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

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 HindIII 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 phi (narL-lacZ) operon fusions was weakly induced by nitrate but was indifferent to aerobiosis and independent of fnr. Expression of phi (narX-lacZ) operon fusions was induced by nitrate and was decreased by narL and fnr mutations. A phi (narK-lacZ) operon fusion was induced by nitrate, and its expression was fully dependent on narL+ and fnr+. 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.     

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

narL and narX mediate nitrate induction of nitrate reductase synthesis and nitrate repression of fumarate reductase synthesis. We report here the nucleotide sequences of narL and narX. The deduced protein sequences aid in defining distinct subclasses of regulators and sensors in the family of two-component regulatory proteins.     

narI region of the Escherichia coli nitrate reductase (nar) operon contains two genes.

In previous studies it has been established that in Escherichia coli the three known subunits of anaerobic nitrate reductase are encoded by the narGHI operon. From the nucleotide sequence of the narI region of the operon we conclude that, in addition to the narG and narH genes, the nar operon contains two other open reading frames (ORFs), ORF1 and ORF2, that encode proteins of 26.5 and 25.5 kilodaltons, respectively. Protein fusions to each of the genes in the operon showed that expression of all four genes was similarly regulated. The reading frames of ORF1 and ORF2 were verified, and the N-terminal sequence for the ORF1 fusion protein was determined. The nar operon therefore contains four genes designated and ordered as narGHJI.     

Requirement of Fnr and NarL functions for nitrate reductase expression in Escherichia coli K-12.

I used a chlC-lac operon fusion to study regulatory mutations which affect nitrate reductase expression in Escherichia coli. A NarL- mutant apparently lacks a nitrate-specific positive regulatory component. Furthermore, an fnr (nirR) mutation prevented enzyme induction under any conditions. These data are consistent with a two-step, positive control model for nitrate reductase regulation.     

chlC (nar) operon of Escherichia coli includes structural genes for alpha and beta subunits of nitrate reductase.

The synthesis of the alpha and beta subunits of nitrate reductase by 20 chlC::Tn5 insertion mutants of Escherichia coli was determined by immune precipitation of the subunits from fractions of cell extracts. Only two of the mutants produced either subunit in detectable amounts; these two accumulated the alpha subunit, but no beta subunit. In both cases the alpha subunit was present in the cytosolic fraction, in contrast to wild-type cells, in which both subunits are present mainly in the membrane fraction. EcoRI restriction fragments containing the Tn5 inserts from five of the mutants were cloned into pBR322. The insertions were localized on two contiguous EcoRI fragments spanning a 5.6-kilobase region that overlapped the contiguous ends of the two fragments. An insertion that permitted alpha subunit formation defined one end of the 5.6-kilobase region. The results indicated that the genes encoding the alpha and beta subunits of nitrate reductase were part of a chlC (nar) operon that is transcribed in the direction alpha leads to beta.     

Periplasmic nitrate reductase (NapABC enzyme) supports anaerobic respiration by Escherichia coli K-12

Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especially Paracoccus pantotrophus. Whole-genome sequencing of Escherichia coli revealed the structural genes napFDAGHBC, which encode NapABC enzyme and associated electron transfer components. E. coli also expresses two membrane-bound proton-translocating nitrate reductases, encoded by the narGHJI and narZYWV operons. We measured reduced viologen-dependent nitrate reductase activity in a series of strains with combinations of nar and nap null alleles. The napF operon-encoded nitrate reductase activity was not sensitive to azide, as shown previously for the P. pantotrophus NapA enzyme. A strain carrying null alleles of narG and narZ grew exponentially on glycerol with nitrate as the respiratory oxidant (anaerobic respiration), whereas a strain also carrying a null allele of napA did not. By contrast, the presence of napA+ had no influence on the more rapid growth of narG+ strains. These results indicate that periplasmic nitrate reductase, like fumarate reductase, can function in anaerobic respiration but does not constitute a site for generating proton motive force. The time course of phi(napF-lacZ) expression during growth in batch culture displayed a complex pattern in response to the dynamic nitrate/nitrite ratio. Our results are consistent with the observation that phi(napF-lacZ) is expressed preferentially at relatively low nitrate concentrations in continuous cultures (H. Wang, C.-P. Tseng, and R. P. Gunsalus, J. Bacteriol. 181:5303-5308, 1999). This finding and other considerations support the hypothesis that NapABC enzyme may function in E. coli when low nitrate concentrations limit the bioenergetic efficiency of nitrate respiration via NarGHI enzyme.     

Autoregulation of the nar operon encoding nitrate reductase in Escherichia coli

Summary Nitrate reductase is demonstrated to exert an autogenous control on its own synthesis. This effect requires the participation of the molybdenum cofactor. Use of strains in which the control region of the nar operon is mutated reveals two loci in this region: one, affected in strain LCB94, is common to both autoregulation and induction by nitrate while the other, mutated in strain LCB188, is specific for the induction by nitrate. It is proposed that the autogenous control prevents the unnecessary accumulation of the nitrate reductase subunits in the cytoplasm.     

Molecular cloning of the N-acetylneuraminate lyase gene in Escherichia coli K-12

Summary The gene for N-acetylneuraminate lyase [N-acetylneuraminate pyruvate-lyase; NPL] of Escherichia coli C600 was cloned onto pBR322 as a 9.8 kilobase HindIII fragment of chromosomal DNA and the hybrid plasmid was designated pMK2. The gene in the hybrid plasmid was subcloned in pBR322 as a 1.2 kilobase HindIII-EcoRI fragment and the resultant hybrid plasmid was designated pMK6. NPL activity level was increased more than 5-fold in the pMK6-bearing strain compared with that of the wild type, when the cells were grown on a medium containing inducer (N-acetylneuraminate: NANA). The transformants harbouring pMK6 also showed higher activity even in the absence of inducer. The NPL produced by pMK6-bearing cells was structurally and immunologically the same as that purified from E. coli C600.     

Molybdenum effector of fumarate reductase repression and nitrate reductase induction in Escherichia coli.

In Escherichia coli the presence of nitrate prevents the utilization of fumarate as an anaerobic electron acceptor. The induction of the narC operon encoding the nitrate reductase is coupled to the repression of the frd operon encoding the fumarate reductase. This coupling is mediated by nitrate as an effector and the narL product as the regulatory protein (S. Iuchi and E. C. C. Lin, Proc. Natl. Acad. Sci. USA 84:3901-3905, 1987). The protein-ligand complex appears to control narC positively but frd negatively. In the present study we found that a molybdenum coeffector acted synergistically with nitrate in the regulation of frd and narC. In chlD mutants believed to be impaired in molybdate transport (or processing), full repression of phi(frd-lac) and full induction of phi(narC-lac) by nitrate did not occur unless the growth medium was directly supplemented with molybdate (1 microM). This requirement was not clearly manifested in wild-type cells, apparently because it was met by the trace quantities of molybdate present as a contaminant in the mineral medium. In chlB mutants, which are known to accumulate the Mo cofactor because of its failure to be inserted as a prosthetic group into proteins such as nitrate reductase, nitrate repression of frd and induction of narC were also intensified by molybdate supplementation. In this case a deficiency of the molybdenum coeffector might have resulted from enhanced feedback inhibition of molybdate transport (or processing) by the elevated level of the unutilized Mo cofactor. In addition, mutations in chlE, which are known to block the synthesis of the organic moiety of the Mo cofactor, lowered the threshold concentration of nitrate (< 1 micromole) necessary for frd repression and narC induction. These changes could be explained simply by the higher intracellular nitrate attainable in cells lacking the ability to destroy the effector.     

Mutational analysis of nitrate regulatory gene narL in Escherichia coli K-12.

The narL gene product, NarL, is the nitrate-responsive regulator of anaerobic respiratory gene expression. We used genetic analysis of narL mutants to better understand the mechanism of NarL-mediated gene regulation. We selected and analyzed seven nitrate-independent narL mutants. Each of three independent, strongly constitutive mutants had changes of Val-88 to Ala. The other four mutants were weakly constitutive. The narL505(V88A) allele was largely dominant to narL+, while narX+ had a negative influence on its constitutive phenotype, suggesting that NarX may play a negative role in nitrate regulation. We also constructed two narL mutations that are analogous to previously characterized constitutive degU alleles. The first, narL503(H15L), was a recessive null allele. The second, narL504(D110K), functioned essentially as wild type but was dependent on narX+ for full activity. We changed Asp-59 of NarL, which corresponds to the site of phosphorylation of other response regulators, to Asn. This change, narL502(D59N), was a recessive null allele, which is consistent with the hypothesis that NarL requires phosphorylation for activation. Finally, we tested the requirement for molybdate on regulation in a narL505(V88A) strain. Although narL505(V88A) conferred some nitrate-independent expression of fdnGHI (encoding formate dehydrogenase-N) in limiting molybdate, it required excess molybdate for full induction both in the absence and in the presence of nitrate. This finding suggests that narL505(V88A) did not confer molybdate-independent expression of fdnGHI.     

An ultraviolet light sensitive target in nitrate reductase of Escherichia coli K-12

Ultraviolet light was shown to inactivate purified nitrate reductase in the presence of reduced benzyl viologen. Loss of activity was not complete, reaching 60 to 70%. Photolysis was maximum at 345 nm. The differential spectrum between native and irradiated enzyme exhibited absorption bands at 216, 275, 314 and 365 nm. The photosensitive electron carrier could be extracted by organic solvents. It had the following absorption bands: 225, 275 and 285 nm. It was reduced by Nile blue A but not by methylene blue. The precise nature of this light sensitive molecule could not be determined although the results support the idea that this chromophore might be a naphthoquinone.     

Effect of rfaH (sfrB) and temperature on expression of rfa genes of Escherichia coli K-12.

In order to study the regulation of a large block of contiguous genes at the rfa locus of Escherichia coli K-12 which are involved in synthesis and modification of the lipopolysaccharide core, the transposon TnlacZ was used to generate in-frame lacZ fusions to the coding regions of five genes (rfaQ, -G, -P, -B and -J) within this block. The beta-galactosidase activity of strains in which these fusions had been crossed into the chromosomal rfa locus was significantly decreased when the rfaH11 (sfrB11) allele was introduced and was restored to wild-type levels when these strains were lysogenized with a lambda phage carrying wild-type rfaH. This indicates that the positive regulatory function encoded by rfaH is required throughout this block of genes. In addition, expression of the lacZ fusion to rfaJ was reduced by growth at 42 degrees C, and this correlated with a temperature-induced change in the electrophoretic profile of the core lipopolysaccharide.     

Cloning and expression of the Escherichia coli K-12 sad gene.

The Escherichia coli K-12 sad gene, which encodes an NAD-dependent succinic semialdehyde dehydrogenase, was cloned into a high-copy-number vector. Minicells carrying a sad+ plasmid produced a 55,000-dalton peptide, the probable sad gene product.     

Cloning of the 3'-phosphoadenylyl sulfate reductase and sulfite reductase genes from Escherichia coli K-12

The structural genes for 3'-phosphoadenylyl sulfate (PAPS) reductase (cysH) and sulfite reductase (alpha and beta subunits; EC 1.8.1.2)(cysI and cysJ) of Escherichia coli K-12 have been cloned by complementation. pCYSI contains two PstI fragments (18.3 and 2.9 kb) which complement cysH-, cysI-, and cysJ- mutants. Subcloning showed that the cysH gene is located on a 1.6-kb ClaI subfragment (pCYSI-3) whereas cysI and most of cysJ are carried on a 3.7-kb ClaI subfragment (pCYSI-5). The PAPS reductase gene is closely linked to the sulfite reductase genes, but its expression is regulated by a unique promoter. The cysI and cysJ genes, on the other hand, are transcribed as an operon and the promoter precedes the cysI gene. Maxicell analysis demonstrated that pCYSI encodes three polypeptides of Mr 27,000, 57,000, and 60,000, in addition to the tetracycline-resistance determinant. The 60- and 57-kDa proteins are most likely the alpha and beta subunits, respectively, of E. coli sulfite reductase while the 27-kDa protein is putatively identified as PAPS reductase. Preliminary data suggest that the alpha and beta subunits of sulfite reductase are encoded by cysI and cysJ, respectively.     

Cloning of alginate lyase gene (alxM) and expression in Escherichia coli.

The alxM gene encoding a D-mannuronan-specific alginate lyase has been cloned from a marine bacterium isolated as an epiphyte on the brown alga, Sargassum fluitans. Expression of this gene in Escherichia coli provides a source of this enzyme for probing alginate structure and modifying the mannuronan-rich alginate polymers produced by bacterial pathogens.     

Genome-wide expression profiling in Escherichia coli K-12.

We have established high resolution methods for global monitoring of gene expression in Escherichia coli. Hybridization of radiolabeled cDNA to spot blots on nylon membranes was compared to hybridization of fluorescently-labeled cDNA to glass microarrays for efficiency and reproducibility. A complete set of PCR primers was created for all 4290 annotated open reading frames (ORFs) from the complete genome sequence of E.coli K-12 (MG1655). Glass- and nylon-based arrays of PCR products were prepared and used to assess global changes in gene expression. Full-length coding sequences for array printing were generated by two-step PCR amplification. In this study we measured changes in RNA levels after exposure to heat shock and following treatment with isopropyl-beta-D-thiogalactopyranoside (IPTG). Both radioactive and fluorescence-based methods showed comparable results. Treatment with IPTG resulted in high level induction of the lacZYA and melAB operons. Following heat shock treatment 119 genes were shown to have significantly altered expression levels, including 35 previously uncharacterized ORFs and most genes of the heat shock stimulon. Analysis of spot intensities from hybridization to replicate arrays identified sets of genes with signals consistently above background suggesting that at least 25% of genes were expressed at detectable levels during growth in rich media.