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 [14C]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 Δalr2310 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 [14C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the Δalr2310 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.

Homospermidine biosynthesis in the cyanobacterium Anabaena requires a deoxyhypusine synthase homologue and is essential for normal diazotrophic growth

“…The genome of Anabaena (Kaneko et al, ) contains a gene that encodes agmatinase ( alr2310 , speB ) (Burnat and Flores, ) and genes putatively encoding arginine decarboxylase ( all3401 ) and a DHS‐like protein ( alr3804 ), that could potentially constitute a homospermidine biosynthetic pathway. Anabaena mutants of these genes that were used in this work are shown in Fig.…”

“…. The agmatinase deletion mutant CSMI11 (∆ alr2310 or ∆ speB ), mutant CSMI11 complemented with a replicating plasmid that bears a construct in which the speB gene is expressed from the promoter of the glnA gene (CSMI11‐C or ∆ speB + speB ), and a strain producing a SpeB protein with the green fluorescent protein (GFP) added to its C‐terminus (SpeB‐C‐GFP) have been described previously (Burnat and Flores, ). Here, an Anabaena strain carrying the plasmid with the P glnA ‐ speB construct to overexpress SpeB was constructed (WT+ speB ).…”

“…The Anabaena agmatinase mutant, strain CSMI11 (∆ speB ), has previously been shown in [ 14 C]arginine catabolism assays to accumulate an agmatine spot mostly absent in the wild type (Burnat and Flores, ). Here, we analyzed polyamines in extracts of wild‐type Anabaena and of the ∆ speB strain, the complemented ∆ speB strain (∆ speB + speB ), and the SpeB‐overexpressing strain (WT+ speB ).…”

“…The Anabaena ∆ speB mutant has been previously shown to be specifically impaired in diazotrophic growth (Burnat and Flores, ). Here, we tested growth of the Δ all3401 ::C.S3 ( speA ) and alr3804 ::pCSV3 ( speY ) mutants in photoautotrophic shaken liquid cultures with different nitrogen sources, and we also included strain ∆ potADB ::C.S3 ( potADB ) in the analysis.…”

“…In particular, homospermidine can be synthesized from putrescine that is produced from decarboxylation of ornithine or indirectly from the decarboxylation of arginine, e.g., via arginine decarboxylase and agmatinase (Michael, ). Whereas cyanobacterial genomes generally appear to lack a gene encoding ornithine decarboxylase, they contain genes putatively encoding arginine decarboxylase and agmatinase (https://genome.annotation.jp/cyanobase), and these genes or enzymes have been characterized in a few cases (Schriek et al, ; Burnat and Flores, ; Kera et al, ). The cyanobacteria do not appear to have, however, a typical homospermidine synthase, and a DHS‐like protein has instead been suggested as a possible alternative homospermidine synthesizing enzyme (Shaw et al, ).…”