A Novel Gene ( narM ) Required for Expression of Nitrate Reductase Activity in the Cyanobacterium Synechococcus elongatus Strain PCC7942

Abstract

A new class of mutants deficient in nitrate assimilation was obtained from the cyanobacterium Synechococcus elongatus strain PCC7942 by means of random insertional mutagenesis. A 0.5-kb genomic region had been replaced by a kanamycin resistance gene cassette in the mutant, resulting in inactivation of two genes, one of which was homologous to the recently characterized cnaT gene of Anabaena sp. strain PCC7120 (J. E. Frías, A. Herrero, and E. Flores, J. Bacteriol. 185:5037-5044, 2003). While insertional mutation of the cnaT homolog did not affect expression of the nitrate assimilation operon or the activity of the nitrate assimilation enzymes in S. elongatus, inactivation of the other gene, designated narM, resulted in specific loss of the cellular nitrate reductase activity. The deduced NarM protein is a hydrophilic protein consisting of 161 amino acids. narM was expressed constitutively at a low level. The narM gene has its homolog only in the cyanobacterial strains that are capable of nitrate assimilation. In most of the cyanobacterial strains, narM is located downstream of narB, the structural gene of the cyanobacterial nitrate reductase, suggesting the functional link between the two genes. NarM is clearly not the structural component of the cyanobacterial nitrate reductase. The narM insertional mutant normally expressed narB, indicating that narM is not the transcriptional regulator of the structural gene of nitrate reductase. These results suggested that narM is required for either synthesis of the prosthetic group of nitrate reductase or assembly of the prosthetic groups to the NarB polypeptide to form functional nitrate reductase in cyanobacteria.

Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120

“…The coordinated expression of narM and the nirA operon observed in Anabaena is in contrast to the situation in S. elongatus, in which narM is constitutively expressed at low levels (11). This may be related to the nitrate-dependent induction of the nirA operon, which is more stringent in Anabaena than in S. elongatus (see the introduction).…”

“…Because a cyanobacterial narB gene alone provides E. coli with nitrate reductase activity (44), NarM does not appear to be a catalytic subunit of nitrate reductase. Instead, NarM appears to support the maturation of NarB (11). The coordinated expression of narM and the nirA operon would ensure the simultaneous production of NarM and the nitrate reductase enzyme, NarB, thus facilitating functional interactions between the two proteins, which may involve transient protein-protein interactions, as suggested by BACTH analysis.…”

“…A third positive regulatory element of nirA operon expression in Anabaena is CnaT, which does not appear to be a DNA-binding protein and, consequently, may affect nirA operon expression indirectly (25). As is the case for NtcB, whereas CnaT is strictly required for nitrate assimilation in Anabaena, any phenotype related to nitrate assimilation was observed in an S. elongatus cnaT mutant (11). In addition to the positive elements described above, two elements negatively affecting the expression of the nirA operon have been identified in Anabaena.…”

“…ORF alr0613 encodes a 155-amino-acid hypothetical protein with four possible transmembrane segments that shows some similarity to the phosphate starvation-inducible E (PsiE) protein from Escherichia coli, which is of unknown function. ORF alr0614, on the other hand, encodes a 153-amino-acid protein that exhibits 47% identity to the S. elongatus NarM protein, which is required for the proper development of nitrate reductase activity in this unicellular cyanobacterium (11).…”

Nitrite-Responsive Activation of the Nitrate Assimilation Operon in Cyanobacteria Plays an Essential Role in Up-Regulation of Nitrate Assimilation Activities under Nitrate-Limited Growth Conditions

“…1), suggesting that nitrate limitation due to the lack of NRT led to the high-level expression from the nirA operon promoter in YKA1. While the YKA2 strain, which differs from YKA1 in (12,13). Since the nucleotide sequence of L1 conforms to the structure of the binding sites for LysR-type proteins, it is likely that L1 constitutes the binding site for NtcB (12).…”