Differential transcriptional analysis of the cyanobacterium Cyanothece sp. strain ATCC 51142 during light-dark and continuous-light growth

Abstract

We analyzed the metabolic rhythms and differential gene expression in the unicellular, diazotrophic cyanobacterium Cyanothece sp. strain ATCC 51142 under N(2)-fixing conditions after a shift from normal 12-h light-12-h dark cycles to continuous light. We found that the mRNA levels of approximately 10% of the genes in the genome demonstrated circadian behavior during growth in free-running (continuous light) conditions. The genes for N(2) fixation displayed a strong circadian behavior, whereas photosynthesis and respiration genes were not as tightly regulated. One of our main objectives was to determine the strategies used by these cells to perform N(2) fixation under normal day-night conditions, as well as under the greater stress caused by continuous light. We determined that N(2) fixation cycled in continuous light but with a lower N(2) fixation activity. Glycogen degradation, respiration, and photosynthesis were also lower; nonetheless, O(2) evolution was about 50% of the normal peak. We also demonstrated that nifH (encoding the nitrogenase Fe protein), nifB, and nifX were strongly induced in continuous light; this is consistent with the role of these proteins during the assembly of the enzyme complex and suggested that the decreased N(2) fixation activity was due to protein-level regulation or inhibition. Many soluble electron carriers (e.g., ferredoxins), as well as redox carriers (e.g., thioredoxin and glutathione), were strongly induced during N(2) fixation in continuous light. We suggest that these carriers are required to enhance cyclic electron transport and phosphorylation for energy production and to maintain appropriate redox levels in the presence of elevated O(2), respectively.

FIG. 1.

Plot of the physiological parameters of Cyanothece sp. strain ATCC 51142 during growth in a 6-liter bioreactor during 12-h light-12-h dark periods followed by 36 h of light and a final 12-h dark period. Grey areas represent dark periods. The parameters monitored were pH (left ordinate); nitrogenase activity (acetylene reduction); and respiration and photosynthesis, measured as O₂ uptake and O₂ evolution, respectively. Photosynthesis is represented as O₂ evolution (positive activity), whereas respiration is represented as O₂ uptake (negative activity). The relative rates of the later three parameters are shown (right ordinate), and the maximum values were nitrogenase, 196 nmol N₂ 10⁸ cells⁻¹ h⁻¹; respiration, 275 μmol O₂ mg Chl a⁻¹ h⁻¹; and photosynthesis, 250 μmol O₂ mg Chl a⁻¹ h⁻¹. rel, relative.

FIG. 2.

Diagram of differential gene expression of Cyanothece sp. strain ATCC 51142 grown in 12-h light and 12-h dark periods followed by 24 h of LL; the times are the same as in Fig. 1. Dark grey represents the dark period. Data for the key functional categories of photosynthesis, respiration, N₂ fixation, cell division, and ribosomal proteins are depicted. The bars represent the time periods when many genes within a category are up-regulated twofold or more.

FIG. 3.

Transcriptional kinetics of specific genes during diazotrophic growth in LD and LL in the cyanobacterium Cyanothece sp. strain ATCC 51142. Grey represents the dark period. (A) Class of genes whose expression increased in the subjective dark but less than in the dark: gnd and glpP1. (B) Genes that were up-regulated in the light and also in the subjective dark: ATP synthase genes. (C) Class of genes whose expression was increased more in the subjective dark than in the dark: groELS and groEL2. (D) Class of genes that were greatly induced in the dark and much less so in the subjective dark: hupLS, psbA4, and a gene encoding a CheY-like response regulator (cce_1982) that was near the dark-inducible cox operon.

FIG. 4.

Transcriptional kinetics of some key nitrogenase genes during diazotrophic growth in LD and LL in the cyanobacterium Cyanothece sp. strain ATCC 51142. Grey represents the dark period. Results for the genes encoding NifH and NifDK, along with some of the genes important in the assembly of the FeMo cofactor, are displayed. The levels of NifH were fourfold higher than the levels on the left ordinate, but the curve was compressed to better highlight the transcriptional kinetics of the other genes.

FIG. 5.

Determination of glycogen content as measured by FTIR spectroscopy. The difference spectrum between cells from D10 (containing no granules) and L10 (filled with granules) is compared to a pure glycogen reference. The qualitative comparison between these curves indicated that the measurements of the integrated area at wave numbers between 1,200 and 1,000 cm⁻¹ were a good monitor of intracellular glycogen. The inset demonstrates these measurements every 4 h through an LD cycle followed by a 36-h period of LL. Grey represents the dark period. Error bars show standard deviations. gly, glycogen.

FIG. 6.

Validation of the microarray results by comparison with the results from RT-PCR experiments. The genes chosen are examples of different categories of differential expression and include nifD (♦), highly expressed in the dark; coxC1 (▴), highly expressed at the end of the light period and into the dark period; psbA (▪) and psbD (✳), highly expressed in the light; and rnpA (×), little change in expression throughout the experiment. Grey represents the dark period.