Identification and expression of genes narL and narX of the nar (nitrate reductase) locus in Escherichia coli K-12

Abstract

Previous studies have shown that narL+ is required for nitrate induction of nitrate reductase synthesis and for nitrate inhibition of fumarate reductase synthesis in Escherichia coli. We cloned narL on a 5.1-kilobase HindlIl fragment. Our clone also contained a previously unidentified gene, which we propose to designate as narX, as well as a portion of narK. Maxicell experiments indicated that narL and narX encode proteins with approximate Mrs of 28,000 and 66,000, respectively. narX insertion mutations reduced nitrate reductase structural gene expression by less than twofold. Expression of 40(narL-lacZ) operon fusions was weakly induced by nitrate but was indifferent to aerobiosis and independent offnr. Expression of 'F(narX-lacZ) operon fusions was induced by nitrate and was decreased by narL and fnr mutations. A 4(narK-lacZ) operon fusion was induced by nitrate, and its expression was fully dependent on narL+ andfnr'. Analysis of these operon fusions indicated that narL and narX are transcribed counterclockwise with respect to the E. coli genetic map and that narK is transcribed clockwise.Escherichia coli will use several terminal electron acceptors for respiration, including oxygen, nitrate (NO3-), and fumarate. Use of these electron acceptors is hierarchical: aerobiosis prevents nitrate and fumarate reductase synthesis, and nitrate prevents the anaerobic formation of fumarate reductase (18,21,46,53; V. Stewart, Microbiol. Rev., in press).The nitrate reductase enzyme complex consists of three membrane-bound subunits and also contains molybdenum cofactor and heme (Stewart, in press). The structural genes for the polypeptide subunits are termed narC (formerly narG [2]), narH, and narI and are organized in an operon at the nar (formerly chlC) locus at 27 min on the E. coli genetic map (2,9,16,32,38,51; Stewart, in press). Genetic studies indicate that the organization of this operon is hemApromoter-narC-narH-narl -trp (9, 51).Nitrate reductase synthesis is induced by anaerobiosis, and during anaerobic growth, it is further induced by nitrate (46). Studies with F(nar-lac) operon fusions (14, 50) and measurements of nar mRNA synthesis (28) indicate that both anaerobiosis and nitrate act to control narCHI operon transcription. The effect of anaerobiosis is thought to be mediated through FNR (the fnr gene product), a positiveacting transcription factor required for the synthesis of several anaerobic respiratory enzymes (26, 43; Stewart, in press) including nitrate reductase (14, 50) and fumarate reductase (18, 21). The physiological signal for FNR-mediated transcription activation is unknown (56).A mutation termed narL215::TnlO prevents nitrate induction of nitrate reductase synthesis and of F(narC-lacZ) expression (50) but has no effect on control by anaerobiosis. This mutation is complemented in trans by a specialized transducing bacteriophage, k pchlC3 (50). Genetic mapping indicates that narL is located upstream of the nar operon, between hemA and narCHI; a gene of unknown function, narK, maps between narL and...

The narK gene product participates in nitrate transport induced in Escherichia coli nitrate‐respiring cells

“…The narK gene product was reported to be fully induced by nitrate under anaerobic conditions [16], as in the case of the narCHJZ gene products. The common character in these two operons was confirmed in our promoter analysis of the nar gene [13].…”

“…We assumed that the gene product is a nitrate transporter on the basis of: (i) the presence of a nitrate transporter has been demonstrated [ 1,7]; (ii) except for the narK gene, no coding capacity for the transport protein is apparently sustained in either the narCHJ1 operon or the downstream [9,10] and upstream regions [13,16]; (iii) the narK gene product is neither involved directly in the electron-transfer system [12] nor qualified as a regulatory protein [14-161; and (iv) the narK gene product shows remarkable characteristics as a typical transmembrane protein, as mentioned above.…”

Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K‐12 depends on DNA binding site arrangement

“…For the experiments in Table 1, anaerobic cultures were grown in 3-[N-morpholino]-propanesulphonic acid (MOPS)-buffered minimal medium with glucose as the sole carbon source (Stewart and Parales, 1988). The initial pH of this medium was 8.0 and NaNO 3 (40 mM) or NaNO 2 (5 mM) were added as indicated.…”

Role of the Escherichia coli nitrate transport protein, NarU, in survival during severe nutrient starvation and slow growth

“…Although narK is optimally expressed during anaerobic growth in the presence of excess nitrate (Stewart & Parales, 1988;Kolesnikow et al, 1992), analysis of either wholecell or membrane proteins by SDS-PAGE failed to reveal a protein band that could reliably be identified as NarK. Western analysis was therefore used to develop a method to detect both NarK and NarU and to provide an indication of the relative quantities of the two proteins in different strains and after growth under different conditions.…”

“…The narK and narU genes are both located immediately upstream from four structural genes encoding the alternative nitrate reductases, nitrate reductase A and nitrate reductase Z, respectively. It is well established that narK and narGHJI are organized as two operons transcribed in the same orientation, but with 500 bp of intergenic DNA that includes a rho-independent narK transcription terminator, and binding sites for regulation by the oxygen-responsive transcription factor, FNR, and the nitrate-nitrite-responsive two-component regulatory system, NarX-NarL (Stewart & Parales, 1988;Kolesnikow et al, 1992). In contrast, narU and narZ are separated by only 81 bp of DNA that lacks recognizable transcription termination or promoter sequences.…”