1 Department of Botany, Stockholm University, 10691 Stockholm, Sweden
2 Department of Zoology and Marine Biology, University of Dar es Salaam, PO Box 35064, Dar es Salaam, Tanzania
3 NIOO Centre for Estuarine and Marine Ecology (NIOO-CEME), 4400 AC, Yerseke, The Netherlands
Correspondence
B. Bergman
Birgitta.Bergman{at}botan.su.se
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ABSTRACT |
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Present address: Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Canada M5S 1A8.
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INTRODUCTION |
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In the heterocystous cyanobacterium Anabaena PCC 7120, differentiation of a vegetative cell into a heterocyst requires the activation of several genes in a cascade-like mechanism of transcriptional activations (Cai & Wolk, 1997). This differentiation process depends on both the global nitrogen regulator NtcA and the cell differentiation regulatory protein HetR (Wolk, 2000
). ntcA encodes a transcriptional regulator that belongs to the CRP family of bacterial regulators (Herrero et al., 2001
), which activates the expression of genes involved in nitrogen assimilation upon ammonium withdrawal by binding to specific sites in their promoters. NtcA is required for development and function of mature heterocysts, and for the expression of nitrogen transport and assimilation systems as well as N2 fixation (Herrero et al., 2001
). HetR is a key regulator of heterocyst differentiation (Buikema & Haselkorn, 1991
) and is an autoregulated serine-type protease that can degrade itself and possibly other proteins (Zhou et al., 1998
). HetR shows enhanced induction within 12 h of nitrogen stepdown, and within 3·5 h hetR expression is clearly localized to spaced cells, presumably developing pro-heterocysts (Black et al., 1993
). Induction of hetR upon nitrogen step-down depends on NtcA (Muro-Pastor et al., 2002
). The presence of hetR in filamentous non-heterocystous strains, including Trichodesmium (Janson et al., 1998
; Orcutt et al., 2002
; Schiefer et al., 2002
), and the requirement of hetR for akinete differentiation (Leganés et al., 1994
) suggest that hetR is not exclusively involved in heterocysts differentiation (see Wolk, 2000
).
Like heterocysts, diazocytes are the exclusive carriers of nitrogenase and fix nitrogen aerobically in the light, and show morphological and physiological changes. These include a decrease in cyanophycin granules and synthesis of additional membranes (Fredriksson & Bergman, 1997), an increase in glutamine synthetase levels (Carpenter et al., 1992
), and an increase in cytochrome oxidase levels (Bergman et al., 1993
). Such changes suggest that differential gene expression may be required to generate diazocytes.
As cell differentiation is a fundamental biological process we initiated a study on identification of the molecular basis for the differentiation of the novel N2-fixing cell type (diazocytes) recently discovered in Trichodesmium (Fredriksson & Bergman, 1997; Lin et al., 1998
; Berman-Frank et al., 2001
). For this purpose, we followed the occurrence and distribution pattern of diazocytes in trichomes and applied the sensitive real-time RT-PCR and SYBR Green I assay to monitor the expression of ntcA, hetR and nifH in cultures of Trichodesmium IMS101 grown under a 24 h light/dark regime, in the absence and presence of ammonia. A comparative search between the Trichodesmium IMS101 and Anabaena PCC 7120 genomes was also performed. Possible mechanisms involved in diazocyte development are discussed.
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METHODS |
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In situ immunolocalization/light microscopy.
Trichomes were mounted on poly-L-lysine-coated glass slides (Sigma, product no. P0425), fixed and permeablized in 100 % ethanol overnight at -20 °C (Lin et al., 1998). A rabbit anti-Rhodospirillum rubrum NifH antibody (100-fold dilution) was used as the primary antibody in conjunction with a fluorescein-conjugated secondary anti-rabbit antibody (200-fold dilution) (Molecular Probes, product no. A-11069). Trichomes were examined by light microscopy using an Olympus microscope fitted with U-MWU filters (BP330385, BA420, DM400).
Acetylene reduction assay.
Samples (2 ml) of Trichodesmium cultures were placed in 10 ml serum vials, 10 % (v/v) acetylene was injected and vials were incubated for 1 h under the same growth conditions as those given above. Head-space gas samples were withdrawn, and the ethylene production was measured using a Perkin-Elmer F11 gas chromatograph. Ethylene concentrations were calculated by reference to calibrated ethylene gas standards (Lundgren et al., 2001).
Sampling and RNA extraction.
At each sampling time during the 12 h/12 h L/D cycle, cells were collected into sterile plastic tubes and quickly filtered through 5 µm Millipore filters (Whatman, cat. no. 110613). The filters were immediately vortexed in RLT buffer containing -mercaptoethanol (Qiagen, cat. no. 74104). The filters were discarded and the samples were stored at -80 °C. RNA was extracted using RNeasy Mini Extraction Kit (Qiagen, cat. no. 74104). Relative quantification (see below) of 16S rRNA (GenBank accession no. AF013030) was performed to verify that equal amounts of total mRNA were loaded in the real time RT-PCR runs (results presented in the figure legends).
PCR and cloning.
Primer sequences used to amplify part of the sequences of ntcA (AF169961), hetR (AF091323) and nifH (U90952) from Trichodesmium IMS101 were designed accordingly (NCBI database): pntcAP, 5'-TGA TGA TTG AAA CCT TGG CA-3'; pntcAM, 5'-CTC TGC GAT TGC TTG ATG AG-3'; phetRP, 5'-TGA ACC CAA ACG GGT TAA AG-3'; phetRM, 5'-GCT TCA CTT AGA GGC ATC CG-3'; pnifHP, 5'-CGG TGG CAT TAA GTG TGT TG-3'; pnifHM, 5'-ACC TAA ACG GAC ACC ACC AG-3'.
Trichodesmium IMS101 DNA was extracted using the Genomic DNA isolation kit (Qiagen, buffer set cat. no. 19060, QIAprep spin cat. no. 27104) and PCR was carried using HotStar Taq polymerase (Qiagen, cat. no. 203203). PCR products with ntcA, hetR and nifH primers were cloned using the TOPO 2.1 TA cloning kit (Invitrogen, cat. no. 45-0641) and sequenced by MWG Biotech (Germany) to verify that the primers bound to the right targets. These primers were then used for the SYBR Green I assay below.
Real-time RT-PCR and SYBR Green I assay.
The real-time PCR reactions were carried out in an iCycler iQ (Bio-Rad) using a QuantiTect SYBR Green RT-PCR kit (Qiagen, cat. no. 204243). The reaction mixture was prepared to contain 1x QuantiTect SYBR Green RT-PCR Master mix, 0·5 µl QuantiTect RT Mix, 0·5 µl fluorescein (1 µM; Bio-Rad, 170-8780), 0·5 µM of each primer, and RNase-free water to 50 µl. The RT-PCR programme was as follows: 50 °C for 30 min, 95 °C for 15 min, 40 cycles at 94 °C for 30 s, 54 °C (55 °C for nifH) for 30 s and 72 °C for 30 s. The fluorescence values were collected at 72 °C. The relative cDNA quantities of ntcA, hetR and nifH, and hence mRNA, were determined using serial dilutions of Trichodesmium IMS101 DNA (330 µg ml-1) as a standard (at least four dilutions were included in each measurement). Following each run, a melt curve analysis step was performed according to the iCycler iQ manual to verify that the PCR products from the mRNA and DNA were the same, and that primer dimers were absent or if present that they were not stable at 72 °C and hence not incorporated into the datasets. PCR products were visualized on 2 % agarose gels stained with ethidium bromide.
Genome analysis.
A comparative genomic analysis between the heterocystous Anabaena PCC 7120 (http://www.kazusa.or.jp/cyano/Anabaena/index.html) and T. erythraeum IMS101 (http://genome.jgi-psf.org/draft_microbes/trier/trier.home.html) was undertaken using BLAST.
Effects of PatS.
The synthetic PatS-5 peptide (a generous gift from Dr J. W. Golden, Texas A & M, Texas, USA), known to prevent heterocyst development in Anabaena PCC 7120 (Yoon & Golden, 1998), was added to Trichodesmium IMS101 cultures at concentrations ranging from 1 to 10 µm. After 36 h, the occurrence of diazocytes was analysed using in situ immunolabelling of NifH and the trichomes were examined by light microscopy (as above).
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RESULTS AND DISCUSSION |
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In order to search for the role of hetR and its possible connection to diazocyte formation in Trichodesmium we made use of the real-time RT-PCR methodology and the SYBR Green I assay. This is a powerful technique for detecting and quantifying low mRNA expression levels (Gibson et al., 1996; Heid et al., 1996
; Lie & Petropoulos, 1998
). We first examined the expression of hetR and the global regulatory gene involved in nitrogen control and heterocyst differentiation, ntcA (Herrero et al., 2001
), in N2-fixing cultures. As nitrogenase is confined to diazocytes, the expression of nifH, the gene encoding dinitrogenase reductase, was simultaneously followed. The specificity of the three primer pairs used was checked by amplification and cloning of the fragments generated, followed by sequencing and comparisons with already published cyanobacterial sequences using BLAST provided at the NCBI database site (http://www.ncbi.nlm.nih.gov/BLAST/). The characteristic Tm of the amplified fragments were easily distinguished from the Tm of non-specific products and primer dimers on a melt curve chart (data not shown). Measurements of fluorescence intensity were only performed at temperatures above the Tm of the primer dimers and below that of the specific PCR products. Product identification was performed by comparing the respective Tm on the melt curve charts and the respective molecular size on agarose gels.
The expression of ntcA, hetR and nifH in samples of Trichodesmium IMS101 was followed over 12 h/12 h L/D periods. As seen in Fig. 4, ntcA was constitutively expressed at moderate levels through the diurnal cycle. In contrast, hetR expression showed a distinct diurnal rhythmicity. It increased sharply from 20 : 00 h and reached a fourfold higher expression 2 h into the dark phase. Expression then decreased to a minimum just before 08 : 00 h in the morning, when the light was again switched on. The data also show that hetR is still expressed, albeit at low levels, under the more nitrogen-replete periods (daytime). The timing in the hetR expression pattern was persistent in all experiments (n=10), although expression levels at the peak varied between 3- and 10-fold.
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To investigate the possible role of ntcA and hetR in relation to N2 fixation and diazocyte formation a source of combined nitrogen was added to the N2-fixing Trichodesmium IMS101 cultures (Fig. 5). When 100 µM NH4Cl was added at 20 : 00 h (n=4), ntcA expression decreased and the up-shift in hetR expression, seen under N2-fixing conditions at 22 : 00 (Fig. 4
), was diminished and followed by a slow continuous decrease. Likewise, nifH expression was negatively affected (Fig. 5
), and no acetylene reduction activity was recorded. The upshift seen at the end of the light period (Fig. 5
) was probably due to exhaustion of
from the growth medium. Higher concentrations of
were avoided as they may negatively affect Trichodesmium growth. A repression of ntcA, hetR and nifH expression and heterocyst differentiation by combined nitrogen is well known for heterocystous cyanobacteria (Herrero et al., 2001
; Adams & Duggan, 1999
), and the expression of ntcA and hetR are mutually dependent on each other (Muro-Pastor et al., 2002
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bergman, B. & Carpenter, E. J. (1991). Nitrogenase confined to randomly distributed trichomes in the marine cyanobacterium Trichodesmium thiebautii. J Phycol 27, 158165.
Bergman, B., Siddiqui, P. J. A., Carpenter, E. J. & Peschek, G. A. (1993). Cytochrome oxidase: subcellular distribution and relation to nitrogenase expression in the nonheterocystous marine cyanobacterium Trichodesmium thiebautii. Appl Environ Microbiol 59, 32393244.[Abstract]
Berman-Frank, I., Lundgren, P., Chen, Y.-B., Küpper, H., Kolber, Z., Bergman, B. & Falkowski, P. (2001). Segregation of nitrogen fixation and oxygenic photosynthesis in the marine cyanobacterium Trichodesmium. Science 294, 15341537.
Black, T. A., Cai, Y. & Wolk, C. P. (1993). Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena. Mol Microbiol 9, 7784.[Medline]
Bryceson, I. & Fay, P. (1981). Nitrogen fixation in Oscillatoria (Trichodesmium) in relation to bundle formation and trichome differentiation. Mar Biol 61, 159166.
Buikema, W. J. & Haselkorn, R. (1991). Characterization of a gene controlling heterocyst differentiation in the cyanobacterium Anabaena 7120. Genes Dev 5, 321330.[Abstract]
Cai, Y. & Wolk, C. P. (1997). Anabaena sp. strain PCC 7120 responds to nitrogen deprivation with a cascade-like sequence of transcriptional activations. J Bacteriol 179, 267271.[Abstract]
Campbell, E. L., Hagen, K. D., Cohen, M. F., Summers, M. L. & Meeks, J. C. (1996). The devR gene product is characteristic of receivers of two-component regulatory systems and is essential for heterocyst development in the filamentous cyanobacterium Nostoc sp. strain ATCC 29133. J Bacteriol 178, 20372043.[Abstract]
Capone, D. G., Zehr, J. P., Paerl, H. W., Bergman, B. & Carpenter, E. J. (1997). Trichodesmium: a globally significant marine cyanobacterium. Science 276, 12211229.
Carpenter, E. J. & Price, C. C. (1976). Marine Oscillatoria (Trichodesmium): explanation for aerobic nitrogen fixation without heterocysts. Science 191, 12781280.[Medline]
Carpenter, E. J., Bergman, B., Dawson, R., Siddiqui, P. J., Soderback, E. & Capone, D. G. (1992). Glutamine synthetase and nitrogen cycling in colonies of the marine diazotrophic cyanobacteria Trichodesmium spp. Appl Environ Microbiol 58, 31223129.[Abstract]
Chen, Y.-B., Dominic, B., Mellon, M. T. & Zehr, J. P. (1998). Circadian rhythm of nitrogenase gene expression in the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp. strain IMS 101. J Bacteriol 180, 35983605.
Dominic, B., Chen, Y.-B. & Zehr, J. P. (1998). Cloning and transcriptional analysis of the nifUHDK genes of Trichodesmium sp. IMS 101 reveals stable nifD, nifDK and nifK transcripts. Microbiology 144, 33593368.[Abstract]
Fredriksson, C. & Bergman, B. (1995). Nitrogenase varies diurnally in a subset of cells within colonies of the non-heterocystous cyanobacteria Trichodesmium spp. Microbiology 141, 24712478.
Fredriksson, C. & Bergman, B. (1997). Ultrastructural characterization of cells specialized for nitrogen fixation in a non-heterocystous cyanobacterium, Trichodesmium. Protoplasma 197, 7685.
Gibson, U. E., Heid, C. A. & Williams, P. M. (1996). A novel method for real time quantitative RT-PCR. Genome Res 6, 9951001.[Abstract]
Heid, C. A., Stevens, J., Livak, K. J. & Williams, P. M. (1996). Real time quantitative PCR. Genome Res 6, 986994.[Abstract]
Herrero, A., Muro-Pastor, A. M. & Flores, E. (2001). Nitrogen control in cyanobacteria. J Bacteriol 183, 411425.
Janson, S., Carpenter, E. J. & Bergman, B. (1994). Compartmentalization of nitrogenase in a non-heterocystous cyanobacterium, Trichodesmium contortum. FEMS Microbiol Lett 118, 914.
Janson, S., Matveyev, A. & Bergman, A. (1998). The presence and expression of hetR in the non-heterocystous cyanobacterium Symploca PCC 8002. FEMS Microbiol Lett 168, 173179.[CrossRef][Medline]
Janson, S., Bergman, B., Carpenter, E. J., Giovannani, S. J. & Vergin, K. (1999). Genetic analysis of natural populations of the marine diazotrophic cyanbacterium Trichodesmium. FEMS Microbiol Ecol 30, 5765.[CrossRef]
Karl, D., Michaels, A., Bergman, B. & 7 other authors (2002). Dinitrogen fixation in the world's oceans. Biogeochem 57/58, 4798.[CrossRef]
Khudyakov, I. & Wolk, C. P. (1996). Evidence that hanA gene coding for HU protein is essential for heterocyst differentiation in, and cyanophage A-4(L) sensitivity of, Anabaena sp. strain PCC 7120. J Bacteriol 178, 35723577.[Abstract]
Leganés, F., Fernández-Piñas, F. & Wolk, C. P. (1994). Two mutations that block heterocyst differentiation have different effects on akinete differentiation in Nostoc ellipsosporum. Mol Microbiol 12, 679684.[Medline]
Liang, J., Scappino, L. & Haselkorn, R. (1992). The patA gene product, which contains a region similar to CheY of Escherichia coli, controls heterocyst pattern formation in the cyanobacterium Anabaena 7120. Proc Natl Acad Sci U S A 89, 56555659.[Abstract]
Liang, J., Scappino, L. & Haselkorn, R. (1993). The patB gene product, required for growth of the cyanobacterium Anabaena spp. strain PCC 7120 under nitrogen-limiting conditions, contains ferredoxin and helix-turn-helix domains. J Bacteriol 175, 16971704.[Abstract]
Lie, Y. S. & Petropoulos, C. J. (1998). Advances in quantitative PCR technology: 5' nuclease assays. Curr Opin Biotechnol 9, 4348.[CrossRef][Medline]
Lin, S., Henze, S., Lundgren, P., Bergman, B. & Carpenter, E. J. (1998). Whole-cell immunolocalization of nitrogenase in marine diazotrophic cyanobacteria, Trichodesmium spp. Appl Environ Microbiol 64, 30523064.
Lugomela, C., Lyimo, T., Bryceson, I., Semesi, A. K. & Bergman, B. (2002). Trichodesmium in coastal waters of Tanzania: diversity, seasonality, nitrogen and carbon fixation. Hydrobiologia 477, 113.[CrossRef]
Lundgren, P., Söderbäck, E., Carpenter, E. J. & Bergman, B. (2001). Katagnymene: characterization of a novel marine diazotroph. J Phycol 37, 10521062.[CrossRef]
Mulholland, M. & Capone, D. G. (1999). Nitrogen fixation, uptake and metabolism in natural and collected populations of Trichodesmium spp. Mar Ecol Prog Ser 188, 3349.
Muro-Pastor, A. M., Valladares, A., Flores, E. & Herrero, A. (2002). Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development. Mol Microbiol 44, 13771385.[CrossRef][Medline]
Nagaraja, R. & Haselkorn, R. (1994). Protein HU from the cyanobacterium Anabaena. Biochimie 76, 10821089.[CrossRef][Medline]
Orcutt, K. M., Rasmussen, U., Webb, E. A., Waterbury, J. B., Gundersen, K. & Bergman, B. (2002). Characterization of Trichodesmium spp. by genetic techniques. Appl Environ Microbiol 68, 22362245.
Prufert-Bebout, L. E., Pearl, H. W. & Lassen, C. (1993). Growth, nitrogen fixation and spectral attenuation in cultivated Trichodesmium. Appl Environ Microbiol 59, 13501359.
Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. & Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111, 161.
Schiefer, W., Schutz, K., Hachtel, W. & Happe, T. (2002). Molecular cloning and characterization of hetR genes from filamentous cyanobacteria. Biochim Biophys Acta 1577, 139143.[Medline]
Wilcox, M., Mitchison, G. J. & Smith, R. J. (1973). Pattern formation in the blue-green alga, Anabaena. I. Basic mechanisms. J Cell Sci 12, 707725.[Medline]
Wolk, C. P. (2000). Heterocyst formation in Anabaena. In Prokaryotic Development, pp. 83104. Edited by Y. V. Brun & L. J. Shimkets. Washington, DC: American Society for Microbiology.
Yoon, H.-S. & Golden, J. W. (1998). Heterocyst pattern formation controlled by a diffusible peptide. Science 282, 935938.
Yoon, H.-S. & Golden, J. W. (2001). PatS and products of nitrogen fixation control heterocyst pattern. J Bacteriol 183, 26052613.
Zehr, J. P., Wyman, M., Miller, V., Duguay, L. & Capone, D. G. (1993). Modification of the Fe protein of nitrogenase in natural populations of Trichodesmium thiebautii. Appl Environ Microbiol 59, 669676.[Abstract]
Zhou, R., Wei, X., Jian, N., Li, H., Dong, Y., His, K.-L. & Zhao, J. (1998). Evidence that HetR protein is an unusual serine-type protease. Proc Natl Acad Sci U S A 95, 49594963.
Received 5 December 2002;
revised 7 February 2003;
accepted 14 February 2003.
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