Nitrogen Fixation Analysis Of The Genome Of The Cyanobacterium Nostoc Punctiforme

T. Thiel1, J.C. Meeks2, J. Elhai3, M. Potts4, F. Larimer5, J. Lamerdin6, P. Predki7, R. Atlas8

department of Biology, University of Missouri, St. Louis, MO 63121, USA 2Section of Microbiology, University of California, Davis, CA 95616, USA department of Biology, University of Richmond, Richmond, VA 23173, USA department of Biochemistry, Virginia Polytechnic Institute and State University,

Blacksburg,VA 24061, USA Computational Biology, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA 6Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory,

Livermore, CA 94551, USA 7DOE Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA

Department of Biology, University of Louisville, Louisville, KY 40292, USA

1. Introduction

Nostoc punctiforme ATCC 29133 is a diazotrophic, filamentous cyanobacterium with oxygen-evolving photosynthesis. Although this strain is primarily an autotroph, it can grow heterotrophically in the dark using sucrose, glucose or fructose (Summers et al. 1995). Like all cyanobacteria it has chlorophyll a as well as light harvesting pigments called phycobiliproteins. This strain chromatically adapts to light by varying the amounts of one biliprotein, phycoerythrin, in response to the presence or absence of green light. Cyanobacteria, which form a monophyletic group, are classified on the basis of cell division (resulting in unicellular or filamentous growth) and the differentiation of specialized cells, such as heterocysts, akinetes, or hormogonia (Rippka et al. 1979). Heterocysts are specialized for nitrogen fixation in an oxic environment, hormogonia are motile filaments, and akinetes are resistant to many environmental stresses. N. punctiforme has one of largest bacterial genomes sequenced to date, which is not surprising since it encompasses virtually every characteristic of the cyanobacterial group. N. punctiforme has a complex life cycle that includes, at different times, the differentiation of vegetative cells into motile hormogonia, nitrogen-fixing heterocysts, or resistant akinetes. The patterning of spaced heterocysts in filaments as well as the interdependence between vegetative cells that supply fixed carbon and heterocysts that supply fixed nitrogen to the filament indicate cell-to-cell communication, suggesting that these and other heterocyst-forming cyanobacteria function as multicellular organisms (Thiel, Pratte 2001).

N. punctiforme forms symbiotic associations with a variety of species including the cycad Macrozamia sp., the angiosperm Gunnera sp., and the bryophyte hornwort Anthoceros punctatus. Cultured cells of this strain can establish a nitrogen-fixing symbiosis with Anthoceros punctatus (Enderlin, Meeks 1983) and Gunnera spp. (Johansson, Bergman 1994). In the association, photosynthesis in the symbiotic Nostoc species decreases and the rate of nitrogen fixation increases. In addition, heterotrophic metabolism, which supports nitrogen fixation, increases. The plant partner produces molecules that control the differentiation of hormogonia and heterocysts (Meeks 1998). The functional analysis of the N. punctiforme genome will help in understanding these microbe-plant interactions that promote a stable nitrogen-fixing association.

2. Procedures

2.1. Genome analysis. The genomes of N. punctiforme and Rhodopseudomonas palustris were sequenced using a whole genome shotgun strategy (Fleischmann et al. 1995). DNA preparation protocols are described on the web site: http://www.jgi.doe.gov/ under "Production protocols". Raw sequence data were assembled with PHRAP and through "auto-finishing" using software written by

Matt Nolan (JGI/Lawrcnce Livermore National Laboratory) and David Gordon (University of Washington). Open reading frames (ORFs) were identified using Critica, Glimmer and Generation. The set was searched against the KEGG GENES, Pfam, PROSITE, PRINTS, ProDom and COGS databases, in addition to BLASTP versus the non-redundant database. Putative genes were organized into functional categories based on KEGG categories and COGs hierarchies. Additional analyzes are based on sequence comparisons to Cyanobase, Genbank and Swissprot.

2.2. Phylogenic analysis. Phylogenetic analysis used the program Clustal W for amino acid sequence alignment and the Phylip programs Seqboot, Protdist, Neighbor, and Consense to produce distance trees. Trees were visualized in Treeview. Bootstrap values were based on 1000 replicates.

3. Results and Discussion

3.1. Genome analysis. The genome of N. punctiforme is significantly larger than any other sequenced cyanobacterial genome. In addition to the three genomes shown in Table 1, two additional cyanobacterial genomes are near completion: the 1.7 Mb genome of Prochlorococcus marinus MED4 and the 2.4 Mb genome of Synechococcus sp. strain WH 8102. The current N. punctiforme genome size is 9,757,495 bases (11X sequencing coverage); however, the annotation is based on only about 92% of the genome (8X coverage; 8,941,326 bases). Of the more than 5000 recognized ORFs only about 62% of those encode proteins with a known or probable known function, while the remainder encode conserved hypothetical proteins with no known function (Table 1). Interestingly, almost a quarter of the genome encodes ORFs that cannot be associated with a previously recognized ORF.

Table 1. Comparison of three cyanobacterial genomes

Strain

Genome

ORFs

Recognized

Known or

ORFs

Unique to

size

ORFs

probable

present in

strain

function

Nostoc

Nostoc

9.78 Mb

7,432

5,314

3,328

7,432

1,578 (23%)

punctiforme

Synechocystis

3.57 Mb

3,215

3,215

1,521

2,547 (80%)

668 (20%)

6803

Anabaena 7120

7.20 Mb

5,610

4,327

4,814 (86%)

797 (14%)

Comparison of the genomes of N. punctiforme and Anabaena sp. PCC 7120 reveal that they share about 80% of their genetic information, implying a close phylogenetic relationship between the two strains. N. punctiforme contains multiple copies of many genes in Anabaena 7120 and/or in Synechocystis 6803. Anabaena 7120 and Synechocystis 6803 each have about 700-800 ORFs that are unique, i.e. not present in any of the other strains, whereas N. punctiforme has about 1500 such ORFs. About 500 ORFs are shared by N. punctiforme and Anabaena sp. PCC 7120, but not with other cyanobacteria, suggesting that these ORFs may encode proteins involved in common phenotypic characteristics such as heterocyst differentiation.

The recognized genes encode proteins involved in all the aspects of cyanobacterial metabolism that are expected. The largest proportion of genes (about 5%) are associated with signal transduction mechanisms such as protein kinases and response regulators. Genes required for cell envelope synthesis, cell division, and chromosome segregation comprise almost 4% of all the ORFs as do genes involved in amino acid transport and metabolism. Almost 3% of the ORFs encode genes involved in organic carbon metabolism, presumably reflecting the heterotrophic capability of this strain. About 10% of the ORFs are probable enzymes or structural proteins; however, they cannot be assigned to a functional category. Clearly there is much to learn about N. punctiforme from a functional analysis of this large and complex genome.

In the N. punctiforme genome many of the gene categories contain a surprisingly large number of genes (Table 2). Among the largest categories are those representing the response regulator/sensor histidine kinase groups. There are also many copies of transcriptional regulators. This suggests a high level of regulation for the many processes in N. punctiforme that are required for cellular differentiation and for establishment and maintenance of symbiosis.

Table 2. Gene categories with multiple gene copies

Gene category based on COG assignments

Number of putative copies

Response regulator receiver domain

168

Transposases

148

Sensor histidine kinase

146

Transcriptional regulators (helix-turn-helix

100

domain, AraC, ArsR, LuxR, LysR, TetR)

ATPase component of ABC Transporter

99

Tetratrico-peptide repeat protein

96

3.2. Nitrogenase genes. Nitrogen fixation is mediated by an enzyme complex, containing dinitrogenase (encoded by nifD and nifK) and dinitrogenase reductase (encoded by ni/H), whose assembly requires many nif gene products (Dean, Jacobson 1992). A large cluster of nif and nif-related genes from nifB to fdxH is highly conserved in cyanobacteria (Thiel et al. 1997, 1998) including N. punctiforme and Anabaena sp. PCC 7120 (Figure 1). A major difference between the latter two clusters is the excision elements that interrupt both fdxN and nifD in Anabaena sp. PCC Nostoc punctiforme

23-kb excision element nilB fdxN nils nifU glbN nlfH

orf nifX 2 1 nifW hesA hesB fdxH mop n II 4

Anabaena sp. PCC 7120

55-kb excision element 11-kb excision element nifB fdxN \ nifS nifU nifH nifD I nifK orf nifE nifN nilX 2 AnifWhesA hesB fäxH mop

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