Identification of Nostoc punctiforme akinete-expressed genes using differential display.
TL;DR: To identify genes associated with akinete development, differential display was used to amplify and compare cDNA from a wild‐type and zwf mutant strain of N. punctiforme following a switch to dark heterotrophic conditions and three novel akinete‐expressed genes were identified.
Abstract: Akinetes are spore-like resting cells formed by certain filamentous cyanobacteria that have increased resistance to environmental stress. They can be found at low frequencies in dense cultures experiencing low light or phosphate limitation, but also form at high frequencies in a zwf mutant strain of Nostoc punctiforme following dark incubation in the presence of fructose. The wild-type strain is capable of facultative heterotrophic growth under these conditions and does not form akinetes. To identify genes associated with akinete development, differential display was used to amplify and compare cDNA from a wild-type and zwf mutant strain of N. punctiforme following a switch to dark heterotrophic conditions. Screening of candidate genes by reverse transcriptase real-time quantitative PCR and subsequent testing for akinete-specific expression using GFP transcriptional reporter plasmids lead to the identification of three novel akinete-expressed genes. The genes identified from the screening encoded for proteins homologous to an aminopeptidase (aapN), a zinc protease (hap) and an ATP-binding cassette (ABC)-type transporter (aet). Expression of hap was also increased in developing hormogonia, a transient type of differentiated filament capable of gliding motility. Transcriptional start sites for akinete-expressed genes were determined using random amplification of cDNA ends (RACE), and promoter regions were compared with orthologues in other filamentous cyanobacteria to identify putative regulatory sequences.
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TL;DR: This Review addresses cyanob bacterial intercellular communication, the supracellular structure of the cyanobacterial filament and the basic principles that govern the process of heterocyst differentiation.
Abstract: Within the wide biodiversity that is found in the bacterial world, Cyanobacteria represents a unique phylogenetic group that is responsible for a key metabolic process in the biosphere - oxygenic photosynthesis - and that includes representatives exhibiting complex morphologies. Many cyanobacteria are multicellular, growing as filaments of cells in which some cells can differentiate to carry out specialized functions. These differentiated cells include resistance and dispersal forms as well as a metabolically specialized form that is devoted to N(2) fixation, known as the heterocyst. In this Review we address cyanobacterial intercellular communication, the supracellular structure of the cyanobacterial filament and the basic principles that govern the process of heterocyst differentiation.
400 citations
Cites background from "Identification of Nostoc punctiform..."
...punctiforme PCC 73102, which is impaired in heterotrophy, akinetes differentiate when the filaments are placed in the dark in the presence of fructos...
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TL;DR: The role of allelopathy in cyanobacteria ecology is still not well understood, and its clarification should benefit from carefully designed field studies, chemical characterization of allelochemicals and new methodological approaches at the “omics” level.
Abstract: Freshwater cyanobacteria produce several bioactive secondary metabolites with diverse chemical structure, which may achieve high concentrations in the aquatic medium when cyanobacterial blooms occur. Some of the compounds released by cyanobacteria have allelopathic properties, influencing the biological processes of other phytoplankton or aquatic plants. These kinds of interactions are more easily detectable under laboratory studies; however their ecological relevance is often debated. Recent research has discovered new allelopathic properties in some cyanobacteria species, new allelochemicals and elucidated some of the allelopathic mechanisms. Ecosystem-level approaches have shed some light on the factors that influence allelopathic interactions, as well as how cyanobacteria may be able to modulate their surrounding environment by means of allelochemical release. Nevertheless, the role of allelopathy in cyanobacteria ecology is still not well understood, and its clarification should benefit from carefully designed field studies, chemical characterization of allelochemicals and new methodological approaches at the "omics" level.
134 citations
Cites background from "Identification of Nostoc punctiform..."
...…error in the outcome of the assay—for discussion on this subject please refer to Bustin (2002) and Ginzinger (2002)—and a survey of the literature reveals that only a few studies which employ this methodology with cyanobacteria have been published (Argueta et al. 2006; Woodger et al. 2003)....
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TL;DR: The differentiation of heterocysts (steady state, N(2) grown), akinetes, and hormogonia appears to involve the up-regulation of genes distinct for each state, consistent with entry into a nongrowth state.
Abstract: 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 NH4+-grown transcriptome was enriched in genes encoding core metabolic functions. A steady-state N2-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 N2 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 N2-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, N2 grown), akinetes, and hormogonia appears to involve the up-regulation of genes distinct for each state.
118 citations
Cites background from "Identification of Nostoc punctiform..."
...NpR4070 was up-regulated in steady-state N2-grown cultures at a significant but low level....
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...and NpR4070) (6) was present in the akinete transcriptome or...
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...Transcription of avaK (NpF5452), encoding an akinete marker protein (6, 48), was enhanced, as was transcription of patA and hetF, whose products are assigned to heterocyst differentiation....
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...None of the four genes determined by differential display to be up-regulated during akinete development (NpF0062, NpF5999, NpF6000, and NpR4070) (6) was present in the akinete transcriptome or in the NH4 -grown transcriptome or proteome....
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...Five genes that are differentially transcribed during akinete differentiation have recently been identified (6, 48)....
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01 Jan 2010
TL;DR: This review focuses on akinetes of Nostocales, emphasizing environmental triggers and cellular responses involved in differentiation, maturation, dormancy, and germination of these resting cells and special attention is given to genetic regulation of the differentiation process.
Abstract: Cyanobacteria are an ancient and morphologically diverse group of photosynthetic prokaryotes, which were the first to evolve oxygenic photosynthesis. Cyanobacteria are widely distributed in diversed environments. In the case of members of the orders Nostocales and Stigonematales, their persistence and success were attributed to their ability to form specialized cells: heterocysts, capable of fixing atmospheric nitrogen and spore-like cells, the akinetes. This review focuses on akinetes of Nostocales, emphasizing environmental triggers and cellular responses involved in differentiation, maturation, dormancy, and germination of these resting cells. Morphological and structural changes, variation in akinete composition, and metabolism are summarized. Special attention is given to the genetic regulation of the differentiation process in an attempt to close gaps in our understanding of the dormancy phenomenon in cyanobacteria and to identify open questions for future research.
100 citations
Cites background or result from "Identification of Nostoc punctiform..."
...These results are consistent with the expression of avaK in A. variabilis (Zhou and Wolk 2002), with N. punctiforme differential display results for aet ( Argueta et al. 2006 ), and with microarray results from zwf akinetes for nblA and avaK (Campbell et al. 2007)....
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...Argueta et al. (2006) reported the detection of three novel genes involved in akinete differentiation....
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TL;DR: The clear differences in the metabolic and morphological adaptations of akinetes of the two species could be related to their different lifestyles and pave the way for genetic and functional studies of akinete differentiation in these species.
Abstract: Akinetes are resting spore-like cells formed by some heterocyst-forming filamentous cyanobacteria for surviving long periods of unfavourable conditions. We studied the development of akinetes in two model strains of cyanobacterial cell differentiation, the planktonic freshwater Anabaena variabilis ATCC 29413 and the terrestrial or symbiotic Nostoc punctiforme ATCC 29133, in response to low light and phosphate starvation. The best trigger of akinete differentiation of Anabaena variabilis was low light; that of N. punctiforme was phosphate starvation. Light and electron microscopy revealed that akinetes of both species differed from vegetative cells by their larger size, different cell morphology and large number of intracellular granules. Anabaena variabilis akinetes had a multilayer envelope; those of N. punctiforme had a simpler envelope. During akinete development of Anabaena variabilis, the amount of the storage compounds cyanophycin and glycogen increased transiently, whereas in N. punctiforme, cyanophycin and lipid droplets increased transiently. Photosynthesis and respiration decreased during akinete differentiation in both species, and remained at a low level in mature akinetes. The clear differences in the metabolic and morphological adaptations of akinetes of the two species could be related to their different lifestyles. The results pave the way for genetic and functional studies of akinete differentiation in these species.
40 citations
Cites background from "Identification of Nostoc punctiform..."
...Here, we showed that low light was the best inducer of akinete formation in Anabaena variabilis and confirmed the earlier finding (Argueta & Summers, 2005; Argueta et al., 2006) that phosphate starvation was the best inducer in N. punctiforme....
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...Here, we showed that low light was the best inducer of akinete formation in Anabaena variabilis and confirmed the earlier finding (Argueta & Summers, 2005; Argueta et al., 2006) that phosphate starvation was the best inducer in N....
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...However, only a few genes involved in akinete formation have been so far identified (e.g. Argueta et al., 2006; Campbell et al., 1996; Leganés et al., 1994, 1998; Lehner et al., 2011; Zhou &Wolk, 2002; see also review by Maldener et al., 2014)....
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References
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TL;DR: The transposon was found within a gene whose translation product is similar in amino acid sequence to the arginine-biosynthetic protein N-acetylglutamate semialdehyde dehydrogenase encoded by argC of Bacillus subtilis, and the newly identified gene from N. ellipsosporum was denoted argL, which renders certain processes in strain NE1 conditionally dependent on provision of L-arginine.
Abstract: In strain NE1 of Tn5-1058-mutagenized Nostoc ellipsosporum, the transposon was found within a gene whose translation product is similar in amino acid sequence to the arginine-biosynthetic protein N-acetylglutamate semialdehyde dehydrogenase encoded by argC of Bacillus subtilis. The argC reported from Anabaena sp. strain PCC 7120 hybridized to a sequence different from the one interrupted by the transposon in NE1. The newly identified gene from N. ellipsosporum was denoted argL. The argL mutation renders certain processes in strain NE1 conditionally dependent on provision of L-arginine. Heterocysts and apparent akinetes that formed in the absence of added L-arginine failed to fix dinitrogen or to germinate, respectively, and lacked granules of cyanophycin, composed of copolymers of arginine and aspartic acid. However, apparent akinetes that differentiated upon growth of the mutant in the presence of L-arginine plus nitrate formed cyanophycin granules and could regenerate a new culture.
33 citations
"Identification of Nostoc punctiform..." refers background in this paper
...The importance of cyanophycin for akinete germination has been demonstrated in a Nostoc ellipsosporum arginine biosynthesis mutant incapable of accumulating this polymer (Leganes et al., 1998)....
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TL;DR: Although akinetes of Nostoc PCC 7524 lost little of their main photosynthetic pigments, phycocyanin and chlorophyll, with increasing age after the onset of sporulation, they lost at least 90% of their photosynthesis and respiratory capacities, and the energetic requirement for germination was not efficiently fulfilled by cyclic photosynthesis on PSI alone or respiration alone.
Abstract: Although akinetes ofNostoc PCC 7524 lost little of their main photosynthetic pigments, phycocyanin and chlorophyll, with increasing age after the onset of sporulation, they lost at least 90% of their photosynthetic and respiratory capacities. Germination needed the supply of light throughout the process, though previous dark metabolism accelerated the following light process. In standard conditions, both respiratory and photosynthetic capacities increased markedly during the first 9–10 h, a time sufficient for the first doublets to appear, but when pigment contents had not yet changed. However, while respiratory capacity could be reacquired without de nove metabolism, resumption of photosynthetic capacity needed RNA and protein synthesis. The energetic requirement for germination was not efficiently fulfilled by cyclic photosynthesis on PSI alone or respiration alone. In the presence of both PSI and respiratory activities only 21% of the akinetes germinated, their endogenous carbon reserves thus being inadequate to support the process to completion. The addition of sucrose to such cultures permitted all of the akinetes to germinate, but at a very slow rate. Rapid and complete germination was only observed when both photosystem operated.
31 citations
"Identification of Nostoc punctiform..." refers background in this paper
...Although the germination of akinetes has received much attention (Chauvat et al., 1982; Sutherland et al., 1985; Sili et al., 1994; van Dok and Hart, 1996), the molecular genetic study of akinete induction has been hindered by their normally slow and asynchronous formation....
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TL;DR: This chapter discusses cellular differentiation in Akinetes, and describes some practical aspects of akinetes identification, production in pure culture; isolation, and properties.
Abstract: Publisher Summary This chapter discusses cellular differentiation in Akinetes. The chapter describes some practical aspects of akinetes identification, production in pure culture; isolation, and properties. Although the nomenclature of cyanobacteria is under discussion at present, and will certainly continue to evolve, for convenience the generic and specific names employed in the publications discussed are retained; however, many of these taxonomic assignments are clearly incorrect. The formation of akinetes only by heterocystous cyanobacteria might be taken to imply that the presence of heterocysts is a prerequisite for akinete differentiation. Techniques used for the separation of akinetes from vegetative cells take advantage of the size of akinetes and their resistance to cold, lysozyme, or mechanical stress. The resistance of akinetes to desiccation, which is not tolerated by vegetative cells, has apparently not yet been employed for their purification. Like baeocytes and hormogonia, akinetes can be induced to develop relatively synchronously. Under favorable conditions, they can also be made to germinate synchronously, and are therefore useful for the study of the biochemical changes which accompany the development of the mature filament.
27 citations
"Identification of Nostoc punctiform..." refers background in this paper
...Akinetes are only found in heterocyst-forming cyanobacteria, although not all heterocyst-forming bacteria can produce akinetes (Castenholz and Waterbury, 1989), especially those in long-term culture (Herdman, 1988)....
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TL;DR: The Akinetes of Nostoc PCC 7524 germinated synchronously when diluted into fresh medium in the light in the absence of a source of combined nitrogen as mentioned in this paper.
Abstract: Summary: Akinetes of Nostoc PCC 7524 germinated synchronously when diluted into fresh medium in the light in the absence of a source of combined nitrogen. The akinetes and their daughter cells were unable to fix N2 until the first heterocysts differentiated in the young three-celled filaments after 19 h. The large glycogen reserves of the mature akinetes were not necessary for germination, since fixation of CO2 commenced immediately and 70% of the fixed carbon accumulated as glycogen. Although cyanophycin was degraded after 6 h, this reserve material was subsequently resynthesized with concomitant breakdown of phycocyanin. These nitrogen reserves were not required for protein synthesis, which was initiated immediately after the induction of germination, apparently at the expense of another, non-proteinaceous, reserve material. Although RNA synthesis occurred without lag, DNA synthesis commenced only after 80 min. Quantitatively similar changes were observed during germination in the presence of combined nitrogen except that neither cyanophycin nor phycocyanin were degraded and balanced growth was achieved more rapidly.
27 citations
"Identification of Nostoc punctiform..." refers background in this paper
...Although the germination of akinetes has received much attention (Chauvat et al., 1982; Sutherland et al., 1985; Sili et al., 1994; van Dok and Hart, 1996), the molecular genetic study of akinete induction has been hindered by their normally slow and asynchronous formation....
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TL;DR: Akinetes of both strains proved to be highly resistant to desiccation, being able to germinate, in the presence of either N2 or nitrate as nitrogen source, with a germination frequency of more than 90% after seven years of storage in a dried state.
Abstract: Morphological and biochemical changes associated with synchronous germination of mature, aged and desiccated akinetes of two alkaliphilic cyanobacteria, Cyanospira rippkae and Cyanospira capsulata, are described. Akinetes of both strains proved to be highly resistant to desiccation, being able to germinate, in the presence of either N2 or nitrate as nitrogen source, with a germination frequency of more than 90% after seven years of storage in a dried state. The first cell division occurred after 8–10 h of incubation, thereafter the germlings of the two strains followed a different pattern of cell differentiation. Heterocysts were first noted, in a terminal position, at 16–18 h in three-celled germlings of C. capsulata and at 21–24 h in C. rippkae, when germlings were at least seven cells in length. Akinetes of both species possessed, on a per cell basis, almost identical amounts of all photosynthetic pigments but, under nitrogen fixing conditions, photosynthetic activity (oxygen evolution) was detected only after new proteins had been synthesized, before a functional heterocyst was developed and while total nitrogen remained constant. With energy provided by aerobic respiration, a wide range of intracellular amino acids characteristic of proteins was utilised to sustain the new protein synthesis. The end of this biosynthetic activity coincided with the timing of the first cell division. From this stage on, no changes in protein concentration occurred until mature heterocysts were developed. In the presence of nitrate, no significant changes in the major germination events were observed.
26 citations
"Identification of Nostoc punctiform..." refers background in this paper
...…like the endospores of Gram-positive bacteria, cyanobacterial akinetes have been reported to survive 5–7 years of desiccation (Yamamoto, 1975; Sili et al., 1994), months of cold (4°C) dark conditions (Sutherland et al., 1979), and have been isolated from sediments as old as 64 years…...
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...Although the germination of akinetes has received much attention (Chauvat et al., 1982; Sutherland et al., 1985; Sili et al., 1994; van Dok and Hart, 1996), the molecular genetic study of akinete induction has been hindered by their normally slow and asynchronous formation....
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...Although not heat-resistant like the endospores of Gram-positive bacteria, cyanobacterial akinetes have been reported to survive 5–7 years of desiccation (Yamamoto, 1975; Sili et al., 1994), months of cold (4°C) dark conditions (Sutherland et al....
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