The amt gene cluster of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120

Abstract

The genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 bears a gene cluster including three amt genes that, based on homology of their protein products, we designate amt4, amt1, and amtB. Expression of the three genes took place upon ammonium withdrawal in combined nitrogen-free medium and was NtcA dependent. The genes were transcribed independently, but an amt4-amt1 dicistronic transcript was also produced, and expression was highest for the amt1 gene. A mutant with the whole amt region removed could grow under laboratory conditions using ammonium, nitrate, or dinitrogen as the nitrogen source.

FIG. 1.

Phylogenetic relationships of some Amt proteins (named MEP in Saccharomyces cerevisiae and NrgA in Bacillus subtilis). The analysis was restricted to some proteins that have been experimentally characterized, and a distantly related human (Homo sapiens) Rh protein was used as an outgroup. The Amt proteins were from the following species: Arabidopsis thaliana, Lotus japonicus, Corynebacterium glutamicum, Synechococcus elongatus, Anabaena sp. strain PCC 7120, Bacillus subtilis, Rhodopseudomonas palustris, Azotobacter vinelandii, Escherichia coli, Archaeoglobus fulgidus, Saccharomyces cerevisiae, Hebeloma cylindrosporum, and Synechocystis sp. strain PCC 6803. Bootstrap values based on 1,000 trials are indicated. The arrows point to the Anabaena sp. strain PCC 7120 (A. PCC 7120) Amt proteins. The bar represents 0.1 substitution per amino acid position. S. PCC 6803, Synechocystis sp. strain PCC 6803.

FIG. 2.

Expression of the amt genes in Anabaena sp. strains PCC 7120 and CSE2 (ntcA::C.S3). (A) Scheme of the Anabaena amt genomic region (15). Arrowheads represent primers used in RT-PCR analysis (RT1, RT2, RT3, and RT4) and in Northern analysis to generate the amt4 probe (primers 41 and 42), the amt1 probe (primers 11 and 12), and the amtB probe (B1 and B2). The arrows at the bottom of the panel represent transcripts. (B) Northern analysis was performed with RNA isolated from cells of strain PCC 7120 (wild type [WT]) or CSE2 grown with ammonium (lanes A) or grown with ammonium and incubated for the indicated number of hours with no combined nitrogen. The filter was successively hybridized with the amt4 probe, the amt1 probe, an rnpB probe (that was used as a loading and transfer control), and the amtB probe. (C) RT-PCR analysis of cotranscription in the amt region. RNA isolated from cells of Anabaena sp. strain PCC 7120 grown with ammonium and incubated for 2 h without any source of combined nitrogen was used for retrotranscription with primer RT2 or RT4, and the products of these reactions were subjected to PCR with oligonucleotide RT1 (forward primer) and the same primer used for retrotranscription as reverse primer. The products of RT4 retrotranscription were also subjected to PCR with oligonucleotide RT3 (forward primer) and RT4. Amplification (lanes R) was observed only for the reaction with RT1 and RT2, which gave rise to a DNA fragment of the expected size, 1.1 kb. Control assays in which the RNA preparation was treated with RNase I (lanes R-) or in which the PCR was carried out with the same primers and strain PCC 7120 DNA (lanes D) are also presented. Lanes λ, phage lambda DNA digested with HindIII.

FIG. 3.

Deletion of amt genes and uptake of [¹⁴C]methylammonium by Anabaena sp. strain PCC 7120 and mutant CSP19 (Δamt::C.K3). (A) Scheme showing the Anabaena amt region replaced by the C.K3 cassette in the generation of mutant strain CSP19 (see the text for details). The arrowheads labeled 1, 2, 7, and 8 indicate the approximate locations of deoxyoligonucleotide primers amts-7120-1, amts-7120-2, amts-7120-7, and amts-7120-8, respectively. (B) Ammonium-grown cells incubated for 90 min in BG11₀C medium with a high supply of CO₂ were used in uptake assays in phosphate-bicarbonate buffer with 1 μM [¹⁴C]methylammonium ([¹⁴C]MA). Uptake in wild-type strain PCC 7120 (circles) and mutant strain CSP19 (squares) is depicted.

FIG. 4.

Growth of Anabaena sp. strain PCC 7120 and mutant CSP19 with different nitrogen sources. (A) Cells of strain PCC 7120 (open symbols) and strain CSP19 (closed symbols) were incubated in liquid medium with ammonium (circles), nitrate (triangles), or dinitrogen (squares) as nitrogen source (see “Methods”). X₀, protein concentration at time zero; X, protein concentration at sample time. (B) PCR analysis carried out on DNA from strain PCC 7120 (wild type [WT]) or CSP19 grown with dinitrogen as a nitrogen source. The locations of primers used for PCR (primer pairs 41/42, 11/12, and B1/B2) are shown in Fig. 2A. Positive controls for PCR amplification with DNA from strain CSP19 were carried out with primers of two unrelated genes, all4294 and alr2394 (not shown). S, phage lambda DNA digested with HindIII used as a size standard.