Alanine dehydrogenase activity is required for adequate progression of phycobilisome degradation during nitrogen starvation in Synechococcus elongatus PCC 7942

Abstract

Degradation of the cyanobacterial light-harvesting antenna, the phycobilisome, is a general acclimation response that is observed under various stress conditions. In this study we identified a novel mutant of Synechococcus elongatus PCC 7942 that exhibits impaired phycobilisome degradation specifically during nitrogen starvation, unlike previously described mutants, which exhibit aberrant degradation under nitrogen, sulfur, and phosphorus starvation conditions. The phenotype of the new mutant, AldOmega, results from inactivation of ald (encoding alanine dehydrogenase). AldOmega is deficient in transcription induction of a number of genes during nitrogen starvation. These genes include the "general nutrient stress-related" genes, nblA and nblC, the products of which are essential for phycobilisome degradation. Furthermore, transcripts of several specific nitrogen-responsive genes accumulate at lower levels in AldOmega than in the wild-type strain. In contrast, ald inactivation did not decrease the accumulation of transcripts during sulfur starvation. Transcription of ald is induced upon nitrogen starvation, which is consistent with the ability of wild-type cells to maintain a low cellular content of alanine under these conditions. Unlike wild-type cells, AldOmega accumulates alanine upon nitrogen starvation. Our analyses suggest that alanine dehydrogenase activity is necessary for an adequate cellular response to nitrogen starvation. Decomposition of alanine may be required to provide a sufficient amount of ammonia. Furthermore, the accumulated alanine, or a related metabolite, may interfere with the cues that modulate acclimation during nitrogen starvation. Taken together, our results provide novel information regarding cellular responses to nitrogen starvation and suggest that mechanisms related to nitrogen-specific responses are involved in modulation of a general acclimation process.

FIG. 1.

(A) Physical map of the genomic region bearing ald in S. elongatus PCC 7942 (encoding alanine dehydrogenase). PstI, restriction sites used for cloning the relevant genomic region. The SmaI site was used for insertional inactivation of ald by a spectinomycin (spc) cassette. The arrow indicates the transposon insertion site in the original mutant, M7. The numbers indicate open reading frames neighboring ald, as follows: 1, predicted open reading frame (containing Fe-S cluster); 2, high-light-inducible protein-related open reading frame; 3, hypothetical protein open reading frame. (B) PCR analysis confirming complete chromosome segregation in the alanine dehydrogenase mutant AldΩ. Lane WT, S. elongatus PCC 7942; lane M, molecular weight markers. See Materials and Methods for details.

FIG. 2.

Phenotype of AldΩ impaired in alanine dehydrogenase. (A) Cultures of S. elongatus PCC 7942 (WT), alanine dehydrogenase mutants AldΩ and M7, and NblR mutant NblRΩ grown in complete medium (+) or under nitrogen starvation conditions (−N) or sulfur starvation conditions (−S) for 24 and 72 h. (B) Absorbance spectra of the wild type (red) and AldΩ (green) grown in complete medium or starved for 24 h. The absorbance maxima of phycocyanin (PC), the major pigment of the phycobilisome, and chlorophyll a (Chl) are indicated. Absorbance spectra were obtained for cultures diluted with growth medium to obtain an optical density at 750 nm of 0.01. Groups of spectra were shifted along the y axis for clarity. (C) Amount of phycocyanin as a function of time in nitrogen-starved cells (triangles) or sulfur-starved cells (squares). The different colors represent different strains as described above. Wild-type and AldΩ cultures normalized to the same optical density at 750 nm contained the same number of cells; hence, the amount of phycocyanin reflects the pigment level per cell.

FIG. 3.

Expression of genes affecting the phycocyanin level in S. elongatus PCC 7942 (WT) and alanine dehydrogenase mutant AldΩ: Northern analysis of nblA (A), nblC (B) and cpcBA (C). Cultures were grown in complete medium (+) or were starved for nitrogen (−N) or sulfur (−S) for the times indicated above the lanes (in hours). The bar graphs are quantitative representations of the hybridization results.

FIG. 4.

Expression of ald in S. elongatus PCC 7942. For details see the legend to Fig. 3.

FIG. 5.

Alanine levels in S. elongatus PCC 7942 (WT) and alanine dehydrogenase mutant AldΩ. Cultures were grown in nutrient-sufficient medium (+) or under nitrogen starvation conditions (−N) or sulfur starvation conditions (−S) for 24 h. The data are averages and standard deviations of three samples. Differences between wild-type and mutant cells were statistically significant under nitrogen and sulfur starvation conditions (P = 0.0004 and P = 0.0002, respectively). No significant difference (P = 0.02) was observed in replete medium (as determined by a t test, α = 0.01).

FIG. 6.

Expression of nitrogen starvation-responsive genes in S. elongatus PCC 7942 (WT) and alanine dehydrogenase mutant AldΩ. For details see the legend to Fig. 3.

FIG. 7.

Expression of rhdA, a sulfur starvation-responsive gene, in S. elongatus PCC 7942 (WT) and alanine dehydrogenase mutant AldΩ. For details see the legend to Fig. 3.