Construction in vitro of a cloned nar operon from Escherichia coli

Abstract

To clone the nar operon of Escherichia coli without an effective selection procedure for the nar+ phenotype, a strategy utilizing nar::TnS mutants was employed. Partial segments of the nar operon containing TnS insertions were cloned into plasmid pBR322 by using the transposon resistance character for selection. A hybrid plasmid was constructed in vitro from two of these plasmids and isolated by a procedure that involved screening a population of transformed nar(Ts) mutant TS9A for expression of thermal stable nitrate reductase activity. A detailed restriction site map of the resulting plasmid, pSR95, corresponded closely to the composite restriction endonuclease map deduced for the nar region from maps of the cloned nar::TnS fragments. When transformed with pSR95, wild-type strain PK27 overproduced the a, 0, and-y subunits of nitrate reductase, although nitrate reductase activity was only slightly increased. The a and B subunits were overproduced about 5to 10fold and accumulated mostly as an inactive aggregate in the cytoplasm; they subunit overproduction was detected as a threefold increase in the specific content of cytochrome b555 in the membrane fraction. Functional nitrate reductase and the cytochrome spectrum associated with functional nitrate reductase were restored in the nar::TnS mutant EEI after transformation with pSR95. Although the specific activity of nitrate reductase in this case was less than that of the wild type, both the a and , subunits appeared to be overproduced in an inactive form. In both strains PK27(pSR95) and EE1(pSR95), the formation of nitrate reductase activity and the accumulation of inactive subunits were repressed during aerobic growth. From these observations and the demonstration that pSR95 transformed narG::TnlO and narl::TnlO mutants to a nar+ phenotype, we conclude that plasmid pSR95 contains a functional nar operon that encodes the a, 1, and-y subunits of nitrate reductase.

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

“…Therefore, the NarK protein thus characterized as a strongly hydrophobic 140 transmembrane protein appears to be qualified enough to' form a channel-like structure. 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.…”

“…The mutants exhibit a normal level of nitrate reductase activity and regulate the expression of the nar operon [12]. However, Rondeau et al [9] reported that the narK mutant did not accuinulate nitrite in the overlay assay, assuming that either the nitrate reductase produced by this mutant is not functional or the ability for the cells to remove nitrite is derepressed. Based upon our observations, however, their result is consistently interpreted as a lack of normal transport of nitrate.…”

The cytochromes ofEscherichia coli

“…It has been characterized both as a component of the active enzyme and as an isolated species [41]. It is encoded by the narl (previously chlI) gene which is part of the nitrate reductase nar operon [43]. The se-quence is not yet known, although the nar operon has been cloned [43].…”

“…It is encoded by the narl (previously chlI) gene which is part of the nitrate reductase nar operon [43]. The se-quence is not yet known, although the nar operon has been cloned [43]. Of particular interest is the role of this cytochrome in the assembly of the enzyme in the cytoplasmic membrane, as revealed by the examination of mutants which lack this subunit or cannot synthesize heme (hemA mutants) [41].…”

Delineation of two distinct regulatory domains in the 5' region of the nar operon of Escherichia coli

“…The genes which encode the three subunits of nitrate reductase are components of an operon, designated nar, which is transcribed in the order narG (ot subunit), narH (3 subunit), and narn (y subunit) (2,6,8,25). We have recently constructed in vitro a hybrid plasmid containing an intact nar operon which, in transformed strains, overproduces the three subunits of nitrate reductase under the control of the same factors which regulate the expression of the chromosomal operon (19).…”