RESUMEN
Endosymbiotic associations between hornworts and nitrogen-fixing cyanobacteria form when the plant is limited for combined nitrogen (N). We generated RNA-seq data to examine temporal gene expression patterns during the culturing of N-starved Anthoceros punctatus in the absence and the presence of symbiotic cyanobacterium Nostoc punctiforme. In symbiont-free A. punctatus gametophytes, N starvation caused downregulation of chlorophyll content and chlorophyll fluorescence characteristics as well as transcription of photosynthesis-related genes. This downregulation was reversed in A. punctatus cocultured with N. punctiforme, corresponding to the provision by the symbiont of N2-derived NH4+, which commenced within 5 days of coculture and reached a maximum by 14 days. We also observed transient increases in transcription of ammonium and nitrate transporters in a N. punctiforme-dependent manner as well as that of a SWEET transporter that was initially independent of N2-derived NH4+. The temporal patterns of differential gene expression indicated that N. punctiforme transmits signals that impact gene expression to A. punctatus both prior to and after its provision of fixed N. This study is the first illustrating the temporal patterns of gene expression during establishment of an endosymbiotic nitrogen-fixing association in this monophyletic evolutionary lineage of land plants. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Asunto(s)
Compuestos de Amonio , Anthocerotophyta , Nostoc , Anthocerotophyta/genética , Clorofila , Expresión Génica , Nitrógeno , Nostoc/genética , Simbiosis/genéticaRESUMEN
Hornworts comprise a bryophyte lineage that diverged from other extant land plants >400 million years ago and bears unique biological features, including a distinct sporophyte architecture, cyanobacterial symbiosis and a pyrenoid-based carbon-concentrating mechanism (CCM). Here, we provide three high-quality genomes of Anthoceros hornworts. Phylogenomic analyses place hornworts as a sister clade to liverworts plus mosses with high support. The Anthoceros genomes lack repeat-dense centromeres as well as whole-genome duplication, and contain a limited transcription factor repertoire. Several genes involved in angiosperm meristem and stomatal function are conserved in Anthoceros and upregulated during sporophyte development, suggesting possible homologies at the genetic level. We identified candidate genes involved in cyanobacterial symbiosis and found that LCIB, a Chlamydomonas CCM gene, is present in hornworts but absent in other plant lineages, implying a possible conserved role in CCM function. We anticipate that these hornwort genomes will serve as essential references for future hornwort research and comparative studies across land plants.
Asunto(s)
Anthocerotophyta/genética , Evolución Biológica , Embryophyta/fisiología , Genoma de Planta , Rasgos de la Historia de VidaRESUMEN
Plant endosymbiosis with nitrogen-fixing cyanobacteria has independently evolved in diverse plant lineages, offering a unique window to study the evolution and genetics of plant-microbe interaction. However, very few complete genomes exist for plant cyanobionts, and therefore little is known about their genomic and functional diversity. Here, we present four complete genomes of cyanobacteria isolated from bryophytes. Nanopore long-read sequencing allowed us to obtain circular contigs for all the main chromosomes and most of the plasmids. We found that despite having a low 16S rRNA sequence divergence, the four isolates exhibit considerable genome reorganizations and variation in gene content. Furthermore, three of the four isolates possess genes encoding vanadium (V)-nitrogenase (vnf), which is uncommon among diazotrophs and has not been previously reported in plant cyanobionts. In two cases, the vnf genes were found on plasmids, implying possible plasmid-mediated horizontal gene transfers. Comparative genomic analysis of vnf-containing cyanobacteria further identified a conserved gene cluster. Many genes in this cluster have not been functionally characterized and would be promising candidates for future studies to elucidate V-nitrogenase function and regulation.
Asunto(s)
Cianobacterias/genética , Familia de Multigenes/genética , Nitrogenasa/genética , Nitrogenasa/clasificación , Filogenia , Plásmidos/genética , ARN Ribosómico 16S/genéticaRESUMEN
Some cyanobacteria are capable of differentiating a variety of cell types in response to environmental factors. For instance, in low nitrogen conditions, some cyanobacteria form heterocysts, which are specialized for N2 fixation. Many heterocyst-forming cyanobacteria have DNA elements interrupting key N2 fixation genes, elements that are excised during heterocyst differentiation. While the mechanism for the excision of the element has been well-studied, many questions remain regarding the introduction of the elements into the cyanobacterial lineage and whether they have been retained ever since or have been lost and reintroduced. To examine the evolutionary relationships and possible function of DNA sequences that interrupt genes of heterocyst-forming cyanobacteria, we identified and compared 101 interruption element sequences within genes from 38 heterocyst-forming cyanobacterial genomes. The interruption element lengths ranged from about 1 kb (the minimum able to encode the recombinase responsible for element excision), up to nearly 1 Mb. The recombinase gene sequences served as genetic markers that were common across the interruption elements and were used to track element evolution. Elements were found that interrupted 22 different orthologs, only five of which had been previously observed to be interrupted by an element. Most of the newly identified interrupted orthologs encode proteins that have been shown to have heterocyst-specific activity. However, the presence of interruption elements within genes with no known role in N2 fixation, as well as in three non-heterocyst-forming cyanobacteria, indicates that the processes that trigger the excision of elements may not be limited to heterocyst development or that the elements move randomly within genomes. This comprehensive analysis provides the framework to study the history and behavior of these unique sequences, and offers new insight regarding the frequency and persistence of interruption elements in heterocyst-forming cyanobacteria.
Asunto(s)
Proteínas Bacterianas/genética , Cianobacterias/genética , ADN/genética , Fijación del Nitrógeno/genética , FilogeniaRESUMEN
In filamentous cyanobacteria, the mechanism of gliding motility is undefined but posited to be driven by a polysaccharide secretion system known as the junctional pore complex (JPC). Recent evidence implies that the JPC is a modified type IV pilus-like structure encoded for in part by genes in the hps locus. To test this hypothesis, we conducted genetic, cytological and comparative genomics studies on hps and pil genes in Nostoc punctiforme, a species in which motility is restricted to transiently differentiated filaments called hormogonia. Inactivation of most hps and pil genes abolished motility and abolished or drastically reduced secretion of hormogonium polysaccharide, and the subcellular localization of several Pil proteins in motile hormogonia corresponds to the site of the junctional pore complex. The non-motile ΔhpsE-G strain, which lacks three glycosyltransferases that synthesize hormogonium polysaccharide, could be complemented to motility by the addition of medium conditioned by wild-type hormogonia. Based on this result, we speculate that secretion of hormogonium polysaccharide facilitates but does not provide the motive force for gliding. Both the Hps and Pil homologs characterized in this study are almost universally conserved among filamentous cyanobacteria, with the Hps homologs rarely found in unicellular strains. These results support the theory that Hps and Pil proteins compose the JPC, a type IV pilus-like nanomotor that drives motility and polysaccharide secretion in filamentous cyanobacteria.
Asunto(s)
Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/fisiología , Nostoc/fisiología , Polisacáridos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Medios de Cultivo Condicionados , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Genómica , Glicosiltransferasas/metabolismo , Movimiento , Nostoc/genética , Nostoc/crecimiento & desarrollo , Nostoc/ultraestructuraRESUMEN
In cyanobacterial Nostoc species, substratum-dependent gliding motility is confined to specialized nongrowing filaments called hormogonia, which differentiate from vegetative filaments as part of a conditional life cycle and function as dispersal units. Here we confirm that Nostoc punctiforme hormogonia are positively phototactic to white light over a wide range of intensities. N. punctiforme contains two gene clusters (clusters 2 and 2i), each of which encodes modular cyanobacteriochrome-methyl-accepting chemotaxis proteins (MCPs) and other proteins that putatively constitute a basic chemotaxis-like signal transduction complex. Transcriptional analysis established that all genes in clusters 2 and 2i, plus two additional clusters (clusters 1 and 3) with genes encoding MCPs lacking cyanobacteriochrome sensory domains, are upregulated during the differentiation of hormogonia. Mutational analysis determined that only genes in cluster 2i are essential for positive phototaxis in N. punctiforme hormogonia; here these genes are designated ptx (for phototaxis) genes. The cluster is unusual in containing complete or partial duplicates of genes encoding proteins homologous to the well-described chemotaxis elements CheY, CheW, MCP, and CheA. The cyanobacteriochrome-MCP gene (ptxD) lacks transmembrane domains and has 7 potential binding sites for bilins. The transcriptional start site of the ptx genes does not resemble a sigma 70 consensus recognition sequence; moreover, it is upstream of two genes encoding gas vesicle proteins (gvpA and gvpC), which also are expressed only in the hormogonium filaments of N. punctiforme.
Asunto(s)
Proteínas Bacterianas/metabolismo , Extensiones de la Superficie Celular/metabolismo , Nostoc/genética , Nostoc/efectos de la radiación , Fotorreceptores Microbianos/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Extensiones de la Superficie Celular/genética , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Luz , Datos de Secuencia Molecular , Familia de Multigenes , Nostoc/citología , Nostoc/metabolismo , Fotorreceptores Microbianos/genéticaRESUMEN
Filamentous cyanobacteria are capable of gliding motility, but the mechanism of motility is not well defined. Here we present a detailed characterization of the hmp locus from Nostoc punctiforme, which encodes chemotaxis-like proteins. Deletions of hmpB, C, D and E abolished differentiation of hormogonia under standard growth conditions, but, upon addition of a symbiotic partner exudate, the mutant strains differentiated hormogonium-like filaments that lacked motility and failed to secrete hormogonium specific polysaccharide. The hmp locus is expressed as two transcripts, one originating 5' of hmpA and encompassing the entire hmp locus, and the other 5' of hmpB and encompassing hmpBCDE. The CheA-like HmpE donates phosphate to its own C-terminal receiver domain, and to the CheY-like HmpB, but not to the PatA family CheY-like HmpA. A GFP-tagged variant of each hmp locus protein localized to a ring adjacent to the septum on each end of the rod-shaped cell. Immunofluorescence demonstrated that PilA localizes to a ring at the junction between cells. The phenotype of the deletion strains, and the localization of the Hmp proteins and the putative PilA protein to rings at the cell junctions are consistent with the hypothesis that these proteins are part of the junctional pore complex observed in a number of filamentous cyanobacteria.
Asunto(s)
Quimiotaxis , Locomoción , Familia de Multigenes , Nostoc/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Eliminación de Gen , Sitios Genéticos , Nostoc/fisiología , Transporte de Proteínas , Transcripción GenéticaAsunto(s)
Bryopsida/fisiología , Fijación del Nitrógeno , Nitrógeno , Nostoc/fisiología , Simbiosis , ÁrbolesRESUMEN
In cyanobacteria-plant symbioses, the symbiotic nitrogen-fixing cyanobacterium has low photosynthetic activity and is supplemented by sugars provided by the plant partner. Which sugars and cyanobacterial sugar uptake mechanism(s) are involved in the symbiosis, however, is unknown. Mutants of the symbiotically competent, facultatively heterotrophic cyanobacterium Nostoc punctiforme were constructed bearing a neomycin resistance gene cassette replacing genes in a putative sugar transport gene cluster. Results of transport activity assays using (14)C-labeled fructose and glucose and tests of heterotrophic growth with these sugars enabled the identification of an ATP-binding cassette-type transporter for fructose (Frt), a major facilitator permease for glucose (GlcP), and a porin needed for the optimal uptake of both fructose and glucose. Analysis of green fluorescent protein fluorescence in strains of N. punctiforme bearing frt::gfp fusions showed high expression in vegetative cells and akinetes, variable expression in hormogonia, and no expression in heterocysts. The symbiotic efficiency of N. punctiforme sugar transport mutants was investigated by testing their ability to infect a nonvascular plant partner, the hornwort Anthoceros punctatus. Strains that were specifically unable to transport glucose did not infect the plant. These results imply a role for GlcP in establishing symbiosis under the conditions used in this work.
Asunto(s)
Anthocerotophyta/microbiología , Proteínas Bacterianas/metabolismo , Metabolismo de los Hidratos de Carbono , Proteínas de Transporte de Membrana/metabolismo , Nostoc/metabolismo , Simbiosis/fisiología , Técnicas de Cocultivo , Fructosa/metabolismo , Genoma Bacteriano/genética , Glucosa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Procesos Heterotróficos , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/genética , Nostoc/genética , Nostoc/crecimiento & desarrollo , FenotipoRESUMEN
Many filamentous cyanobacteria are capable of gliding motility by an undefined mechanism. Within the heterocyst-forming clades, some strains, such as the Nostoc spp. and Fisherella spp., are motile only as specialized filaments termed hormogonia. Here we report on the phenotype of inactivation of a methyl-accepting chemotaxis-like protein in Nostoc punctiforme, designated HmpD. The gene hmpD was found to be essential for hormogonium development, motility and polysaccharide secretion. Comparative global transcriptional profiling of the ΔhmpD strain demonstrated that HmpD has a profound effect on the transcriptional programme of hormogonium development, influencing approximately half of the genes differentially transcribed during differentiation. Utilizing this transcriptomic data, we identified a gene locus, designated here as hps, that appears to encode for a novel polysaccharide secretion system. Transcripts for the genes in the hps locus are upregulated in two steps, with the second step dependent on HmpD. Deletion of hpsA, hpsBCD or hpsEFG resulted in the complete loss of motility and polysaccharide secretion, similar to deletion of hmpD. Genes in the hps locus are highly conserved in the filamentous cyanobacteria, but generally absent in unicellular strains, implying a common mechanism of motility unique to the filamentous cyanobacteria.
Asunto(s)
Sistemas de Secreción Bacterianos , Perfilación de la Expresión Génica , Nostoc/citología , Nostoc/metabolismo , Polisacáridos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Mutación , Nostoc/genética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Heterocysts, cells specialized for nitrogen fixation in certain filamentous cyanobacteria, appear singly in a nonrandom spacing pattern along the chain of vegetative cells. A two-stage, biased initiation and competitive resolution model has been proposed to explain the establishment of this spacing pattern. There is substantial evidence that competitive resolution of a subset of cells initiating differentiation occurs by interactions between a self-enhancing activator protein, HetR, and a diffusible pentapeptide inhibitor PatS-5 (RGSGR). Results presented here show that the absence of a unique membrane protein, PatN, in Nostoc punctiforme strain ATCC 29133 leads to a threefold increase in heterocyst frequency and a fourfold decrease in the vegetative cell interval between heterocysts. A PatN-GFP translational fusion shows a pattern of biased inheritance in daughter vegetative cells of ammonium-grown cultures. Inactivation of another heterocyst patterning gene, patA, is epistatic to inactivation of patN, and transcription of patA increases in a patN-deletion strain, implying that patN may function by modulating levels of patA. The presence of PatN is hypothesized to decrease the competency of a vegetative cell to initiate heterocyst differentiation, and the cellular concentration of PatN is dependent on cell division that results in cells transiently depleted of PatN. We suggest that biased inheritance of cell-fate determinants is a phylogenetic domain-spanning paradigm in the development of biological patterns.
Asunto(s)
Proteínas Bacterianas/fisiología , Cianobacterias/metabolismo , Proteínas de la Membrana/fisiología , Nostoc/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Diferenciación Celular , Linaje de la Célula , ADN Complementario/metabolismo , Epistasis Genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/genética , Microscopía Fluorescente/métodos , Fijación del Nitrógeno , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistemas de Lectura Abierta , Fenotipo , Filogenia , Estructura Terciaria de Proteína , Factores de Tiempo , Transcripción GenéticaRESUMEN
The filamentous cyanobacterium Nostoc punctiforme differentiates from vegetative cells into three distinct cell types, heterocysts, hormogonia, and akinetes, in response to different stimuli. Cultures growing with ammonium can be induced to form hormogonia or heterocysts upon removal of the combined nitrogen. A DNA microarray consisting of 94% of the open reading frames predicted from the 9.059-Mb N. punctiforme genome was used to generate a global transcription data set consisting of seven time points over a 24-h period of nitrogen deprivation, which results in heterocyst formation. This data set was compared to a similarly generated data set of nitrogen-starved N. punctiforme resulting in hormogonium formation that had previously been published (E. L. Campbell, H. Christman, and J. C. Meeks, J. Bacteriol. 190:7382-7391, 2008). The transition from vegetative cells to either heterocysts or hormogonia resulted in rapid and sustained expression of genes required for utilization of alternate nitrogen sources. Overall, 1,036 and 1,762 genes were found to be differentially transcribed during the heterocyst and hormogonium time courses, respectively, as analyzed with the Bayesian user-friendly software for analyzing time series microarray experiments (BATS). Successive transcription of heterocyst regulatory, structural, and functional genes occurred over the 24 h required to form a functional heterocyst. During hormogonium differentiation, some heterocyst structural and functional genes were upregulated, while the heterocyst master regulator hetR was downregulated. There are commonalities in differential expression between cells bound for differentiation into heterocysts or hormogonia, yet the two paths are distinguished by their developmentally specific transcription profiles.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Nitrógeno/metabolismo , Nostoc/crecimiento & desarrollo , Nostoc/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Nostoc/fisiología , Estrés FisiológicoRESUMEN
OBJECTIVE: To evaluate outcome of treatment with a combination of azathioprine and prednisone in dogs with meningoencephalomyelitis of undetermined etiology (MUE). DESIGN: Retrospective case series. ANIMALS: 40 dogs. PROCEDURES: Medical records of dogs with MUE treated with prednisone and azathioprine were evaluated with regard to response, survival, and adverse effects. RESULTS: All dogs improved during treatment. Twenty-four (60%) dogs had a complete response (resolution of clinical signs), and the other 16 (40%) dogs had a partial response (improvement but not resolution of signs). Most dogs that achieved a complete response remained neurologically normal. Six dogs remained stable after a partial response. Eleven dogs had a relapse of clinical signs. Twenty dogs died during the study period, 18 survived, and 2 were lost to follow-up monitoring. Median survival time was 1,834 days (range, 50 to 2,469 days). Survival time was significantly longer for dogs that had a complete response than for those that did not. Survival time was significantly shorter for dogs that relapsed than for those that did not. The most common adverse effects included weight gain, thinning of the hair, and elevated activities of liver enzymes, all of which may have been attributed to concurrent corticosteroid administration. Less common adverse effects included diabetes mellitus, keratoconjunctivitis sicca, mammary gland adenoma, lymphoma, and hepatic masses. CONCLUSIONS AND CLINICAL RELEVANCE: Azathioprine appeared to be a safe and potentially effective adjunct to prednisone for treatment of dogs with MUE. Prospective, double-blinded, controlled studies with histologic confirmation are warranted to substantiate these findings.
Asunto(s)
Azatioprina/uso terapéutico , Enfermedades de los Perros/tratamiento farmacológico , Meningoencefalitis/veterinaria , Prednisona/uso terapéutico , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/uso terapéutico , Azatioprina/administración & dosificación , Perros , Femenino , Inmunosupresores/administración & dosificación , Inmunosupresores/uso terapéutico , Masculino , Meningoencefalitis/tratamiento farmacológico , Prednisona/administración & dosificación , Estudios RetrospectivosRESUMEN
Hormogonia are nongrowing filaments, motile by means of a gliding mechanism, that are produced by certain cyanobacteria. Their differentiation is induced by positive and negative factors for growth, such as deprivation of combined nitrogen (nitrogen stress induction [NSI]). In Nostoc punctiforme, they are also induced by the exudate (hormogonium-inducing factor [HIF]) of a symbiotic plant partner. Time course (0.5 to 24 h) transcription profiles were determined by DNA microarray assays for hormogonia of N. punctiforme following induction by HIF and NSI. Clustering analysis revealed both common and distinct transcriptional patterns for the two methods of induction. By 24 h, a common set of 1,328 genes was identified. This 24-h common set of genes arose by the transition of 474 genes from an 819-member common set of genes at 1 h after induction; 405 and 51 genes unique to the HIF and NSI groups at 1 h, respectively; and 398 genes differentially transcribed at later time points. The NSI hormogonia showed a transcriptional checkpoint at 12 h following induction in which up- and downregulated genes were transiently down- or upregulated, respectively. The transient changes in these 1,043 genes appeared to reflect a switch back to a vegetative growth state. Such a checkpoint was not seen in HIF hormogonia. Genes uniquely upregulated in HIF hormogonia included those encoding proteins hypothesized to synthesize a metabolite repressor of hormogonium differentiation. Approximately 34 to 42% of the 6,893 printed genes were differentially transcribed during hormogonium differentiation; about half of those genes were upregulated, and 1,034 genes responded within 0.5 h after induction. These collective results indicate extensive and rapid global changes in the transcription of specific genes during the differentiation of these specialized filaments.
Asunto(s)
Perfilación de la Expresión Génica , Nitrógeno/metabolismo , Nostoc/genética , Plantas/microbiología , Proteínas Bacterianas/genética , Northern Blotting , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Modelos Biológicos , Nitrógeno/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Simbiosis/genética , Transcripción Genética/efectos de los fármacosRESUMEN
The vegetative cells of the filamentous cyanobacterium Nostoc punctiforme can differentiate into three mutually exclusive cell types: nitrogen-fixing heterocysts, spore-like akinetes, and motile hormogomium filaments. A DNA microarray consisting of 6,893 N. punctiforme genes was used to identify the global transcription patterns at single time points in the three developmental states, compared to those in ammonium-grown time zero cultures. Analysis of ammonium-grown cultures yielded a transcriptome of 2,935 genes, which is nearly twice the size of a soluble proteome. The NH(4)(+)-grown transcriptome was enriched in genes encoding core metabolic functions. A steady-state N(2)-grown (heterocyst-containing) culture showed differential transcription of 495 genes, 373 of which were up-regulated. The majority of the up-regulated genes were predicted from studies of heterocyst differentiation and N(2) fixation; other genes are candidates for more detailed genetic analysis. Three days into the developmental process, akinetes showed a similar number of differentially expressed genes (497 genes), which were equally up- and down-regulated. The down-regulated genes were enriched in core metabolic functions, consistent with entry into a nongrowth state. There were relatively few adaptive genes up-regulated in 3-day akinetes, and there was little overlap with putative heterocyst developmental genes. There were 1,827 differentially transcribed genes in 24-h hormogonia, which was nearly fivefold greater than the number in akinete-forming or N(2)-fixing cultures. The majority of the up-regulated adaptive genes were genes encoding proteins for signal transduction and transcriptional regulation, which is characteristic of a motile filament that is poised to sense and respond to the environment. The greatest fraction of the 883 down-regulated genes was involved in core metabolism, also consistent with entry into a nongrowth state. The differentiation of heterocysts (steady state, N(2) grown), akinetes, and hormogonia appears to involve the up-regulation of genes distinct for each state.
Asunto(s)
Proteínas Bacterianas/fisiología , Perfilación de la Expresión Génica , Nitrógeno/farmacología , Nostoc/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos , Genoma Bacteriano , Nostoc/citología , Nostoc/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Compuestos de Amonio Cuaternario/farmacologíaRESUMEN
Nostoc punctiforme is an oxygenic photoautotrophic cyanobacterium with multiple developmental states, which can form nitrogen-fixing symbioses with a variety of terrestrial plants. 3D LC/MS/MS shotgun peptide sequencing was used to analyze the proteome when N. punctiforme is grown in continuous moderate light with ammonia as the nitrogen source. The soluble proteome includes 1575 proteins, 50% of which can be assigned to core metabolic and transport functions. Another 39% are assigned to proteins with no known function, a substantially higher fraction than in the Escherichia coli proteome. Many expressed proteins protect against oxidative and light stress. Seventy-one sensor histidine kinases, response regulators, and serine/threonine kinases, individually and as hybrid, multidomain proteins, were identified, reflecting a substantial capacity to sense and respond to environmental change. Proteins encoded by each of the five N. punctiforme plasmids were identified, as were 10 transposases, reflecting the plasticity of the N. punctiforme genome. This core proteome sets the stage for comparison with that of other developmental states.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Nostoc/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cromatografía Liquida , Electroforesis Capilar/métodos , Espectrometría de Masas , Nostoc/genética , Plásmidos , Proteoma , Proteómica/métodosRESUMEN
Nostoc punctiforme is an example of a filamentous cyanobacterium that is capable of differentiating non-growing cells that constitute gliding filaments termed hormogonia. These gliding filaments serve in short distance dispersal and as infective units in establishing a symbiosis with plants, such as the bryophyte Anthoceros punctatus. Mutants of N. punctiforme exist which show elevated levels of initial infection of A. punctatus as a consequence of repeated cycles of hormogonium differentiation. Such mutations occur within the hrmA and hrmU genes. Further characterization of the hrm locus revealed several genes with an organizational and predicted protein sequence similarity to genes of heterotrophic bacteria that are involved in hexuronic acid metabolism. Genes in the N. punctiforme locus are transcribed in response to the presence of a plant extract containing hormogonium-repressing factors. A predicted transcriptional repressor encoded in the locus, HrmR, was shown herein to be a specific DNA binding protein that regulates the transcription of its own gene and that of hrmE, a nearby gene. The ability of HrmR to bind DNA was abolished upon addition of either galacturonate or lysate from specifically induced N. punctiforme cells, implying that the in vivo HrmR binding activity is modulated via an internal compound, most likely a sugar molecule.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cianobacterias/crecimiento & desarrollo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Plantas/microbiología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Cianobacterias/genética , Cianobacterias/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Simbiosis , Transcripción GenéticaRESUMEN
Nostoc punctiforme is a phenotypically complex, filamentous, nitrogen-fixing cyanobacterium, whose vegetative cells can mature in four developmental directions. The particular developmental direction is determined by environmental signals. The vegetative cell cycle is maintained when nutrients are sufficient. Limitation for combined nitrogen induces the terminal differentiation of heterocysts, cells specialized for nitrogen fixation in an oxic environment. A number of unique regulatory events and genes have been identified and integrated into a working model of heterocyst differentiation. Phosphate limitation induces the transient differentiation of akinetes, spore-like cells resistant to cold and desiccation. A variety of environmental changes, both positive and negative for growth, induce the transient differentiation of hormogonia, motile filaments that function in dispersal. Initiation of the differentiation of heterocysts, akinetes and hormogonia are hypothesized to depart from the vegetative cell cycle, following separate and distinct events. N. punctiforme also forms nitrogen-fixing symbiotic associations; its plant partners influence the differentiation and behavior of hormogonia and heterocysts. N. punctiforme is genetically tractable and its genome sequence is nearly complete. Thus, the regulatory circuits of three cellular differentiation events and symbiotic interactions of N. punctiforme can be experimentally analyzed by functional genomics.