TEXT

Top Abstract Text References

Nitrogen-fixing cyanobacteria are phototrophic microorganisms that use the energy of sunlight to reduce CO₂ and N₂ from the air, utilizing water as the reductant. Oxygen derived from photooxidation of water is highly damaging to nitrogenase, the enzyme responsible for the reduction of molecular nitrogen. Some genera (e.g., Anabaena) of multicellular cyanobacteria respond to deprivation of combined nitrogen by differentiation of about every 10th cell of a filament into a heterocyst, a cell specialized for the task of N₂ fixation (25). Developing heterocysts lose the capacity to fix CO₂, enhance respiration, and form a special envelope that limits the entrance of O₂. The inner laminated layer of this envelope is composed of heterocyst-specific glycolipids that are derivatives of hexoses containing long-chain polyhydroxyl alcohols (3). The outer homogeneous layer is built of specific polysaccharides (6). Adjacent vegetative cells supply heterocysts with photosynthates that are then oxidized to provide reductants required for N₂ fixation and respiration. In turn, heterocysts provide vegetative cells with fixed nitrogen.

The devBCA operon, encoding the subunits of an ATP-binding cassette (ABC) transporter, is essential for formation of the laminated layer of heterocysts in Anabaena sp. strain PCC 7120 and Anabaena variabilis ATCC 29413-FD (7-9, 15). Based on mutational and biochemical analysis, the DevBCA transporter was proposed to be an exporter of heterocyst-specific glycolipids or of an enzyme that is required for assembly of the laminated layer (7).

Heterocyst development requires the product of the regulatory gene hetR (2, 4) and is also dependent on NtcA (12, 24). NtcA, a transcriptional regulator exerting global nitrogen control in cyanobacteria, activates the expression of genes involved in nitrogen assimilation in response to ammonium withdrawal (10, 11, 14, 20, 21, 23). NtcA interacts in vitro with DNA bearing the consensus sequence of cyanobacterial NtcA-regulated promoters: GTAN₈TACN₂₂TAN₃T (10, 14). Expression of hetC (17), encoding an ABC transporter that acts early in heterocyst differentiation, as well as of petH (19), encoding ferredoxin NADP⁺ reductase, has been shown to be regulated by NtcA. Although expression of neither hetC nor petH requires HetR, expression of several other genes in heterocyst development or function is HetR dependent (5). Because hetR expression is itself NtcA dependent (12), it is possible that the requirement for NtcA for the expression of some genes related to heterocyst differentiation or function is indirect, via HetR. In this work we have investigated the expression of the devBCA operon of Anabaena sp. strain PCC 7120 and A. variabilis strain ATCC 29413-FD. Although expression of devBCA in strain PCC 7120 is dependent on HetR (5), the results presented in this work suggest a direct role of NtcA as an activator of this operon.

Methods. Growth conditions and media for cyanobacterial strains, procedures for induction experiments, and DNA and RNA isolation were as previously described (17). Methods of molecular biology were standard (1, 18). Northern blot analysis was performed with samples of 70 µg of RNA; as a probe, a DNA fragment generated by PCR with oligonucleotides O34 (5'-ATG TCA AGG GTG ACG GAA G-3', corresponding to positions +1 to +19 relative to the translational start site of devB) and O18 (5'-ATT TAT TAA TGT CAA CCA CTA CC-3', complementary to positions +1423 to +1400 relative to the translational start site of devB), and plasmid pIM11 (7) as a template. Primer extension analysis was carried out as previously described (17) with oligonucleotides O100 (5'-TTG AAG AGG TTC TAT CAA AAG TT-3', complementary to positions 575 to 597 relative to the translational start site of devB of A. variabilis), OdevB7120 (5'-GAA GAG GTT CTA TCA AAG G-3', complementary to positions 519 to 537 relative to the translational start site of devB of strain PCC 7120), and O69 (5'-ATA ACA TAA CAT TTC CCC AAG TCT-3', complementary to positions 576 to 599 relative to the translational start site of devB from strain 7120). Band shift assays were performed with DNA fragments generated by PCR using pIM35 (8) as the template and oligonucleotides O113 (5'-TTA CCC GCT AGC GAC TGG-3', corresponding to positions 830 to 813 relative to the translational start site of devB of A. variabilis) and O100 (see above) or with pIM23 (7) as the template and oligonucleotides O113 (see above; corresponding also to positions 795 to 778 relative to the translational start site of devB of strain PCC 7120) and O69 (see above). The purified PCR fragments were used for nonradioactive (16) and radioactive (14, 17) binding assays.

Expression experiments with the devBCA operon. To determine the time course of activation of the devBCA gene cluster and to test the involvement of the transcription factor NtcA in the control of the expression of the gene cluster, Northern blot analysis was done with a devB probe. Results obtained with RNA isolated from wild-type A. variabilis strain ATCC 29413-FD and Anabaena sp. strain PCC 7120 cells deprived of combined nitrogen were compared to those obtained with RNAs isolated from cells of the ntcA mutant strain CSE2 (12) and the hetR mutant strain 216 (4). Hybridization could be observed with RNA isolated from A. variabilis cells, which were grown on ammonium and then deprived of combined nitrogen for 6 and 10 h (data not shown). Hybridization was also detectable with RNA from wild-type Anabaena sp. strain PCC 7120 cells grown on ammonium and then incubated in combined-nitrogen-free medium for 6, 7.5, 9, and 24 h but was not observed with RNAs from the mutants or in RNA from wild-type cells grown on ammonium or deprived of combined nitrogen for less than 6 h (Fig. 1). The observed signals correspond mainly to degradation products of the devBCA transcripts, which should be at least 3.4 kb in length. Repeated attempts to isolate intact transcripts from the devBCA operon were unsuccessful. Nevertheless, the sizes of the degraded devBCA transcripts (well above 2.9 kb) indicate polycistronic transcription of the dev genes and, together with the sequence data (7, 15), imply an operon structure. The absence of devBCA transcripts in the ntcA mutant CSE2 indicates that NtcA protein is necessary for activation of expression of the devBCA operon. Dependence on HetR confirms previously reported data (5).

View larger version (58K): [in this window] [in a new window]   FIG. 1.   Northern blot analysis of expression of the devBCA operon in Anabaena sp. strain PCC 7120 and the ntcA mutant strain CSE2 (A) and in Anabaena sp. strain PCC 7120 and the hetR mutant strain 216 (B). RNA was isolated from ammonium-grown cells (lanes 0) or from ammonium-grown cells incubated in combined-nitrogen-free medium for 1, 4.5, 7.5, 9, or 24 h (A) and 3, 6, 9, or 24 h (B) in two independent experiments. Hybridization to a devB probe (upper panel in each case) or to an rnpB probe (22) as an internal control (lower panel in each case) was performed. WT, wild-type strain PCC 7120. Molecular size markers (in kilobases) are noted at the left.

Initiation of transcription of devBCA. To study the transcriptional regulation of the devBCA operon, primer extension analysis was done with RNAs isolated from cells of both Anabaena strains grown under different conditions of nitrogen supply. A nitrogen-dependent transcriptional start point (tsp) could be identified at position 762 in strain ATCC 29413-FD (Fig. 2A) using oligonucleotide O100. The signal of the extension product was clearly detectable in RNA isolated from cells grown on N₂ or incubated in combined-nitrogen-free medium for 6 and 10 h but not in RNA from ammonium-grown cultures of A. variabilis.

View larger version (100K): [in this window] [in a new window]   FIG. 2.   Primer extension analysis of the expression of the devBCA operon was performed with RNAs isolated from A. variabilis strain ATCC 29413-FD cells grown on N2 or ammonium (lane 0) or grown on ammonium and deprived of combined nitrogen for 6 and 10 h (A); Anabaena sp. strain PCC 7120 cells grown on ammonium (lane 0) or grown on ammonium and incubated in combined-nitrogen-free medium for 0.5, 1, 2, 6, 8, and 10 h (B); and wild-type Anabaena sp. strain PCC 7120 and ntcA mutant strain CSE2 cells grown on ammonium (lane 0) or grown on ammonium and deprived of combined nitrogen for 24 h (C). Oligonucleotides used as primers were O100 (A) and OdevB7120 (B and C). Sequencing ladders were generated with the corresponding oligonucleotides and plasmids pIM23 (A) and pIM35 (B and C). The putative tsp is indicated by an arrowhead. (D) Comparison of devB promoter sequences with the consensus sequence of NtcA-activated promoters.

Binding of NtcA to the promoter region of the devBCA operon. Sequences upstream from the devBCA tsps show the consensus sequence of cyanobacterial NtcA-regulated promoters (10, 14) in both Anabaena strains (Fig. 2). Because of the observed NtcA dependence of devBCA transcription, the levels of binding of overexpressed NtcA to DNA fragments carrying these promoters from Anabaena sp. strain PCC 7120 and A. variabilis strain ATCC 29413-FD were determined. As a source of NtcA, an extract of an Escherichia coli strain containing plasmid pCSAM70, overexpressing the ntcA gene from Anabaena sp. strain PCC 7120 (17), was used. NtcA-dependent band shifts could be clearly observed in nonradioactive assays with the devBCA promoters of A. variabilis (data not shown) and Anabaena sp. strain PCC 7120 (Fig. 3A). The control extract from E. coli BL21(DE3)(pREP4, pQE9) (17), instead of the NtcA-containing extract, did not result in a band shift of the promoter-containing fragments. In addition to the nonradioactive method, a highly sensitive radioactive assay was carried out with the PCR fragment containing the devBCA promoter of strain PCC 7120 (Fig. 3B). A shift of the ³²P-labeled promoter-containing fragment could be detected only when the DNA was incubated with E. coli extracts expressing the NtcA protein. Retardation of the labeled fragment was competed to a certain extent by the same unlabeled DNA fragment. These results indicate that NtcA binds to the N-regulated promoter of the devBCA operon.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   Band shift assays of DNA fragments from the devBCA promoter of Anabaena sp. strain PCC 7120 with Anabaena NtcA protein. Assays were carried out with a 220-bp PCR fragment containing the devBCA promoter (C) and no extract (lanes 1) or extracts from E. coli BL21(DE3)(pREP4) containing either pQE9 (lanes 2) or the NtcA expression plasmid pCSAM70 (lanes 3 and 4). A 25-fold molar excess of an unlabeled dev promoter fragment was included in the assay for the results shown in lane 4. (A) Nonradioactive assay; (B) radioactive assay with labeled DNA. Arrowheads point to retarded fragments.

Conclusions. In this work, we demonstrated that a promoter for the devBCA operon shows the structure of the cyanobacterial NtcA-activated promoters: an NtcA-binding site in the form GTAN₈TAC followed, at a distance of 22 bp, by a 10 Pribnow box in the form TAN₃T, which is located 5 bp upstream from the tsp. This tsp is located far upstream from devB, at 762 (strain ATCC 29413-FD) or 704 (strain PCC 7120) bp. Because the transcripts detected with a devB probe were also NtcA dependent and no sequences matching those of the NtcA-activated promoters are found between the detected promoter and the translation start site of devB, the presence of additional promoters upstream of devB is unlikely. Examination of the genomic sequence of Nostoc punctiforme (DOE Joint Genome Institute [http://www.jgi.doe.gov/]) indicates that a putative NtcA-binding site is present in approximately the same location upstream from the dev operon as in Anabaena spp. NtcA-activated promoters, which are also located far from the corresponding genes in Anabaena sp. strain PCC 7120, include those of the nir operon (tsp located at 460 [13]) and the hetC gene (tsp located at 571 [17]). The function, if any, of the long, presumably untranslated mRNA fragment is unknown.