Cell-specific gene expression in Anabaena variabilis grown phototrophically, mixotrophically, and heterotrophically

Abstract

Background: When the filamentous cyanobacterium Anabaena variabilis grows aerobically without combined nitrogen, some vegetative cells differentiate into N2-fixing heterocysts, while the other vegetative cells perform photosynthesis. Microarrays of sequences within protein-encoding genes were probed with RNA purified from extracts of vegetative cells, from isolated heterocysts, and from whole filaments to investigate transcript levels, and carbon and energy metabolism, in vegetative cells and heterocysts in phototrophic, mixotrophic, and heterotrophic cultures.

Results: Heterocysts represent only 5% to 10% of cells in the filaments. Accordingly, levels of specific transcripts in vegetative cells were with few exceptions very close to those in whole filaments and, also with few exceptions (e.g., nif1 transcripts), levels of specific transcripts in heterocysts had little effect on the overall level of those transcripts in filaments. In phototrophic, mixotrophic, and heterotrophic growth conditions, respectively, 845, 649, and 846 genes showed more than 2-fold difference (p < 0.01) in transcript levels between vegetative cells and heterocysts. Principal component analysis showed that the culture conditions tested affected transcript patterns strongly in vegetative cells but much less in heterocysts. Transcript levels of the genes involved in phycobilisome assembly, photosynthesis, and CO2 assimilation were high in vegetative cells in phototrophic conditions, and decreased when fructose was provided. Our results suggest that Gln, Glu, Ser, Gly, Cys, Thr, and Pro can be actively produced in heterocysts. Whether other protein amino acids are synthesized in heterocysts is unclear. Two possible components of a sucrose transporter were identified that were upregulated in heterocysts in two growth conditions. We consider it likely that genes with unknown function represent a larger fraction of total transcripts in heterocysts than in vegetative cells across growth conditions.

Conclusions: This study provides the first comparison of transcript levels in heterocysts and vegetative cells from heterocyst-bearing filaments of Anabaena. Although the data presented do not give a complete picture of metabolism in either type of cell, they provide a metabolic scaffold on which to build future analyses of cell-specific processes and of the interactions of the two types of cells.

Figure 1

Verification of cell specificity of RNA extractions by RT-qPCR. rbcL and nifK were used as the probes for genes expressed specifically (see text) in vegetative cells (rbcL, top panel) and in heterocysts (nifK, bottom panel). The internal standard was rnpB, which is expressed constitutively in all cells [40]. Culture conditions are shown in white (phototrophic), gray (mixotrophic), and black (heterotrophic). V: RNA extracted from vegetative cells; Ht: RNA extracted from heterocysts. Means and standard deviations are based on three biological replicates. Transcript levels are normalized to 1 in heterocysts (rbcL) and in vegetative cells (nifK).

Figure 2

Histograms of the log ₂ values of the normalized average signal intensities for all microarray experiments. (P): Phototrophic cultures; (M): mixotrophic cultures; and (H): heterotrophic cultures. Black: signal distribution in whole filaments; gray: signal distribution in vegetative cells; and white: signal distribution in heterocysts. Left panels: all genes; right panels: reduced sets of genes (see Additional files 5 and 6; P: 3,949 genes; M: 3,885 genes, and H: 3,933 genes).

Figure 3

Principal component analysis of gene expression patterns in different cell types and different growth conditions. Component 1 is plotted versus component 2. PCA was performed using the entire normalized data set of 5,657 genes. F: whole filaments; H: heterotrophic conditions; Ht: heterocysts; M: mixotrophic conditions; P: phototrophic conditions; and V: vegetative cells.

Figure 4

Distribution of gene transcript levels in functional categories. Transcript levels of the genes participating in different pathways are represented as percent of total genome transcripts in each experiment. The number of genes in each functional category is given in parentheses. The N₂-fixation genes are represented by a wedge with an enlarged radius.

Figure 5

Transcripts of amino acid biosynthetic genes. White, yellow, orange, red, and crimson represent transcript levels in the ranges ≤ 200, 201–600, 601–2000, 2001–6000, and ≥ 6001 SIU, respectively. Transcript levels of all genes involved in amino acid biosynthesis are shown in Additional file 7. Legends for superscripts “a” to “s” are in Additional file 8.

Figure 6

Transcripts of genes encoding PS I, PS II, and phycobiliproteins. Colors are as in Figure  5.

Figure 7

Transcripts of CO ₂ -fixation genes. Colors are as in Figure  5. Legends for superscripts “a” to “d” are in Additional file 8.