A single channel for nitrate uptake, nitrite export and nitrite uptake by Escherichia coli NarU and a role for NirC in nitrite export and uptake

Two related polytopic membrane proteins of the major facilitator family, NarK and NarU, catalyse nitrate uptake, nitrite export and nitrite uptake across the Escherichia coli cytoplasmic membrane by an unknown mechanism. A 12-helix model of NarU was constructed based upon six alkaline phosphatase and beta-galactosidase fusions to NarK and the predicted hydropathy for the NarK family. Fifteen residues conserved in the NarK-NarU protein family were substituted by site-directed mutagenesis, including four residues that are essential for nitrate uptake by Aspergillus nidulans: arginines Arg(87) and Arg(303) in helices 2 and 8, and two glycines in a nitrate signature motif. Despite the wide range of substitutions studied, in no case did mutation result in loss of one biochemical function without simultaneous loss of all other functions. A NarU+ NirC+ strain grew more rapidly and accumulated nitrite more rapidly than the isogenic NarU+ NirC(-) strain. Only the NirC+ strain consumed nitrite rapidly during the later stages of growth. Under conditions in which the rate of nitrite reduction was limited by the rate of nitrite uptake, NirC+ strains reduced nitrite up to 10 times more rapidly than isogenic NarU+ strains, indicating that both nitrite efflux and nitrite uptake are largely dependent on NirC. Isotope tracer experiments with [15N]nitrate and [14N]nitrite revealed that [15N]nitrite accumulated in the extracellular medium even when there was a net rate of nitrite uptake and reduction. We propose that NarU functions as a single channel for nitrate uptake and nitrite expulsion, either as a nitrate-nitrite antiporter, or more likely as a nitrate/H+ or nitrite/H+ channel.     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

Physicochemical roles of soluble metal cations in the outer membrane of Escherichia coli K-12

Atomic absorption spectroscopy of isolated native and EDTA-modified (lipopolysaccharide-depleted) outer membrane revealed trace amounts of potassium, manganese, and iron (1.0-7.0 nmol/mg dry weight outer membrane). Sodium, magnesium, and calcium were approximately one order of magnitude more plentiful, but EDTA-modified outer membrane was deficient in calcium. When metal-binding assays were conducted to find the binding capacity of native and EDTA-modified outer membrane, potassium bound poorly compared with sodium. However, there was no difference in the binding of these ions between the OM preparations. In contrast, reduced amounts of magnesium, calcium, manganese, and iron III bound to the EDTA-modified OM. Partitioning of intact cells in a biphasic dextran-polyethyleneglycol system indicated that the reduced lipopolysaccharide content of the EDTA-modified outer membrane increased the hydrophobicity of the cell surface. Exposure of control and EDTA-treated cells to divalent metal salt solutions before phase partitioning also increased cell surface hydrophobicity. Freeze-etching showed that sodium ions had no effect on the membrane fractures observed in control cells, but with EDTA-treated cells, this cation increased the occurrence of small outer membrane fractures (plateaus) which are characteristic of EDTA treatment. Both magnesium and manganese increased the frequency of outer membrane cleavage in control cells, whereas calcium did not. In contrast, all three divalent metallic ions increased the frequency and extent of cleavage in the outer membrane of EDTA-treated cells.     

Pullulanase secretion in Escherichia coli K-12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein

The previously uncharacterized third and fourth genes (pulE and pulF) of the pullulanase secretion gene operon of Klebsiella oxytoca strain UNF5023 are, respectively, predicted to encode a 55 kDa polypeptide with a putative nucleotide-binding site, and a highly hydrophobic 44 kDa polypeptide that probably spans the cytoplasmic membrane several times. Expression of pulE in minicells or under the control of a strong bacteriophage T7 promoter resulted in the production of a c. 58 kDa cytoplasmic protein. A representative PulE-beta-galactosidase hybrid protein created by Tnlac mutagenesis was also found mainly in the cytoplasm. These results are in line with the predicted absence from PulE of a region of sufficient hydrophobicity to function as a signal sequence. The PulF polypeptide could not be detected either in minicells or when the gene was transcribed from the T7 promoter, but the acquirement of three pulF-lacZ gene fusions that encoded hybrid proteins with relatively high levels of beta-galactosidase activity indicates that this gene can be transcribed and translated. Gene disruption experiments indicated that both pulE and pulF are required for pullulanase secretion in Escherichia coli K-12. Both proteins exhibit considerable homology throughout their entire lengths with other proteins involved in protein secretion, pilin assembly, conjugation and transformation competence in a variety of bacteria. In addition, PulE protein has consensus sequences found in a wide variety of nucleotide-binding proteins. This study completes the initial characterization of the pullulanase secretion gene operon, which comprises 13 genes that are all essential for the transport of pullulanase across the outer membrane.     

Interdependence of two NarK domains in a fused nitrate/nitrite transporter

Nitrate uptake is essential for various bacterial processes and combines with nitrite export to form the usual initial steps of denitrification, a process that reduces nitrate to dinitrogen gas. Although many bacterial species contain NarK-like transporters that are proposed to function as either nitrate/proton symporters or nitrate/nitrite antiporters based on sequence homology, these transporters remain, in general, poorly characterized. Several bacteria appear to contain a transporter that is a fusion of two NarK-like proteins, although the significance of this arrangement remains elusive. We demonstrate that NarK from Paracoccus denitrificans is expressed as a fusion of two NarK-like transporters. NarK1 and NarK2 are separately capable of supporting anaerobic denitrifying growth but with growth defects that are partially mitigated by coexpression of the two domains. NarK1 appears to be a nitrate/proton symporter with high affinity for nitrate and NarK2 a nitrate/nitrite antiporter with lower affinity for nitrate. Each transporter requires two conserved arginine residues for activity. A transporter consisting of inactivated NarK1 fused to active NarK2 has a dramatically increased affinity for nitrate compared with NarK2 alone, implying a functional interaction between the two domains. A potential model for nitrate and nitrite transport in P. denitrificans is proposed.     

Identification of three genes controlling production of new outer membrane pore proteins in Escherichia coli K-12.

Escherichia coli K-12 strains carrying mutations in the ompB gene or double mutations in the tolF and par genes lack the major outer membrane proteins 1a and 1b. These strains are deficient in the transport of small hydrophylic compounds and are multiply colicin resistant. When revertants of these strains were sought, a number of extragenic pseudorevertants were obtained which produced new outer membrane proteins. These new proteins could be divided into three classes by differences in electrophoretic mobility on polyacrylamide gels, by differing specificities for transport of small molecules, and by the identification of three different genetic loci for genes controlling their production. These genetic loci are designated as nmpA (at approximately 82.5 min on the E. coli K-12 genetic map), nmpB (8.6 min), and nmpC (12 min). The new proteins produced in strains carrying nmpA, nmpB, or nmpC mutations did not cross-react with antiserum against a mixture of proteins 1a and 1b, or with antiserum against phage-directed protein 2. Production of the new membrane proteins restored sensitivity to some of the colicins.     

Characterisation of Escherichia coli K-12 mutants defective in formate-dependent nitrite reduction: essential roles for hemN and the menFDBCE operon

Three Escherichia coli mutants defective in formate-dependent nitrite reduction (Nrf activity) were characterised. Two of the mutants, JCB354 and JCB356, synthesized all five c-type cytochromes previously characterised in anaerobic cultures of E. coli. The third mutant, JCB355, was defective for both cytochrome b and cytochrome c synthesis, but only during anaerobic growth. The insertion sites of the transposon in strains JCB354 and JCB356 mapped to the menFDBCE operon; the hemN gene was disrupted in strain JCB355. The mutation in strain JCB354 was complemented by a plasmid encoding only menD; strain JCB356 was complemented by a plasmid encoding only menBCE. A mutant defective in the methyltransferase activity involved in both ubiquinone synthesis and conversion of demethylmenaquinone to menaquinone expressed the same Nrf activity as the parental strain. The effects of men, ubiA and ubiE mutations on other cytochrome-c-dependent electron transfer pathways were also determined. The combined data establish that menaquinones are essential for cytochrome-c-dependent trimethylamine-N-oxide reductase (Tor) and Nrf activity, but that either menaquinone or ubiquinone, but not demethylmenaquinone, can transfer electrons to a third cytochrome-c-dependent electron transfer chain, the periplasmic nitrate reductase. Received: 9 December 1996 / Accepted: 11 June 1997     

Interaction of the expression of two membrane genes, acrA and plsA, in Escherichia coli K-12.

The mutation acrA1, leading to acriflavine sensitivity through disorganization of the plasma membrane, is located between proC and purE on the Escherichia coli K-12 chromosome. Gene plsA has been reported to determine biosynthesis of membrane phospholipid and to be located very near acrA (1). Genes acrA and plsA fall into different cistrons and are arranged in the order proC-acrA-plasA-purE. The genes were shown to interact with each other. Introduction of acrA mutation into a plsA temperature-sensitive mutant mitigated the heat sensitivity. Plasmid (F-gal+) stability in acrA mutants was restored by introduction of the plasA mutation into the acrA cells. When an Hfr plsA donor was conjugated with an acrA recipient, or when reciprocally conjugated, the exogenotes were eliminated at high frequency during subsequent subcultivation in broth. However, the exogenotes were not eliminated in all other allelic combinations of genes acrA and plsA. When an F-gal+ plasmid was introduced into the unstable heterozygotes (acrA+plsA/acrApls1+), the plasmids were stably hosted, whereas the acrA+ plasA exogenotes were spontaneously lost at a high frequency. On the other hand, when the unstable heterozygotes carrying F-gal+ were cultured in acriflavine-containing medium, the F-gal+ plasmids were preferentially eliminated but the acrA+plasA exogenotes were not affected. The results suggest that the organization of the plasma membrane controls the recombination of the exogenotes introduced into zygotes.     

Suppression of iron uptake deficiency in Escherichia coli K-12 by loss of two major outer membrane proteins.

A novel iron uptake system was observed in pseudorevertants of $\text{Escherichia}$,$\text{coli}$ strains defective in ferrienterochelin transport. The new system is unique in that it is an active transport system that does not utilize any known siderophore. Acquisition of the new uptake system occurs concomitantly with the loss of two major outer membrane proteins (b and c) believed to function as structural components of transmembrane pores.     

Export of FepA::PhoA fusion proteins to the outer membrane of Escherichia coli K-12.

A library of fepA::phoA gene fusions was generated in order to study the structure and secretion of the Escherichia coli K-12 ferric enterobactin receptor, FepA. All of the fusion proteins contained various lengths of the amino-terminal portion of FepA fused in frame to the catalytic portion of bacterial alkaline phosphatase. Localization of FepA::PhoA fusion proteins in the cell envelope was dependent on the number of residues of mature FepA present at the amino terminus. Hybrids containing up to one-third of the amino-terminal portion of FepA fractionated with their periplasm, while those containing longer sequences of mature FepA were exported to the outer membrane. Outer membrane-localized fusion proteins expressed FepA sequences on the external face of the outer membrane and alkaline phosphatase moieties in the periplasmic space. From sequence determinations of the fepA::phoA fusion joints, residues within FepA which may be exposed on the periplasmic side of the outer membrane were identified.     

TolB protein of Escherichia coli K-12 interacts with the outer membrane peptidoglycan-associated proteins Pal, Lpp and OmpA

The Tol–Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. Transmembrane domains of TolQ, TolR and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. TolB and the central domain of TolA interact in vitro with the outer membrane porins. In this study, both genetic and biochemical analyses were carried out to analyse the links between TolB, Pal and other components of the cell envelope. It was shown that TolB could be cross-linked in vivo with Pal, OmpA and Lpp, while Pal was associated with TolB and OmpA. The isolation of pal and tolB mutants disrupting some interactions between these proteins represents a first approach to characterizing the residues contributing to the interactions. We propose that TolB and Pal are part of a multiprotein complex that links the peptidoglycan to the outer membrane. The Tol–Pal proteins might form transenvelope complexes that bring the two membranes into close proximity and help some outer membrane components to reach their final destination.     

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.     

Localization and characterization of cytochromes from membrane vesicles of Escherichia coli K-12 grown in anaerobiosis with nitrate.

Cytochromes b of anaerobically nitrate-grown Escherichia coli cells are analysed. Ascorbate phenazine methosulfate distinguishes low and high potential cytochromes b. Reduction kinetics performed at 559 nm presents a very complex pattern which can be analysed assuming that at least four b-type cytochromes are present. The electron transport chain from formate to oxygen would contain a low potential cytochrome b-556, a cytochrome b-558 associated to the oxidase, and a cytochrome d as the principle oxidase. Cytochrome o is also present, but seems to be functional only at low oxygen concentrations. A cytochrome b-556 associated to nitrate reductase is shown to belong to a branch of the formate-oxidase chain. 2-N-Heptyl-4-hydroxyquinoline-N-oxide affects the reduction kinetics in a very complex way. One inhibition site is in evidence between cytochrome b-558 and cytochrome d; another between the cytochrome associated to nitrate reductase and the nitrate reductase. A third inhibition site is located in the common part of the formate-nitrate and the formate-oxidase systems. Ascorbate phenazine methosulfate is shown to donate electrons near cytochrome b-558.