Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in Anabaena sp. PCC7120

Abstract

The fixation of atmospheric N(2) by cyanobacteria is a major source of nitrogen in the biosphere. In Nostocales, such as Anabaena, this process is spatially separated from oxygenic photosynthesis and occurs in heterocysts. Upon nitrogen step-down, these specialized cells differentiate from vegetative cells in a process controlled by two major regulators: NtcA and HetR. However, the regulon controlled by these two factors is only partially defined, and several aspects of the differentiation process have remained enigmatic. Using differential RNA-seq, we experimentally define a genome-wide map of >10,000 transcriptional start sites (TSS) of Anabaena sp. PCC7120, a model organism for the study of prokaryotic cell differentiation and N(2) fixation. By analyzing the adaptation to nitrogen stress, our global TSS map provides insight into the dynamic changes that modify the transcriptional organization at a critical step of the differentiation process. We identify >900 TSS with minimum fold change in response to nitrogen deficiency of eight. From these TSS, at least 209 were under control of HetR, whereas at least 158 other TSS were potentially directly controlled by NtcA. Our analysis of the promoters activated during the switch to N(2) fixation adds hundreds of protein-coding genes and noncoding transcripts to the list of potentially involved factors. These data experimentally define the NtcA regulon and the DIF(+) motif, a palindrome at or close to position -35 that seems essential for heterocyst-specific expression of certain genes.

Fig. 1.

Genome-wide identification of TSS in Anabaena 7120. (A) Differential RNA-seq identifies single TSS in complex promoter regions as exemplified by the hetR gene. The total number of reads mapped to each 5′ end is indicated for each of the four previously described TSS. (B) Distribution of 3401 TSS with ≥300 reads each along a linear plot of the Anabaena 7120 chromosome. TSS mapped for the forward strand are plotted above the x axis, and for the reverse strand below. The number of sequence reads is given on the y axis. The location of each TSS according to SI Appendix, Fig. S1A served for classification as gTSS (blue), nTSS (green), aTSS (red), or iTSS (gray). Selected TSS for each of the four classes are annotated.

Fig. 2.

Analysis of genes with multiple TSS identified by dRNA-seq. RNA was isolated from ammonium-grown cells (lanes labeled 0) or from ammonium-grown cells incubated in the absence of combined nitrogen for the number of hours indicated. (A) Graphical representation of reads mapped to the promoter of nblA. The histograms correspond to the WT-0 (red), WT-8 (green), hetR-0 (black), and hetR-8 (blue) samples. (B) Primer extension analysis of the nblA mRNA in WT and mutant strains CSE2 (ntcA) and 216 (hetR). Samples contained 20 μg of RNA. The oligonucleotide used was complementary to positions +5 to −18 with respect to the translational start of nblA. The 5′ ends identified by dRNA-seq are numbered 1–5 (for positions, see Dataset 1, Table S1). (C) Graphical representation of reads mapped to the promoter of alr3808. (D) Northern blot analysis of the alr3808 mRNA in Anabaena 7120 and mutant strain 216 (hetR). Samples contained 10 μg of RNA. The probe used was an internal fragment of alr3808. rnpB (38) was used as a loading control.

Fig. 3.

Occurrence of a palindrome, 5′ TCCGGA, in promoters of the DIF⁺ category. (A) Alignment of the heterocyst-specific promoters for NsiR1, the hetR TSS3, sigC, and hetC with selected promoters in the DIF⁺ category (fold change ≥ 8) containing TCCGGA around position −35 (one mismatch allowed). (B) Cell-specific transcription from the wild-type promoter of NsiR1 (P6; Upper) or a mutated version of P6 carrying GAATTC instead of TCCGGA (Lower). Images corresponding to red autofluorescence (Right) and GFP fluorescence (Left) are shown. White triangles point to proheterocysts. (Scale bars: 10 μm.)

Fig. 4.

NtcA-activated and -repressed promoters. (A) Promoter regions of 20 TSS (underlined nucleotide) in the DEF⁺ category with the highest fold change. Possible −10 elements are highlighted in blue, nucleotides matching the consensus for NtcA binding sites in red. (B) Nucleotide frequencies derived for positions 1–14 of 87 putative NtcA binding sites. (C) Corresponding Weblogo. (D) Position of NtcA binding sites identified in a sliding window approach using the PSSM along the promoters in the DEF⁺ category (fold change ≥8). The bars indicate the first nucleotide of a putative NtcA binding site and its position with regard to the TSS (E) Putative NtcA binding sites identified at repression-compatible positions around TSS in the DEF⁻ category.