriobotrya/citologia , Isoformas de Proteínas/isolamento & purificação , Proteômica/métodos , Tilacoides/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Eriobotrya/genética , Eriobotrya/metabolismo , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Isoformas de Proteínas/genética
6.
Front Microbiol ; 7: 857, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27375579

RESUMO

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.

7.
Nucleic Acids Res ; 43(20): 9905-17, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26464444

RESUMO

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-tRNA(Thr) synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNA(Thr). 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.


Assuntos
Genes Duplicados , Treonina-tRNA Ligase/genética , Treonina-tRNA Ligase/metabolismo , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Anabaena/enzimologia , Anabaena/genética , Código Genético , Isoenzimas/genética , Isoenzimas/metabolismo , Multimerização Proteica , Edição de RNA , Estresse Fisiológico/genética , Zinco/metabolismo
8.
Environ Microbiol ; 17(6): 2006-17, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25244409

RESUMO

Iron and zinc are necessary nutrients whose homeostasis is tightly controlled by members of the ferric uptake regulator (FUR) superfamily in the cyanobacterium Anabaena sp. PCC7120. Although the link between iron metabolism and oxidative stress management is well documented, little is known about the connection between zinc homeostasis and the oxidative stress response in cyanobacteria. Zinc homeostasis in Anabaena is controlled by Zur, also named FurB. When overexpressed in Escherichia coli, Zur (FurB) improved cell survival during oxidative stress. In order to investigate the possible correlation between Zur and the oxidative stress response in Anabaena, zur deletion and zur-overexpressing strains have been constructed, and the consequences of Zur imbalance evaluated. The lack of Zur increased sensitivity to hydrogen peroxide (H2 O2 ), whereas an excess of Zur enhanced oxidative stress resistance. Both mutants displayed pleiotropic phenotypes, including alterations on the filament surfaces observable by scanning electron microscopy, reduced content of endogenous H2 O2 and altered expression of sodA, catalases and several peroxiredoxins. Transcriptional and biochemical analyses unveiled that the appropriate level of Zur is required for proper control of the oxidative stress response and allowed us to identify major antioxidant enzymes as novel members of the Zur regulon.


Assuntos
Anabaena/metabolismo , Anabaena/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo/fisiologia , Anabaena/genética , Catalase/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Oxirredução , Estresse Oxidativo/genética , Peroxirredoxinas/metabolismo , Regulon , Superóxido Dismutase/metabolismo , Zinco/metabolismo
9.
Anal Biochem ; 452: 46-53, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24556246

RESUMO

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were>2.0) but also of high yield (up to 720 µg on average [coefficient of variation=21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.


Assuntos
Fracionamento Químico/métodos , Eriobotrya/química , RNA de Plantas/isolamento & purificação , Madeira/química , Cetrimônio , Compostos de Cetrimônio/química , Eriobotrya/genética , Eriobotrya/crescimento & desenvolvimento , Genoma de Planta/genética , RNA de Plantas/química , Madeira/genética , Madeira/crescimento & desenvolvimento
10.
Trends Plant Sci ; 19(2): 63-6, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24388270

RESUMO

CURT1 proteins induce membrane curvature to grana margins in Arabidopsis (Arabidopsis thaliana) thylakoids. A domain sharing sequence and structural features with CURT1 is found in some cyanobacterial aminoacyl-tRNA synthetases (aaRSs) that show an unusual localization to the thylakoid membranes. Evolutionary scenarios and functional implications are discussed in this article.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Tilacoides/enzimologia , Tilacoides/metabolismo
11.
J Proteome Res ; 12(12): 5709-22, 2013 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-24245590

RESUMO

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 external calibrants.


Assuntos
Catecol Oxidase/genética , Eriobotrya/genética , Frutas/genética , Regulação da Expressão Gênica de Plantas , Fragmentos de Peptídeos/isolamento & purificação , Proteínas de Plantas/genética , Sequência de Aminoácidos , Calibragem , Catecol Oxidase/química , Catecol Oxidase/metabolismo , Cromatografia Líquida , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Eriobotrya/enzimologia , Frutas/enzimologia , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Família Multigênica , Fragmentos de Peptídeos/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas em Tandem
12.
Proc Natl Acad Sci U S A ; 110(21): 8597-602, 2013 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-23569224

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glucose/metabolismo , Oceanos e Mares , Prochlorococcus/metabolismo , Microbiologia da Água , Proteínas de Bactérias/genética , Transporte Biológico Ativo/fisiologia , Proteínas Facilitadoras de Transporte de Glucose/genética , Prochlorococcus/genética
13.
J Bacteriol ; 195(6): 1285-93, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23316045

RESUMO

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.


Assuntos
Anabaena/genética , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Transcrição Genética , Zinco/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
J Bacteriol ; 194(10): 2426-36, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22389488

RESUMO

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.


Assuntos
Anabaena/efeitos dos fármacos , Anabaena/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Zinco/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Regulação Bacteriana da Expressão Gênica/fisiologia , Homeostase , Metaloproteínas/genética , Metaloproteínas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica
15.
J Biol Chem ; 286(47): 41057-68, 2011 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-21965654

RESUMO

Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain.


Assuntos
Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Membrana Celular/enzimologia , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/genética , Anabaena/citologia , Anabaena/enzimologia , Membrana Celular/metabolismo , Evolução Molecular , Espaço Intracelular/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Valina-tRNA Ligase/metabolismo
16.
J Bacteriol ; 193(5): 1172-82, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21193608

RESUMO

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.


Assuntos
Anabaena/fisiologia , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Anabaena/classificação , Anabaena/genética , Proteínas de Bactérias/genética , Sequência de Bases , Pegada de DNA , DNA Bacteriano , Desoxirribonuclease I/metabolismo , Dados de Sequência Molecular , Mutação
17.
Mol Biol Evol ; 25(11): 2369-89, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18775898

RESUMO

A comparative genomic analysis of 35 cyanobacterial strains has revealed that the gene complement of aminoacyl-tRNA synthetases (AARSs) and routes for aminoacyl-tRNA synthesis may differ among the species of this phylum. Several genes encoding AARS paralogues were identified in some genomes. In-depth phylogenetic analysis was done for each of these proteins to gain insight into their evolutionary history. GluRS, HisRS, ArgRS, ThrRS, CysRS, and Glu-Q-RS showed evidence of a complex evolutionary course as indicated by a number of inconsistencies with our reference tree for cyanobacterial phylogeny. In addition to sequence data, support for evolutionary hypotheses involving horizontal gene transfer or gene duplication events was obtained from other observations including biased sequence conservation, the presence of indels (insertions or deletions), or vestigial traces of ancestral redundant genes. We present evidences for a novel protein domain with two putative transmembrane helices recruited independently by distinct AARS in particular cyanobacteria.


Assuntos
Aminoacil-tRNA Sintetases/genética , Proteínas de Bactérias/genética , Cianobactérias/genética , Evolução Molecular , Motivos de Aminoácidos , Aminoacil-tRNA Sintetases/metabolismo , Arginina-tRNA Ligase/genética , Arginina-tRNA Ligase/metabolismo , Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/metabolismo , Cianobactérias/classificação , Cianobactérias/enzimologia , Duplicação Gênica , Variação Genética , Genoma Bacteriano , Glutamato-tRNA Ligase/genética , Glutamato-tRNA Ligase/metabolismo , Histidina-tRNA Ligase/genética , Histidina-tRNA Ligase/metabolismo , Filogenia , Estrutura Terciária de Proteína , RNA Bacteriano/metabolismo , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência de Asparagina/metabolismo , RNA de Transferência de Glutamina/metabolismo , Treonina-tRNA Ligase/genética , Treonina-tRNA Ligase/metabolismo
18.
J Proteome Res ; 7(9): 4095-106, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18620449

RESUMO

Here, we approach the problem of obtaining accurate and reliable information about the gene origin of a protein belonging to a multigenic family, polyphenol oxidase, from an underrepresented species, Eriobotrya japonica. De novo sequencing was a key approach to obtain broad sequence coverage. Alignment of peptides on their most similar homologous protein revealed divergent amino acid positions that lead to hypothesize the minimal number of genes encoding for the proteins analyzed.


Assuntos
Catecol Oxidase/metabolismo , Bases de Dados de Proteínas , Eriobotrya/enzimologia , Família Multigênica , Sequência de Aminoácidos , Formação de Anticorpos , Western Blotting , Catecol Oxidase/química , Catecol Oxidase/genética , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Solubilidade
19.
Mol Microbiol ; 60(5): 1276-88, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16689802

RESUMO

The genome of Tolypothrix sp. PCC 7601 carries two copies of a novel insertion sequence, ISTosp1. One of the two copies is located upstream of the gene encoding glutamyl-tRNA synthetase, an enzyme playing a key role in protein and pigment synthesis. The tnpA gene of the IS element and gltX were co-transcribed and their expression was transiently upregulated upon retrieval of the ammonium source irrespective of whether nitrate or no nitrogen source were available. The second copy is also transcribed and shows a similar regulatory pattern. Structural elements of the promoter (-10 and -35 sequences) directing the expression of the tnpA-gltX operon have been localized within the IS. Regulatory sequences involving the NtcA transcription factor in the control of tnpA-gltX expression were found both within and in sequences upstream of the insertion element. The expression of gltX in a closely related cyanobacterium, Nostoc sp. PCC 7120, which lacks the insertion upstream of gltX, decreased upon ammonium retrieval, a regulatory pattern that markedly differs from that observed in Tolypothrix sp. PCC 7601. ISTosp1 constitutes a good example of how cells can make use of a transposable element to evolve an original regulatory mechanism.


Assuntos
Cianobactérias , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Glutamato-tRNA Ligase/metabolismo , Sequências Repetitivas Dispersas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Cianobactérias/enzimologia , Cianobactérias/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glutamato-tRNA Ligase/genética , Dados de Sequência Molecular , Nostoc/genética , Nostoc/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Microb Ecol ; 52(3): 365-77, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16710791

RESUMO

Biological soil crusts are very sensitive to human-induced disturbances and are in a degraded state in many areas throughout their range. Given their importance in the functioning of arid and semiarid ecosystems, restoring these crusts may contribute to the recovery of ecosystem functionality in degraded areas. We conducted a factorial microcosm experiment to evaluate the effects of inoculation type (discrete fragments vs slurry), fertilization (control vs addition of composted sewage sludge), and watering frequency (two vs five times per week) on the cyanobacterial composition, nitrogen fixation, chlorophyll content, and net CO2 exchange rate of biological soil crusts inoculated on a semiarid degraded soil from SE Spain. Six months after the inoculation, the highest rates of nitrogen fixation and chlorophyll a content were found when the biological crusts were inoculated as slurry, composted sewage sludge was added, and the microcosms were watered five times per week. Net CO2 exchange rate increased when biological crusts were inoculated as slurry and the microcosms were watered five times per week. Denaturing gradient gel electrophoresis fingerprints and phylogenetic analyses indicated that most of the cyanobacterial species already present in the inoculated crust had the capability to spread and colonize the surface of the surrounding soil. These analyses showed that cyanobacterial communities were less diverse when the microcosms were watered five times per week, and that watering frequency (followed in importance by the addition of composted sewage sludge and inoculation type) was the treatment that most strongly influenced their composition. Our results suggest that the inoculation of biological soil crusts in the form of slurry combined with the addition of composted sewage sludge could be a suitable technique to accelerate the recovery of the composition and functioning of biological soil crusts in drylands.


Assuntos
Clorofila/metabolismo , Cianobactérias/metabolismo , Ecossistema , Líquens/metabolismo , Microbiologia do Solo , Acetileno/metabolismo , Análise de Variância , Dióxido de Carbono/metabolismo , Clorofila A , Conservação dos Recursos Naturais , Cianobactérias/classificação , Cianobactérias/genética , Cianobactérias/crescimento & desenvolvimento , Clima Desértico , Monitoramento Ambiental , Fertilizantes , Líquens/crescimento & desenvolvimento , Nitrogênio/metabolismo , Fixação de Nitrogênio , Nitrogenase/metabolismo , Filogenia , RNA Ribossômico 16S/química , Esgotos , Água
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