RESUMO
IMPORTANCE: Multicellular organization is a requirement for the development of complex organisms, and filamentous cyanobacteria such as Anabaena represent a paradigmatic case of bacterial multicellularity. The Anabaena filament can include hundreds of communicated cells that exchange nutrients and regulators and, depending on environmental conditions, can include different cell types specialized in distinct biological functions. Hence, the specific features of the Anabaena filament and how they are propagated during cell division represent outstanding biological issues. Here, we studied SepT, a novel coiled-coil-rich protein of Anabaena that is located in the intercellular septa and influences the formation of the septal specialized structures that allow communication between neighboring cells along the filament, a fundamental trait for the performance of Anabaena as a multicellular organism.
Assuntos
Anabaena , Nanoporos , Peptidoglicano/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Anabaena/genética , Anabaena/metabolismo , Citoesqueleto/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Physalis peruviana L. fruits are rich in bioactive compounds with health benefits. Processing of physalis into pulp with further dehydration has been proposed as a method to increase shelf life and preserve bioactive compounds. Here, the effect of three drying methods on the physico-chemical properties, color, antioxidant capacity, tocopherol and fatty acids content of physalis pulp thin layers were evaluated. The radical scavenging activity showed higher antioxidant activity at high temperatures rather than at low temperatures. Both, DPPH and ORAC assay showed a high antioxidant capacity of the physalis pulp. Chromatic parameters as well as Chroma and Hue angle were affected by drying temperature, which contributed to the discoloring of physalis pulp during this process. Based on these results, both convective drying and infrared drying at 80 °C were proved to be viable drying options for physalis pulp.
RESUMO
Nowadays berries are globally recognized to be among the healthiest foods because they contain diverse and abundant bioactive compounds. Among these are phenolic acids, flavonoids, and anthocyanins, which are known to have beneficial health effects. South America, particularly southern Chile, is covered by a diversity of insufficiently studied and underexploited native berry species. In this review we summarize all the available literature on the phenolic composition, antioxidant activity, bioaccessibility, and biological activity of five native South American berries: calafate, maqui, murta, arrayán, and chequén. The potential of these native berries for promoting human health and as source of bioactive substances is remarkable. Bioactive compounds, mainly anthocyanins, and in less abundance flavonoids and phenolic acids, show strong antioxidant effects. Some of these constituents are bioaccessible and bioavailable, and exert anticancer, antimicrobial, and anti-inflammatory activities as well as inhibitory effects against enzymes involved in metabolic syndromes. Given the potential of native South American berries to promote health, more work is still needed to understand fully the potential beneficial effects of the consumption of these berries on human health. © 2020 Society of Chemical Industry.
Assuntos
Antioxidantes/química , Frutas/química , Extratos Vegetais/química , Animais , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/farmacologia , Humanos , Extratos Vegetais/farmacologia , América do SulRESUMO
Polymerizing and filament-forming proteins are instrumental for numerous cellular processes such as cell division and growth. Their function in stabilization and localization of protein complexes and replicons is achieved by a filamentous structure. Known filamentous proteins assemble into homopolymers consisting of single subunits - for example, MreB and FtsZ in bacteria - or heteropolymers that are composed of two subunits, for example, keratin and α/ß tubulin in eukaryotes. Here, we describe two novel coiled-coil-rich proteins (CCRPs) in the filament-forming cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena) that assemble into a heteropolymer and function in the maintenance of the Anabaena multicellular shape (termed trichome). The two CCRPs - Alr4504 and Alr4505 (named ZicK and ZacK) - are strictly interdependent for the assembly of protein filaments in vivo and polymerize nucleotide independently in vitro, similar to known intermediate filament (IF) proteins. A ΔzicKΔzacK double mutant is characterized by a zigzagged cell arrangement and hence a loss of the typical linear Anabaena trichome shape. ZicK and ZacK interact with themselves, with each other, with the elongasome protein MreB, the septal junction protein SepJ and the divisome associate septal protein SepI. Our results suggest that ZicK and ZacK function in cooperation with SepJ and MreB to stabilize the Anabaena trichome and are likely essential for the manifestation of the multicellular shape in Anabaena. Our study reveals the presence of filament-forming IF-like proteins whose function is achieved through the formation of heteropolymers in cyanobacteria.
Assuntos
Anabaena/genética , Proteínas de Bactérias/genética , Proteínas do Citoesqueleto/genética , Citoesqueleto/genética , Regulação Bacteriana da Expressão Gênica , Tricomas/genética , Anabaena/metabolismo , Anabaena/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Divisão Celular , Clonagem Molecular , Sequência Conservada , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/genética , Histidina/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Polimerização , Multimerização Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tricomas/metabolismo , Tricomas/ultraestruturaRESUMO
Cells have to erect and sustain an organized and dynamically adaptable structure for an efficient mode of operation that allows drastic morphological changes during cell growth and cell division. These manifold tasks are complied by the so-called cytoskeleton and its associated proteins. In bacteria, FtsZ and MreB, the bacterial homologs to tubulin and actin, respectively, as well as coiled-coil-rich proteins of intermediate filament (IF)-like function to fulfil these tasks. Despite generally being characterized as Gram-negative, cyanobacteria have a remarkably thick peptidoglycan layer and possess Gram-positive-specific cell division proteins such as SepF and DivIVA-like proteins, besides Gram-negative and cyanobacterial-specific cell division proteins like MinE, SepI, ZipN (Ftn2) and ZipS (Ftn6). The diversity of cellular morphologies and cell growth strategies in cyanobacteria could therefore be the result of additional unidentified structural determinants such as cytoskeletal proteins. In this article, we review the current advances in the understanding of the cyanobacterial cell shape, cell division and cell growth.
RESUMO
Multiseriate and true-branching cyanobacteria are at the peak of prokaryotic morphological complexity. However, little is known about the mechanisms governing multiplanar cell division and morphogenesis. Here, we study the function of the prokaryotic cytoskeletal proteins, MreB and FtsZ in Fischerella muscicola PCC 7414 and Chlorogloeopsis fritschii PCC 6912. Vancomycin and HADA labeling revealed a mixed apical, septal, and lateral trichome growth mode in F. muscicola, whereas C. fritschii exhibits septal growth. In all morphotypes from both species, MreB forms either linear filaments or filamentous strings and can interact with FtsZ. Furthermore, multiplanar cell division in F. muscicola likely depends on FtsZ dosage. Our results lay the groundwork for future studies on cytoskeletal proteins in morphologically complex cyanobacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/química , Proteínas do Citoesqueleto/metabolismo , Proteínas de Bactérias/química , Proteínas do Citoesqueleto/químicaRESUMO
Cyanobacteria are unique among the eubacteria as they possess a hybrid Gram phenotype, having an outer membrane but also a comparably thick peptidoglycan sheet. Furthermore, the cyanobacterial divisome includes proteins specific for both the Gram types as well as cyanobacteria-specific proteins. Cells in multicellular cyanobacteria share a continuous periplasm and their cytoplasms are connected by septal junctions that enable communication between cells in the filament. The localization of septal junction proteins depends on interaction with the divisome, however additional yet unknown proteins may be involved in this process. Here, we characterized Alr3364 (termed SepI), a novel septal protein that interacts with the divisome in the multicellular heterocystous cyanobacterium Anabaena sp. strain PCC 7120. SepI localized to the Z-ring and the intercellular septa but did not interact with FtsZ. Instead, SepI interacted with the divisome proteins ZipN, SepF and FtsI and with the septal protein SepJ. The inactivation of sepI led to a defect in cell filament integrity, colony and cell morphology, septum size, nanopore formation and peptidoglycan biogenesis, and inability to differentiate heterocysts. Our results show that SepI plays a role in intercellular communication and furthermore indicate that SepI functions in the coordination of septal junction localization during cell division.
Assuntos
Anabaena/crescimento & desenvolvimento , Proteínas da Membrana Bacteriana Externa/metabolismo , Divisão Celular/fisiologia , Interações Microbianas/fisiologia , Anabaena/genética , Anabaena/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptidoglicano/biossínteseRESUMO
Filament-forming proteins in bacteria function in stabilization and localization of proteinaceous complexes and replicons; hence they are instrumental for myriad cellular processes such as cell division and growth. Here we present two novel filament-forming proteins in cyanobacteria. Surveying cyanobacterial genomes for coiled-coil-rich proteins (CCRPs) that are predicted as putative filament-forming proteins, we observed a higher proportion of CCRPs in filamentous cyanobacteria in comparison to unicellular cyanobacteria. Using our predictions, we identified nine protein families with putative intermediate filament (IF) properties. Polymerization assays revealed four proteins that formed polymers in vitro and three proteins that formed polymers in vivo. Fm7001 from Fischerella muscicola PCC 7414 polymerized in vitro and formed filaments in vivo in several organisms. Additionally, we identified a tetratricopeptide repeat protein - All4981 - in Anabaena sp. PCC 7120 that polymerized into filaments in vitro and in vivo. All4981 interacts with known cytoskeletal proteins and is indispensable for Anabaena viability. Although it did not form filaments in vitro, Syc2039 from Synechococcus elongatus PCC 7942 assembled into filaments in vivo and a Δsyc2039 mutant was characterized by an impaired cytokinesis. Our results expand the repertoire of known prokaryotic filament-forming CCRPs and demonstrate that cyanobacterial CCRPs are involved in cell morphology, motility, cytokinesis and colony integrity.
Assuntos
Anabaena/citologia , Proteínas de Bactérias/metabolismo , Cianobactérias/citologia , Proteínas do Citoesqueleto/metabolismo , Synechococcus/citologia , Motivos de Aminoácidos/genética , Anabaena/genética , Anabaena/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cianobactérias/genética , Cianobactérias/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/isolamento & purificação , Citoesqueleto/metabolismo , Genes Bacterianos/genética , Mutação , Conformação Proteica em alfa-Hélice/genética , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Synechococcus/genética , Synechococcus/metabolismoRESUMO
This work evaluated nutritional and organoleptic properties of murta, a Chilean native berry, impregnated with Lactobacillus casei var. rhamnosus and dehydrated by different methods: freeze- (FD), convective- (CD) and vacuum- (VD) drying. Scanning electron microscopy revealed that L. casei localized at the peduncle and near the peduncle of the impregnated fruit. Murta enriched with probiotics contained higher L. casei viable counts after dehydration with FD compared to CD and VD methods. Overall, drying resulted in a decrease in crude fibre and phenolic compounds, which was attributed to L. casei metabolic activity suggesting that murta berries could act as prebiotics for L. casei. Among drying methods, L. casei enriched FD murta presented less alterations in the microstructure, less drying-induced damage and obtained a higher sensory acceptability score than CD and VD murta. Taken together, these results will contribute to the development of functional foods from regional products improving local economy.
Assuntos
Frutas/química , Lacticaseibacillus rhamnosus , Myrtaceae/química , Chile , Dessecação , Fibras na Dieta/análise , Liofilização , Frutas/microbiologia , Humanos , Lacticaseibacillus rhamnosus/crescimento & desenvolvimento , Myrtaceae/microbiologia , Fenóis/análise , Prebióticos , Probióticos , Sensação , VácuoRESUMO
The aim of this study was to evaluate dehydrated murta berries enriched with probiotic (Lactobacillus casei var. rhamnosus) bacteria. L. casei was incorporated to fresh murta by vacuum impregnation at alternative conditions (pressure 50, 150 and 300 mbar; time 5, 10 and 15 min; temperature 20 ± 0.2 °C) and impregnated murta samples were dehydrated by two drying methods at 40 °C, vacuum and convective drying. Both drying processes were modeled by three mathematical models (Weibull, Page and modified Page). According to the statistical tests applied, the Weilbull model obtained the best-fit quality on experimental data. Effective moisture diffusivity varied between 1.23-1.75 × 10-10 m2/s and 1.16-1.44 × 10-10 m2/s for vacuum and convective drying, respectively. After impregnation, murta berries contained approximately 107 CFU/g L. casei although maximum counts were found at 150 mbar for 15 min. Drying decreased L. casei viability in 1.5-1.9 log and 0.5-1.2 log for vacuum and convective drying, respectively. Thus, impregnation at 150 mbar for 15 min followed by convective drying at 40 °C appears as the method of choice to produce probiotic enriched murta berries that can be commercialized as probiotic dried snacks.
Assuntos
Temperatura Baixa , Peixes , Conservação de Alimentos/métodos , Músculo Esquelético/química , Alimentos Marinhos/análise , Animais , Cor , Dureza , Pressão Hidrostática , Microscopia Eletrônica de Transmissão , Músculo Esquelético/ultraestrutura , Rigor Mortis , Fatores de Tempo , Água/análiseRESUMO
BACKGROUND: Filamentous cyanobacteria that differentiate multiple cell types are considered the peak of prokaryotic complexity and their evolution has been studied in the context of multicellularity origins. Species that form true-branching filaments exemplify the most complex cyanobacteria. However, the mechanisms underlying the true-branching morphology remain poorly understood despite of several investigations that focused on the identification of novel genes or pathways. An alternative route for the evolution of novel traits is based on existing phenotypic plasticity. According to that scenario - termed genetic assimilation - the fixation of a novel phenotype precedes the fixation of the genotype. RESULTS: Here we show that the evolution of transcriptional regulatory elements constitutes a major mechanism for the evolution of new traits. We found that supplementation with sucrose reconstitutes the ancestral branchless phenotype of two true-branching Fischerella species and compared the transcription start sites (TSSs) between the two phenotypic states. Our analysis uncovers several orthologous TSSs whose transcription level is correlated with the true-branching phenotype. These TSSs are found in genes that encode components of the septosome and elongasome (e.g., fraC and mreB). CONCLUSIONS: The concept of genetic assimilation supplies a tenable explanation for the evolution of novel traits but testing its feasibility is hindered by the inability to recreate and study the evolution of present-day traits. We present a novel approach to examine transcription data for the plasticity first route and provide evidence for its occurrence during the evolution of complex colony morphology in true-branching cyanobacteria. Our results reveal a route for evolution of the true-branching phenotype in cyanobacteria via modification of the transcription level of pre-existing genes. Our study supplies evidence for the 'plasticity-first' hypothesis and highlights the importance of transcriptional regulation in the evolution of novel traits.
Assuntos
Evolução Biológica , Cianobactérias/classificação , Cianobactérias/genética , Sequências Reguladoras de Ácido Nucleico , Cianobactérias/citologia , Cianobactérias/metabolismo , Evolução Molecular , Regulação da Expressão Gênica , Fenótipo , Sacarose/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
Chaperonins promote protein folding and are known to play a role in the maintenance of cellular stability under stress conditions. The group I bacterial chaperonin complex comprises GroEL, that forms a barrel-like oligomer, and GroES that forms the lid. In most eubacteria the GroES/GroEL chaperonin is encoded by a single-copy bicistronic operon, whereas in cyanobacteria up to three groES/groEL paralogs have been documented. Here we study the evolution and functional diversification of chaperonin paralogs in the heterocystous, multi-seriate filament forming cyanobacterium Chlorogloeopsis fritschii PCC 6912. The genome of C. fritschii encodes two groES/groEL operons (groESL1, groESL1.2) and a monocistronic groEL gene (groEL2). A phylogenetic reconstruction reveals that the groEL2 duplication is as ancient as cyanobacteria, whereas the groESL1.2 duplication occurred at the ancestor of heterocystous cyanobacteria. A comparison of the groEL paralogs transcription levels under different growth conditions shows that they have adapted distinct transcriptional regulation. Our results reveal that groEL1 and groEL1.2 are upregulated during diazotrophic conditions and the localization of their promoter activity points towards a role in heterocyst differentiation. Furthermore, protein-protein interaction assays suggest that paralogs encoded in the two operons assemble into hybrid complexes. The monocistronic encoded GroEL2 is not forming oligomers nor does it interact with the co-chaperonins. Interaction between GroES1.2 and GroEL1.2 could not be documented, suggesting that the groESL1.2 operon does not encode a functional chaperonin complex. Functional complementation experiments in Escherichia coli show that only GroES1/GroEL1 and GroES1/GroEL1.2 can substitute the native operon. In summary, the evolutionary consequences of chaperonin duplication in cyanobacteria include the retention of groESL1 as a housekeeping gene, subfunctionalization of groESL1.2 and neofunctionalization of the monocistronic groEL2 paralog.
Assuntos
Proteínas de Bactérias/genética , Chaperoninas/genética , Cianobactérias/genética , Evolução Molecular , Duplicação Gênica , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , FilogeniaRESUMO
Different environmental nitrogen sources play selective roles in the development of cyanobacterial blooms and noxious effects are often exacerbated when toxic cyanobacteria are dominant. Cylindrospermopsis raciborskii CS-505 (heterocystous, nitrogen fixing) and Raphidiopsis brookii D9 (non-N2 fixing) produce the nitrogenous toxins cylindrospermopsin (CYN) and paralytic shellfish toxins (PSTs), respectively. These toxin groups are biosynthesized constitutively by two independent putative gene clusters, whose flanking genes are target for nitrogen (N) regulation. It is not yet known how or if toxin biosynthetic genes are regulated, particularly by N-source dependency. Here we show that binding boxes for NtcA, the master regulator of N metabolism, are located within both gene clusters as potential regulators of toxin biosynthesis. Quantification of intra- and extracellular toxin content in cultures at early stages of growth under nitrate, ammonium, urea and N-free media showed that N-sources influence neither CYN nor PST production. However, CYN and PST profiles were altered under N-free medium resulting in a decrease in the predicted precursor toxins (doCYN and STX, respectively). Reduced STX amounts were also observed under growth in ammonium. Quantification of toxin biosynthesis and transport gene transcripts revealed a constitutive transcription under all tested N-sources. Our data support the hypothesis that PSTs and CYN are constitutive metabolites whose biosynthesis is correlated to cyanobacterial growth rather than directly to specific environmental conditions. Overall, the constant biosynthesis of toxins and expression of the putative toxin-biosynthesis genes supports the usage of qPCR probes in water quality monitoring of toxic cyanobacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/biossíntese , Cianobactérias/metabolismo , Cylindrospermopsis/metabolismo , Regulação Bacteriana da Expressão Gênica , Fixação de Nitrogênio , Venenos/metabolismo , Alcaloides , Compostos de Amônio/metabolismo , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Técnicas de Cultura Celular por Lotes , Brasil , Cianobactérias/crescimento & desenvolvimento , Cianobactérias/isolamento & purificação , Toxinas de Cianobactérias , Cylindrospermopsis/crescimento & desenvolvimento , Cylindrospermopsis/isolamento & purificação , Água Doce/microbiologia , Estrutura Molecular , Nitratos/metabolismo , Venenos/química , Queensland , Uracila/análogos & derivados , Uracila/biossíntese , Uracila/química , Ureia/metabolismoRESUMO
Paralytic shellfish poisoning toxins (PSTs) are a family of more than 30 natural alkaloids synthesized by dinoflagellates and cyanobacteria whose toxicity in animals is mediated by voltage-gated Na(+) channel blocking. The export of PST analogues may be through SxtF and SxtM, two putative MATE (multidrug and toxic compound extrusion) family transporters encoded in PSTs biosynthetic gene cluster (sxt). sxtM is present in every sxt cluster analyzed; however, sxtF is only present in the Cylindrospermopsis-Raphidiopsis clade. These transporters are energetically coupled with an electrochemical gradient of proton (H(+)) or sodium (Na(+)) ions across membranes. Because the functional role of PSTs remains unknown and methods for genetic manipulation in PST-producing organisms have not yet been developed, protein structure analyses will allow us to understand their function. By analyzing the sxt cluster of eight PST-producing cyanobacteria, we found no correlation between the presence of sxtF or sxtM and a specific PSTs profile. Phylogenetic analyses of SxtF/M showed a high conservation of SxtF in the Cylindrospermopsis-Raphidiopsis clade, suggesting conserved substrate affinity. Two domains involved in Na(+) and drug recognition from NorM proteins (MATE family) of Vibrio parahaemolyticus and V. cholerae are present in SxtF/M. The Na(+) recognition domain was conserved in both SxtF/M, indicating that Na(+) can maintain the role as a cation anti-transporter. Consensus motifs for toxin binding differed between SxtF and SxtM implying differential substrate binding. Through protein modeling and docking analysis, we found that there is no marked affinity between the recognition domain and a specific PST analogue. This agrees with our previous results of PST export in R. brookii D9, where we observed that the response to Na(+) incubation was similar to different analogues. These results reassert the hypothesis regarding the involvement of Na(+) in toxin export, as well as the motifs L(398)XGLQD(403) (SxtM) and L(390)VGLRD(395) (SxtF) in toxin recognition.
Assuntos
Proteínas de Bactérias/metabolismo , Cylindrospermopsis/metabolismo , Toxinas Marinhas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico Ativo , Simulação por Computador , Cylindrospermopsis/química , Cylindrospermopsis/genética , Toxinas Marinhas/química , Toxinas Marinhas/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Família Multigênica , Filogenia , Conformação Proteica , Saxitoxina/análogos & derivados , Saxitoxina/genética , Saxitoxina/metabolismoRESUMO
Cyanobacteria display a large diversity of cellular forms ranging from unicellular to complex multicellular filaments or aggregates. Species in the group present a wide range of metabolic characteristics including the fixation of atmospheric nitrogen, resistance to extreme environments, production of hydrogen, secondary metabolites and exopolysaccharides. These characteristics led to the growing interest in cyanobacteria across the fields of ecology, evolution, cell biology and biotechnology. The number of available cyanobacterial genome sequences has increased considerably in recent years, with more than 140 fully sequenced genomes to date. Genetic engineering of cyanobacteria is widely applied to the model unicellular strains Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942. However the establishment of transformation protocols in many other cyanobacterial strains is challenging. One obstacle to the development of these novel model organisms is that many species have doubling times of 48 h or more, much longer than the bacterial models E. coli or B. subtilis. Furthermore, cyanobacterial defense mechanisms against foreign DNA pose a physical and biochemical barrier to DNA insertion in most strains. Here we review the various barriers to DNA uptake in the context of lateral gene transfer among microbes and the various mechanisms for DNA acquisition within the prokaryotic domain. Understanding the cyanobacterial defense mechanisms is expected to assist in the development and establishment of novel transformation protocols that are specifically suitable for this group.
Assuntos
Proteínas de Bactérias/genética , Cianobactérias/genética , DNA Bacteriano/genética , Sequência de Aminoácidos , Sequência de Bases , Técnicas de Transferência de Genes , FilogeniaRESUMO
Cyanobacteria forged two major evolutionary transitions with the invention of oxygenic photosynthesis and the bestowal of photosynthetic lifestyle upon eukaryotes through endosymbiosis. Information germane to understanding those transitions is imprinted in cyanobacterial genomes, but deciphering it is complicated by lateral gene transfer (LGT). Here, we report genome sequences for the morphologically most complex true-branching cyanobacteria, and for Scytonema hofmanni PCC 7110, which with 12,356 proteins is the most gene-rich prokaryote currently known. We investigated components of cyanobacterial evolution that have been vertically inherited, horizontally transferred, and donated to eukaryotes at plastid origin. The vertical component indicates a freshwater origin for water-splitting photosynthesis. Networks of the horizontal component reveal that 60% of cyanobacterial gene families have been affected by LGT. Plant nuclear genes acquired from cyanobacteria define a lower bound frequency of 611 multigene families that, in turn, specify diazotrophic cyanobacterial lineages as having a gene collection most similar to that possessed by the plastid ancestor.
Assuntos
Cianobactérias/genética , Evolução Molecular , Genoma Bacteriano , Genomas de Plastídeos , Fotossíntese/genética , Cianobactérias/metabolismo , Ecossistema , Água Doce , Transferência Genética Horizontal , Família Multigênica , Filogenia , Plantas/genética , Simbiose/genética , Água/metabolismoRESUMO
Cyanobacteria display a large diversity of cellular forms ranging from unicellular to complex multicellular filaments or aggregates. Species in the group present a wide range of metabolic characteristics including the fixation of atmospheric nitrogen, resistance to extreme environments, production of hydrogen, secondary metabolites and exopolysaccharides. These characteristics led to the growing interest in cyanobacteria across the fields of ecology, evolution, cell biology and biotechnology. The number of available cyanobacterial genome sequences has increased considerably in recent years, with more than 140 fully sequenced genomes to date. Genetic engineering of cyanobacteria is widely applied to the model unicellular strains Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942. However the establishment of transformation protocols in many other cyanobacterial strains is challenging. One obstacle to the development of these novel model organisms is that many species have doubling times of 48 h or more, much longer than the bacterial models E. coli or B. subtilis. Furthermore, cyanobacterial defense mechanisms against foreign DNA pose a physical and biochemical barrier to DNA insertion in most strains. Here we review the various barriers to DNA uptake in the context of lateral gene transfer among microbes and the various mechanisms for DNA acquisition within the prokaryotic domain. Understanding the cyanobacterial defense mechanisms is expected to assist in the development and establishment of novel transformation protocols that are specifically suitable for this group.
Assuntos
Proteínas de Bactérias/genética , Cianobactérias/genética , DNA Bacteriano/genética , Sequência de Aminoácidos , Sequência de Bases , Técnicas de Transferência de Genes , FilogeniaRESUMO
Cyanobacteria of subsection V grow as filaments with asymmetrical cell divisions that can generate a true-branching phenotype. Members of the genera Fischerella and Chlorogloeopsis furthermore differentiate akinetes (spore-like resting stages), heterocysts (specialized in nitrogen fixation) and hormogonia (cell aggregates with gliding motility for colonization and dispersal). Genetic approaches to studying the complex morphology and differentiations of these prokaryotes require transformation techniques. For Fischerella and Chlorogloeopsis reliable protocols for introducing foreign genes are lacking. Here, we explored conjugation, electroporation, and biolistic DNA transfer methods in Fischerella and Chlorogloeopsis, using the cyanobacterial replicon pRL25C as a marker. We successfully transformed Fischerella muscicola PCC 7414 and Chlorogloeopsis fritschii PCC 6912 and were able to express the GFP reporter protein under two different promoters: the nitrogen regulated (p) glnA and the strong E. coli hybrid (p) trc. For Fischerella all methods worked, for Chlorogloeopsis electroporation was unsuccessful. For both strains conjugation delivered the most reproducible results, whereby partial removal of the exopolysaccharide sheath by salt washing was a critical step.
Assuntos
Conjugação Genética , Cianobactérias/crescimento & desenvolvimento , Cianobactérias/genética , Transferência Genética Horizontal , Transformação Genética , Cianobactérias/metabolismo , Eletroporação , Técnicas de Transferência de Genes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismoRESUMO
The toxigenic freshwater cyanobacterium Cylindrospermopsis raciborskii T3 has been used as a model to study and elucidate the biosynthetic pathway of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP). There are nevertheless several inconsistencies and contradictions in the toxin profile of this strain as published by different research groups, and claimed to include carbamoyl (STX, NEO, GTX2/3), decarbamoyl (dcSTX), and N-sulfocarbamoyl (C1/2, B1) derivatives. Our analysis of the complete genome of another PSP toxin-producing cyanobacterium, Raphidiopsis brookii D9, which is closely related to C. raciborskii T3, resolved many issues regarding the correlation between biosynthetic pathways, corresponding genes and the T3 toxin profile. The putative sxt gene cluster in R. brookii D9 has a high synteny with the T3 sxt cluster, with 100% nucleotide identity among the shared genes. We also compared the PSP toxin profile of the strains by liquid chromatography coupled to mass spectrometry (LC-MS/MS). In contrast to published reports, our reassessment of the PSP toxin profile of T3 confirmed production of only STX, NEO and dcNEO. We gained significant insights via correlation between specific sxt genes and their role in PSP toxin synthesis in both D9 and T3 strains. In particular, analysis of sulfotransferase functions for SxtN (N-sulfotransferase) and SxtSUL (O-sulfotransferase) enzymes allowed us to propose an extension of the PSP toxin biosynthetic pathway from STX to the production of the derivatives GTX2/3, C1/2 and B1. This is a significantly revised view of the genetic mechanisms underlying synthesis of sulfated and sulfonated STX analogues in toxigenic cyanobacteria.