Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst-forming cyanobacterium Anabaena

Abstract

Arginine decarboxylase produces agmatine, and arginase and agmatinase are ureohydrolases that catalyze the production of ornithine and putrescine from arginine and agmatine, respectively, releasing urea. In the genome of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ORF alr2310 putatively encodes an ureohydrolase. Cells of Anabaena supplemented with [(14) C]arginine took up and catabolized this amino acid generating a set of labeled amino acids that included ornithine, proline, and glutamate. In an alr2310 deletion mutant, an agmatine spot appeared and labeled glutamate increased with respect to the wild type, suggesting that Alr2310 is an agmatinase rather than an arginase. As determined in cell-free extracts, agmatinase activity could be detected in the wild type but not in the mutant. Thus, alr2310 is the Anabaena speB gene encoding agmatinase. The ∆alr2310 mutant accumulated large amounts of cyanophycin granule polypeptide, lacked nitrogenase activity, and did not grow diazotrophically. Growth tests in solid media showed that agmatine is inhibitory for Anabaena, especially under diazotrophic conditions, suggesting that growth of the mutant is inhibited by non-metabolized agmatine. Measurements of incorporation of radioactivity from [(14) C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the ∆alr2310 mutant. Analysis of an Anabaena strain producing an Alr2310-GFP (green fluorescent protein) fusion showed expression in vegetative cells but much less in heterocysts, implying compartmentalization of the arginine decarboxylation pathway in the diazotrophic filaments of this heterocyst-forming cyanobacterium.

Keywords: Agmatinase; Anabaena; arginine catabolism; nitrogenase.

Figure 1

Schematic representation of the arginine decarboxylase, arginase and arginine deiminase pathways. Enzymes and the possible corresponding ORFs in the genome of Anabaena sp. strain PCC 7120 (Kaneko et al. 2001) are as follows: 1, arginine decarboxylase (all3401); 2, agmatinase (alr2310); 3, arginase; 4, ornithine transaminase (alr1080); 5, Δ¹pyrroline-5-carboxylate reductase (alr0488); 6, proline oxidase (alr0540); 7, ornithine carbamoyltransferase (alr4907); 8, arginine deiminase; 9, argininosuccinate synthetase (alr4798); 10, argininosuccinate lyase (alr3887). Note that no gene is annotated as encoding arginase or arginine deiminase. 2-OG, 2-oxoglutarate; [H], reducing power; ≈P, energy-requiring reaction (energy provided by the hydrolysis of ATP).

Figure 2

Characterization of an Anabaena alr2310 mutant. (A) Schematic of the alr2310 genomic region in Anabaena with indication of the DNA fragment removed to create strain CSMI11. (B) Growth tests in solid medium using ammonium (BG11₀ NH₄⁺), nitrate (BG11) or N₂ (BG11₀) as the nitrogen source. Each spot was inoculated with an amount of cells containing the indicated amount of Chl, and the plates were incubated under culture conditions for 7 days and photographed. Strain CSMI11-C is strain CSMI11 complemented with alr2310. (C) Filaments of Anabaena wild type (PCC 7120) and strain CSMI11 from cultures incubated for 5 days in BG11 medium and visualized by light microscopy. Scale bars, 2 μm.

Figure 3

Production of ¹⁴C-labeled metabolites from l-[¹⁴C]arginine in filaments of Anabaena wild type (PCC 7120) and strain CSMI11 grown in BG11₀ NH₄⁺ medium and incubated in BG11 (nitrate-containing) medium for 24 h. Suspensions of filaments containing 5–10 μg of Chl mL⁻¹ were incubated for 10 and 30 min with 1 μmol/L l-[¹⁴C-(U)]arginine. Metabolites in the cell suspensions were extracted and analyzed by TLC and autoradiography as described in Materials and Methods. The amino acids identified were as follows: arginine (Arg), citrulline (Cit), proline (Pro), glutamate (Glu), glutamine (Gln), ornithine (Orn), aspartate (Asp), and agmatine. Two unidentified spots, indicated as #6 and #7, were also detected. Note that the glutamine and citrulline spots overlap. The black triangles point to the origin of the chromatography.

Figure 4

Growth test of Anabaena wild type (PCC 7120) and strain CSMI11 in BG11₀ NH₄⁺-, BG11-, and BG11₀-solid media supplemented with 10 mM TES-NaOH buffer (pH 7.5) and, when indicated, 1 mmol/L agmatine sulfate (Agm). Each spot was inoculated with an amount of cells containing the indicated amount of Chl, and the plates were incubated for 8 days under culture conditions and photographed.

Figure 5

Growth tests of Anabaena sp. strains PCC 7120 (WT), CSMI11 (Δalr2310) and FQ163 (hepP) in BG11₀ solid medium supplemented with 10 mmol/L TES-NaOH buffer (pH 7.5) and, when indicated, arginine (Arg) at the specified concentration. Spots were inoculated with an amount of cells containing the indicated amount of Chl, and the plates were incubated for 14 days under culture conditions and photographed. Strain FQ163 was used as a Fox⁻ control.

Figure 6

Cellular localization of Alr2310-GFP in the Anabaena filaments. (A) Filaments of Anabaena sp. strain CSMI21 grown in bubbled BG11 medium or incubated in bubbled BG11₀ medium (without combined nitrogen) for 48 h were visualized by confocal microscopy as described in Materials and Methods. Bright field and GFP fluorescence images are shown Brightness and contrast were increased to improve visibility. (B) Quantification of GFP fluorescence in cells of strain CSMI21. Average background fluorescence from wild-type cells (which lack GFP) was subtracted. Figures are the mean and standard deviation of the mean of the fluorescence recorded in cells grown in bubbled BG11 medium (487 cells counted) or incubated in bubbled BG11₀ medium for 24 h (309 vegetative cells and 58 heterocysts counted; Student's t test P = 2.7 × 10⁻¹⁸) or 48 h (409 vegetative cells and 67 heterocysts counted; P = 1.3 × 10⁻⁵⁶).

Figure 7

Western blot analysis of the GFP in strain CSMI21 and control strains. The Anabaena strains used were PCC 7120 (WT), mutant CSMI21 (alr2310-gfp) and a strain carrying pAM1954, which is a replicative plasmid bearing the gfp gene expressed from the rbc gene promoter. Cell-free extracts were prepared from whole filaments grown in bubbled cultures with BG11 medium or incubated in BG11₀ medium for 48 h, or from heterocysts (Hets) isolated from the latter. Anabaena sp. [pAM1954] was grown in shaken cultures containing BG11 medium. Proteins were subjected to SDS-PAGE electrophoresis and transferred to membranes. Protein detection was performed with anti-GFP antibodies. Native GFP is 26.9 kDa and the Alr2310-GFP fusion, which includes Alr2310, a tetra-glycine linker peptide, and the GFP is 65.7 kDa. Protein loaded was 40 μg per well (0.6 μg in the case of Anabaena [pAM1954]). As observed with the wild-type extracts, the antibodies marked 3 or 4 unspecific bands (brackets). Strain CSMI21 produced mainly a band corresponding to the fusion protein (black arrow) and only a faint band corresponding to free GFP, which was identified with the extract of Anabaena [pAM1954] that only produced, as expected, free GFP (open arrow). Note that some material from lane BG11/CSMI21 may have contaminated lane BG11/WT.