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Journal ArticleDOI

Identification of Nostoc punctiforme akinete-expressed genes using differential display.

01 Aug 2006-Molecular Microbiology (Blackwell Publishing Ltd)-Vol. 61, Iss: 3, pp 748-757
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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Citations
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Journal ArticleDOI
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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Journal ArticleDOI
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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Journal ArticleDOI
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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Book ChapterDOI
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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Journal ArticleDOI
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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Journal ArticleDOI
TL;DR: Akinetes from species of blue-green algae were recovered from the recent sediments of Rostherne Mere and gas vacuolate populations of Aphanizomenon and Anabaena were cultured from sediment up to 18 and 64 years old respectively.
Abstract: Akinetes from species of blue-green algae were recovered from the recent sediments of Rostherne Mere. Gas vacuolate populations of Aphanizomenon and Anabaena were cultured from sediment up to 18 and 64 years old respectively. Akinetes of blue-green algae may not only have a temporary, or overwintering, function but may also ensure long term survival.

95 citations


"Identification of Nostoc punctiform..." refers background in this paper

  • ...…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 (Livingstone and Jaworski, 1980)....

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  • ..., 1979), and have been isolated from sediments as old as 64 years (Livingstone and Jaworski, 1980)....

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Journal ArticleDOI
TL;DR: Transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium and reaffirms the relevance of the L PS-less lactococcal model for studies on Msb a-mediated drug transport.
Abstract: MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gram-negative Escherichia coli and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive Lactococcus lactis, our recent work in E. coli suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in L. lactis. MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with Ki values of 16 and 11 μM, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a Ki of 57 μM, in a similar range as the Ki for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on MsbA-mediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary E. coli proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium (C. L. Reyes and G. Chang, Science 308:1028-1031, 2005).

86 citations


"Identification of Nostoc punctiform..." refers background in this paper

  • ...When expressed in a Grampositive organism lacking LPS, MsbA conferred resistance to multiple drugs (Woebking et al., 2005)....

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01 Jan 1989

78 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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Journal ArticleDOI
TL;DR: Results confirm that the infection of A. punctatus by hormogonia leading to the establishment of an association is physiologically uncoupled from the development of a functional diazotrophic association and indicate that heterocyst regulatory elements downstream from HetR and HetF are required in both free-living and symbiotic heterocysts differentiation and nitrogenase expression.
Abstract: Three mutant strains (ntcA, hetR, hetF) of the cyanobacterium Nostoc punctiforme unable to differentiate heterocysts were characterized and examined for their ability to form a symbiotic association with the bryophyte Anthoceros punctatus. Previously unknown characteristics of the N. punctiforme hetR mutant include differentiation of chilling-resistant akinetes, while vegetative cells of the ntcA mutant randomly lysed, yielding short filaments, following ammonium deprivation. Strains with mutations in hetF and hetR infected A. punctatus with similar frequency to that of wild-type N. punctiforme but did not support growth of the plant partner. These results confirm that the infection of A. punctatus by hormogonia leading to the establishment of an association is physiologically uncoupled from the development of a functional diazotrophic association. They also indicate that heterocyst regulatory elements downstream from HetR and HetF are required in both free-living and symbiotic heterocyst differentiation and nitrogenase expression. A strain with a mutation in the global nitrogen regulator ntcA did not infect A. punctatus despite its ability to differentiate hormogonia at a low frequency. When complemented with one or more copies of ntcA, the mutant strain infected A. punctatus at a similar frequency as the wild-type and supported growth of the plant partner in the absence of combined nitrogen. These results established a connection between the presence of a functional copy of ntcA and the magnitude of hormogonium differentiation, and the behaviour of the formed hormogonia.

67 citations


"Identification of Nostoc punctiform..." refers background in this paper

  • ...…and Stigonematales, akinete induction by N. punctiforme usually occurs in cultures exposed to light limitation or phosphate starvation (Campbell et al., 1996; Wong and Meeks, 2002), although low intracellular ATP levels may not be the direct signal for their formation (Argueta and Summers, 2005)....

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  • ...punctiforme usually occurs in cultures exposed to light limitation or phosphate starvation (Campbell et al., 1996; Wong and Meeks, 2002), although low intracellular ATP levels may not be the direct signal for their formation (Argueta and Summers, 2005)....

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  • ...…HetR, was shown to be essential for both heterocyst and akinete formation in N. ellipsosporum (Leganes et al., 1994); however, a hetR mutant strain of N. punctiforme was able to form coldresistant akinete-like cells that lacked obvious granulation typical of akinetes (Wong and Meeks, 2002)....

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  • ...punctiforme was able to form coldresistant akinete-like cells that lacked obvious granulation typical of akinetes (Wong and Meeks, 2002)....

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Journal ArticleDOI
TL;DR: On the basis of RNA analysis, devR is constitutively expressed with respect to the nitrogen source for growth and is essential to the development of mature heterocysts and may be involved in a sensory pathway that is not directly responsive to cellular nitrogen status.
Abstract: Strain UCD 311 is a transposon-induced mutant of Nostoc sp. strain ATC C 29133 that is unable to fix nitrogen in air but does so under anoxic conditions and is able to establish a functional symbiotic association with the hornwort Anthoceros punctatus. These properties of strain UCD 311 are consistent with previous observations that protection against oxygen inactivation of nitrogenase is physiologically provided within A. punctatus tissue. Upon deprivation of combined nitrogen, strain UCD 311 clearly differentiates heterocysts and contains typical heterocyst-specific glycolipids; it also makes apparently normal akinetes upon phosphate starvation. Sequence analysis adjacent to the point of the transposon insertion revealed an open reading frame designated devR. Southern analysis established that similar sequences are present in other heterocyst-forming cyanobacteria. devR putatively encodes a protein of 135 amino acids with high similarity to the receiver domains of response regulator proteins characteristics of two-component regulatory systems. On the basis of its size and the absence of other functional domains, DevR is most similar to CheY and Spo0F. Reconstruction of the mutation with an interposon vector confirmed that the transposition event was responsible for the mutant phenotype. The presence of wild-type devR on a plasmid in strain UCD 311 restored the ability to fix nitrogen in air. While devR was not essential for differentiation of akinetes, its presence in trans in Nostoc sp. strain ATCC 29133 stimulated their formation to above normal levels in aging medium. On the basis of RNA analysis, devR is constitutively expressed with respect to the nitrogen source for growth. The devR gene product is essential to the development of mature heterocysts and may be involved in a sensory pathway that is not directly responsive to cellular nitrogen status.

59 citations


"Identification of Nostoc punctiform..." refers background in this paper

  • ...…doi:10.1111/j.1365-2958.2006.05263.x First published online 15 June 2006 © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd involved in heterocyst polysaccharide synthesis, causesincreased akinete induction in N. punctiforme, possibly due to cross-talk (Campbell et al., 1996)....

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  • ...punctiforme, possibly due to cross-talk (Campbell et al., 1996)....

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  • ...punctiforme usually occurs in cultures exposed to light limitation or phosphate starvation (Campbell et al., 1996; Wong and Meeks, 2002), although low intracellular ATP levels may not be the direct signal for their formation (Argueta and Summers, 2005)....

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  • ...…Nostocales and Stigonematales, akinete induction by N. punctiforme usually occurs in cultures exposed to light limitation or phosphate starvation (Campbell et al., 1996; Wong and Meeks, 2002), although low intracellular ATP levels may not be the direct signal for their formation (Argueta and…...

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