Global Gene Expression Patterns ofNostoc punctiformein Steady-State Dinitrogen-Grown Heterocyst-Containing Cultures and at Single Time Points during the Differentiation of Akinetes and Hormogonia

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 NH 4 ؉ -grown transcriptome was enriched in genes encoding core metabolic functions. A steady-state N 2 -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 N 2 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 N 2 -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, N 2 grown), akinetes, and hormogonia appears to involve the up-regulation of genes distinct for each state.

Biased inheritance of the protein PatN frees vegetative cells to initiate patterned heterocyst differentiation

“…Contrasts were computed for the patN-deletion strain using the wild-type strain as a reference at each time point (i.e., the ΔpatN strain: t = 3 h; wild type: t = 3 h) (ΔpatN vs. wild-type contrast), as well as for each time point using t = 0 of the same strain as a reference (i.e., the ΔpatN strain t = 3 h vs. the ΔpatN strain t = 0 h) (ΔpatN or wild-type time-course contrast). Differential expression of genes with B values ≥0 was considered statistically significant, as before (21). Heat maps were generated using Genesis (25), with the M values from top tables for each time point.…”

“…For DNA microarray studies, two 50-mL cultures of strains UCD 153 or UCD 524 at 2-3 μg of Chl a/mL were harvested at t = 0 (supplemented with 2.5 mM NH 4 Cl) and 1, 3, 6, 12, 18, 24, and 120 h after N step-down (n = 3). RNA extraction was performed as described previously (21). Ten micrograms of total RNA were used for cDNA synthesis using SuperScript Double Stranded cDNA Synthesis Kit (Invitrogen).…”

“…S4. N. punctiforme strain UCD 153 and its derivatives were cultured in Allan and Arnon medium diluted fourfold (AA/4), as described previously (21), with the exception that cultures were supplemented with 30 mM fructose to suppress hormogonium differentiation (22). For nitrogen step-down, cultures supplemented with 2.5 mM NH 4 Cl at a chlorophyll (Chl) a concentration of 2-3 μg/mL were harvested at 1,000 × g for 5 min, washed three times with AA/4, and suspended in AA/4 (21).…”

Global transcriptomic analysis ofCyanothece51142 reveals robust diurnal oscillation of central metabolic processes

“…These oxygenic diazotrophs are challenged with maintaining obligate anaerobic conditions because the nitrogenase enzyme is highly sensitive to oxygen. In filamentous cyanobacteria such as Anabaena and Nostoc, nitrogen fixation is spatially separated and occurs in specialized heterocysts (11,12), cells whose differentiation involves the specific expression of numerous genes (13). In contrast, unicellular diazotrophic cyanobacteria such as Cyanothece sp.…”

Genomics of variation in nitrogen fixation activity in a population of the thermophilic cyanobacterium Mastigocladus laminosus

“…N fixation has a complex genetic basis in heterocystous cyanobacteria (Kumar et al, 2010), requiring genes that regulate and specify the morphological and physiological changes that occur during the differentiation process, in addition to N fixation (nif) genes that are common to all diazotrophs. Estimates of the number of genes that are differentially regulated during heterocyst development range from~500 in Nostoc punctiforme (Campbell et al, 2007) to over 1000 in Anabaena PCC 7120 (Ehira et al, 2003). Among these are 100-140 'Fox' genes that are required for N fixation in the presence of oxygen (Wolk, 2000), including those involved in the remodeling of the cell surface to form an envelope of glycolipid and polysaccharide layers that provides a passive gas diffusion barrier to limit the entry of oxygen (Nicolaisen et al, 2009).…”