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Developmental Mechanisms in Filamentous Cyanobacteria

We study the filamentous cyanobacterium Anabaena. This organism provides models of the short-range intercellular interactions that control development in many organisms. When Anabaena grows in the presence of abundant fixed nitrogen, all of its cells appear to be of the same type, referred to as a vegetative cell. But when nitrogen becomes limiting, 5 to 10% of the cells differentiate, at semi-regular intervals along the filaments, into nitrogen-fixing cells called heterocysts. In some species, yet a third type of cell, the akinete (or spore), can differentiate from vegetative cells positioned adjacent to heterocysts. Pre-existing heterocysts inhibit nearby cells from differentiating into heterocysts and (in the species referred to) can induce nearby cells to become akinetes. We wish to know how they do so.

Figure 1. Filaments of Anabaena cylindrica. Heterocysts (H) are nitrogen-fixing cells. Enlarged spores (S), a.k.a. akinetes, here still immature, form by differentiation of vegetative cells adjacent to heterocysts, and can germinate after exposure to harsh conditions. The remaining vegetative cells of the filaments photosynthesize and grow.

To facilitate molecular analysis of development in Anabaena, we developed a genetic methodology. If restriction by cyanobacterial endonucleases is avoided, DNA can be transferred with high efficiency from Escherichia coli to Anabaena by conjugative bacterial plasmids. Mutations can thereupon be complemented and reporter genes, e.g., luciferase or green fluorescent protein, can be inserted downstream from a specific promoter in the genome, permitting visualization of the cells in which a particular promoter is active. Transposons active in Anabaena are available, gene replacement is routine, and the genome has been sequenced.

Using such tools of microbial genetics, we and others are elucidating the genetics and biochemistry of Anabaena development. At the same time, we are illuminating the mechanisms and biochemical processes that permit nitrogen fixation and hydrogen production, oxygen-labile processes, to take place in oxygen-producing filaments. We have begun to try to determine whether genetic manipulation might enable Anabaena, starting with sunlight and water, to become a commercially practicable producer of hydrogen gas as a fuel.

Selected Publications

Zhao J, Wolk CP (2008) Developmental biology of heterocysts, 2006. In D Whitworth, ed, Myxobacteria: Multicellularity and Differentiation. Am Soc Microbiol, Washington, DC., pp 397-418

Fan Q, Lechno-Yossef S, Ehira S, Kaneko T, Ohmori M, Sato N, Tabata S, Wolk CP (2006) Signal transduction genes required for heterocyst maturation in Anabaena sp. strain PCC 7120. J Bacteriol 188: 6688-6693

Fan Q, Huang G, Lechno-Yossef S, Wolk CP, Kaneko T, Tabata S (2005) Clustered genes required for synthesis and deposition of envelope glycolipids in Anabaena sp. strain PCC 7120. Mol Micro 58: 227-243 Full Text

Zhou R, Wolk CP (2002) Identification of an akinete marker gene in Anabaena variabilis. J Bacteriol 184: 2529-2532 Full Text

Kaneko T, Nakamura Y, Wolk CP, Kuritz T, Sasamoto S, Watanabe A, Iriguchi M, Ishikawa A, Kawashima K, Kimura T, Kishida Y, Kohara M, Matsumoto M, Matsuno A, Muraki A, Nakazaki N, Shimpo S, Sugimoto M, Takazawa M, Yamada M,Yasuda M, Tabata S (2001) Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res 8: 205-213 and 8 (Suppl):227-253   Full Text     Supplement


Selected Abstracts

Kaneko T, Nakamura Y, Wolk CP, Kuritz T, Sasamoto S, Watanabe A, Iriguchi M, Ishikawa A, Kawashima K, Kimura T, Kishida Y, Kohara M, Matsumoto M, Matsuno A, Muraki A, Nakazaki N, Shimpo S, Sugimoto M, Takazawa M, Yamada M,Yasuda M, Tabata S (2001) Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res 8: 205-213 and 8 (Suppl):227-253

The nucleotide sequence of the entire genome of a filamentous cyanobacterium, Anabaena sp. strain PCC 7120, was determined. The genome of Anabaena consisted of a single chromosome (6,413,771 bp) and six plasmids, designated pCC7120α (408,101 bp), pCC7120β (186,614 bp), pCC7120γ (101,965 bp), pCC7120δ (55,414 bp), pCC7120ε (40,340 bp), and pCC7120ζ (5,584 bp). The chromosome bears 5368 potential protein-encoding genes, four sets of rRNA genes, 48 tRNA genes representing 42 tRNA species, and 4 genes for small structural RNAs. The predicted products of 45% of the potential protein-encoding genes showed sequence similarity to known and predicted proteins of known function, and 27% to translated products of hypothetical genes. The remaining 28% lacked significant similarity to genes for known and predicted proteins in the public DNA databases. More than 60 genes involved in various processes of heterocyst formation and nitrogen fixation were assigned to the chromosome based on their similarity to the reported genes. One hundred and ninety-five genes coding for components of two-component signal transduction systems, nearly 2.5 times as many as those in Synechocystis sp. PCC 6803, were identified on the chromosome. Only 37% of the Anabaena genes showed significant sequence similarity to those of Synechocystis, indicating a high degree of divergence of the gene information between the two cyanobacterial strains.

Zhou R, Wolk CP (2002) Identification of an akinete marker gene in Anabaena variabilis. J Bacteriol 184: 2529-2532

Cyanobacteria that form akinetes as well as heterocysts present a rare opportunity to investigate the relationships between alternative differentiation processes and pattern formation processes in a single bacterium. Because no akinete marker gene has been identified, akinete formation has been little studied genetically. We report the first identification of an akinete marker gene.

Fan Q, Huang G, Lechno-Yossef S, Wolk CP, Kaneko T, Tabata S (2005) Clustered genes required for synthesis and deposition of envelope glycolipids in Anabaena sp. strain PCC 7120. Mol Microbiol 58: 227-243

Photoreduction of dinitrogen by heterocyst-forming cyanobacteria is of great importance ecologically and for subsistence rice agriculture. Their heterocysts must have a glycolipid envelope layer that limits the entry of oxygen if nitrogenase is to remain active to fix dinitrogen in an oxygen-containing milieu (the Fox⁺ phenotype). Genes alr5354 (hglD), alr5355 (hglC) and alr5357 (hglB) of the filamentous cyanobacterium, Anabaena sp. strain PCC 7120, and hglE of Nostoc punctiforme are required for synthesis of heterocyst envelope glycolipids. Newly identified Fox^- mutants bear transposons in nearby open reading frames (orfs) all5343, all5345–asr5349 and alr5351–alr5358. Complementation and other analysis provide evidence that at least orfs all5343 (or a co-transcribed gene), all5345, all5347, alr5348, asr5350–alr5353 and alr5356, but not asr5349, are also required for a Fox⁺ phenotype. Lipid and sequence analyses suggest that alr5351–alr5357 encode the enzymes that biosynthesize the glycolipid aglycones. Electron microscopy indicates a role of all5345 through all5347 in the normal deposition of the envelope glycolipids. 

Fan Q, Lechno-Yossef S, Ehira S, Kaneko T, Ohmori M, Sato N, Tabata S, Wolk CP (2006) Signal transduction genes required for heterocyst maturation in Anabaena sp. strain PCC 7120. J Bacteriol 188: 6688-6693

How heterocyst differentiation is regulated, once particular cells start to differentiate, remains largely unknown. Using near-saturation transposon mutagenesis and testing of transposon-tagged loci, we identified three presumptive regulatory genes not previously recognized as being required specifically for normal heterocyst maturation. One of these genes has a hitherto unreported mutant phenotype. Two previously identified regulatory genes were further characterized.