Anaerobic growth of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> using nitrate as a terminal electron acceptor

Corynebacterium glutamicum, a gram-positive soil bacterium, has been regarded as an aerobe because its growth by fermentative catabolism or by anaerobic respiration has, to this date, not been demonstrated. In this study, we report on the anaerobic growth of C. glutamicum in the presence of nitrate as a terminal electron acceptor. C. glutamicum strains R and ATCC13032 consumed nitrate and excreted nitrite during growth under anaerobic, but not aerobic, conditions. This was attributed to the presence of a narKGHJI gene cluster with high similarity to the Escherichia coli narK gene and narGHJI operon. The gene encodes a nitrate/nitrite transporter, whereas the operon encodes a respiratory nitrate reductase. Transposonal inactivation of C. glutamicum narG or narH resulted in mutants with impaired anaerobic growth on nitrate because of their inability to convert nitrate to nitrite. Further analysis revealed that in C. glutamicum, narK and narGHJI are cotranscribed as a single narKGHJI operon, the expression of which is activated under anaerobic conditions in the presence of nitrate. C. glutamicum is therefore a facultative anaerobe.     

Impacts of Nitrate and Nitrite on Physiology of Shewanella oneidensis

Shewanella oneidensis exhibits a remarkable versatility in anaerobic respiration, which largely relies on its diverse respiratory pathways. Some of these are expressed in response to the existence of their corresponding electron acceptors (EAs) under aerobic conditions. However, little is known about respiration and the impact of non-oxygen EAs on the physiology of the microorganism when oxygen is present. Here we undertook a study to elucidate the basis for nitrate and nitrite inhibition of growth under aerobic conditions. We discovered that nitrate in the form of NaNO₃ exerts its inhibitory effects as a precursor to nitrite at low concentrations and as an osmotic-stress provider (Na⁺) at high concentrations. In contrast, nitrite is extremely toxic, with 25 mM abolishing growth completely. We subsequently found that oxygen represses utilization of all EAs but nitrate. To order to utilize EAs with less positive redox potential, such as nitrite and fumarate, S. oneidensis must enter the stationary phase, when oxygen respiration becomes unfavorable. In addition, we demonstrated that during aerobic respiration the cytochrome bd oxidase confers S. oneidensis resistance to nitrite, which likely functions via nitric oxide (NO).     

Organization of the nar genes at the chlZ locus

The two membrane-bound respiratory nitrate reductases of Escherichia coli are encoded by distinct operons at two different loci, chlC and chlZ, on the chromosome. The chlZ locus includes a narK homologue, narU, encoding a nitrite extrusion protein, and narZYWV encoding nitrate reductase Z. No apparent homologue to the narXL operon has been found. Homology between narU and narK on the one hand and narZYWV and narGHJI on the other hand is limited to the coding regions.     

Requirement for terminal cytochromes in generation of the aerobic signal for the arc regulatory system in Escherichia coli: study utilizing deletions and lac fusions of cyo and cyd.

Escherichia coli has two terminal oxidases for its respiratory chain: cytochrome o (low O2 affinity) and cytochrome d (high O2 affinity). Expression of the cyo operon, encoding cytochrome o, is decreased by anaerobic growth, whereas expression of the cyd operon, encoding cytochrome d, is increased by anaerobic growth. We show by the use of lac gene fusion that the expressions of cyo and cyd are under the control of the two-component arc system. In a cyo+ cyd+ background, expression of phi(cyo-lac) is higher when the organism is grown aerobically than when it is grown anaerobically. A mutation in either the sensor gene arcB or the pleiotropic regulator gene arcA almost abolishes the anaerobic repression. In the same background, expression of phi(cyd-lac) is higher under anaerobic growth conditions than under aerobic growth conditions. A mutation in arcA or arcB lowers both the aerobic and anaerobic expressions, suggesting that ArcA plays an activating role instead of the typical repressing role. Under aerobic growth conditions, double deletions of cyo and cyd lower phi(cyo-lac) expression but enhance phi(cyd-lac) expression. The double deletions also prevent elevated aerobic induction of the lct operon (encoding L-lactate dehydrogenase), another target operon of the arc system. In contrast, these deletions do not circumvent aerobic repression of the nar operon (encoding the anaerobic respiratory enzyme nitrate reductase) under the control of the pleiotropic fnr gene product. It thus appears that ArcB senses the presence of O2 by level of an electron transport component in reduced form or that of an nonautoxidizable compound linked to the process by a redox reaction, whereas Fnr senses O2 by a different mechanism.     

Dynamics of denitrification activity of Paracoccus denitrificans in continuous culture during aerobic-anaerobic changes.

Induction and repression of denitrification activity were studied in a continuous culture of Paracoccus denitrificans during changes from aerobic to anaerobic growth conditions and vice versa. The denitrification activity of the cells was monitored by measuring the formation of denitrification products (nitrite, nitric oxide, nitrous oxide, and dinitrogen), individual mRNA levels for the nitrate, nitrite, and nitrous oxide reductases, and the concentration of the nitrite reductase enzyme with polyclonal antibodies against the cd1-type nitrite reductase. On a change from aerobic to anaerobic respiration, the culture entered an unstable transition phase during which the denitrification pathway became induced. The onset of this phase was formed by a 15- to 45-fold increase of the mRNA levels for the individual denitrification enzymes. All mRNAs accumulated during a short period, after which their overall concentration declined to reach a stable value slightly higher than that observed under aerobic steady-state conditions. Interestingly, the first mRNAs to be formed were those for nitrate and nitrous oxide reductase. The nitrite reductase mRNA appeared significantly later, suggesting different modes of regulation for the three genes. Unlike the mRNA levels, the level of the nitrite reductase protein increased slowly during the anaerobic period, reaching a stable value about 30 h after the switch. All denitrification intermediates could be observed transiently, but when the new anaerobic steady state was reached, dinitrogen was the main product. When the anaerobic cultures were switched back to aerobic respiration, denitrification of the cells stopped at once, although sufficient nitrite reductase was still present. We could observe that the mRNA levels for the individual denitrification enzymes decreased slightly to their aerobic, uninduced levels. The nitrite reductase protein was not actively degraded during the aerobic period.     

Three classes of Escherichia coli mutants selected for aerobic expression of fumarate reductase

Fumarate reductase (encoded by frd) and succinate dehydrogenase (encoded by sdh) of Escherichia coli are both known to catalyze the interconversion of fumarate and succinate. Fumarate reductase, however, is not inducible aerobically and therefore cannot participate in the dehydrogenation of succinate. Three classes of suppressor mutants, classified as frd oxygen-resistant [frd(Oxr)], constitutive [frd(Con)], and gene amplification [frd(Amp)] mutants, were selected from an sdh strain as pseudorevertants that regained the partial ability to grow aerobically on succinate. All contained increased aerobic levels of fumarate reductase activity. In frd(Oxr) mutants expression of the operon showed increased resistance to aerobic repression. Under anaerobic conditions expression of the operon became less dependent on the fnr+ gene product, a pleiotropic activator protein for genes encoding anaerobic respiratory enzymes. Exogenous fumarate, however, was still required for full induction, and repression by nitrate was undiminished. Thus, aerobic repression and anaerobic nitrate repression appear to involve separate mechanisms. In frd(Con) mutants expression of the operon became highly resistant to aerobic repression. Under anaerobic conditions expression of the operon no longer required the fnr+ gene product or exogenous fumarate and became immune to nitrate repression. In partial diploids bearing an frd(Oxr) or an frd(Con) allele and phi(frd+-lac) there was no mutual regulatory influence between the two genetic loci. Thus, the frd mutations act in cis and hence are probably in the promoter region. In frd(Amp) mutants the frd locus was amplified without significant alteration in the pattern of regulation.     

Nitrate reductases inEscherichia coli

Escherichia coli expresses two different membrane-bound respiratory nitrate reductases, nitrate reductase A (NRA) and nitrate reductase Z (NRZ). In this review, we compare the genetic control, biochemical properties and regulation of these two closely related enzyme systems. The two enzymes are encoded by distinct operons located within two different loci on theE. coli chromosome. ThenarGHJI operon, encoding nitrate reductaseA, is located in thechlC locus at 27 minutes, along with several functionally related genes:narK, encoding a nitrate/nitrite antiporter, and thenarXL operon, encoding a nitrate-activated, two component regulatory system. ThenarZYWV operon, encoding nitrate reductase Z, is located in thechlZ locus located at 32.5 minutes, a region which includes anarK homologue,narU, but no apparent homologue to thenarXL operon. The two membrane-bound enzymes have similar structures and biochemical properties and are capable of reducing nitrate using normal physiological substrates. The homology of the amino acid sequences of the peptides encoded by the two operons is extremely high but the intergenic regions share no related sequences. The expression of both thenarGHJI operon and thenarK gene are positively regulated by two transacting factors Fnr and NarL-Phosphate, activated respectively by anaerobiosis and nitrate, while thenarZYWV operon and thenarU gene are constitutively expressed. Nitrate reductase A, which accounts for 98% of the nitrate reductase activity when fully induced, is clearly the major respiratory nitrate reductase inE. coli while the physiological role of the constitutively expressed nitrate reductase Z remains to be defined.Abbreviations NR nitrate reductase On leave from Department of Biochemistry and Molecular Biology, The University of Texas Medical school at Houston, Houston, Texas, 77225, USA     

Ammonification in Bacillus subtilis Utilizing Dissimilatory Nitrite Reductase Is Dependent on resDE

During anaerobic nitrate respiration Bacillus subtilis reduces nitrate via nitrite to ammonia. No denitrification products were observed. B. subtilis wild-type cells and a nitrate reductase mutant grew anaerobically with nitrite as an electron acceptor. Oxygen-sensitive dissimilatory nitrite reductase activity was demonstrated in cell extracts prepared from both strains with benzyl viologen as an electron donor and nitrite as an electron acceptor. The anaerobic expression of the discovered nitrite reductase activity was dependent on the regulatory system encoded by resDE. Mutation of the gene encoding the regulatory Fnr had no negative effect on dissimilatory nitrite reductase formation.     

Denitrification by Actinomycetes and Purification of Dissimilatory Nitrite Reductase and Azurin from Streptomyces thioluteus

Many actinomycete strains are able to convert nitrate or nitrite to nitrous oxide (N₂O). As a representative of actinomycete denitrification systems, the system of Streptomyces thioluteus was investigated in detail. S. thioluteus attained distinct cell growth upon anaerobic incubation with nitrate or nitrite with concomitant and stoichiometric conversion of nitrate or nitrite to N₂O, suggesting that the denitrification acts as anaerobic respiration. Furthermore, a copper-containing, dissimilatory nitrite reductase (CuNir) and its physiological electron donor, azurin, were isolated. This is the first report to show that denitrification generally occurs among actinomycetes.     

Assimilation of [N]Nitrate and [N]Nitrite in Leaves of Five Plant Species under Light and Dark Conditions

Light dependency of nitrate and nitrite assimilation to reduced-N in leaves remains a controversial issue in the literature. With the objective of resolving this controversy, the light requirement for nitrate and nitrite assimilation was investigated in several plant species. Dark and light assimilation of [¹⁵N]nitrate and [¹⁵N]nitrite to ammonium and amino-N was determined with leaves of wheat, corn, soybean, sunflower, and tobacco. In dark aerobic conditions, assimilation of [¹⁵N]nitrate as a percentage of the light rate was 16 to 34% for wheat, 9 to 16% for tobacco, 26% for corn, 35 to 76% for soybean, and 55 to 63% for sunflower. In dark aerobic conditions, assimilation of [¹⁵N]nitrite as a percentage of the light rate was 11% for wheat, 7% for tobacco, 13% for corn, 28 to 36% for soybeans, and 12% for sunflower. It is concluded that variation among plant species in the light requirement for nitrate and nitrite assimilation explains some of the contradictory results in the literature, but additional explanations must be sought to fully resolve the controversy.

In dark anaerobic conditions, the assimilation of [¹⁵N]nitrate to ammonium and amino-N in leaves of wheat, corn, and soybean was 43 to 58% of the dark aerobic rate while dark anaerobic assimilation of [¹⁵N]nitrite for the same species was 31 to 41% of the dark aerobic rate. In contrast, accumulation of nitrite in leaves of the same species in the dark was 2.5-to 20-fold higher under anaerobic than aerobic conditions. Therefore, dark assimilation of nitrite cannot alone account for the absence of nitrite accumulation in the in vivo nitrate reductase assay under aerobic conditions. Oxygen apparently inhibits nitrate reduction in the dark even in leaves of plant species that exhibit a relatively high dark rate of [¹⁵N]nitrite assimilation.

     

Parallel Pathways for Nitrite Reduction during Anaerobic Growth in Thermus thermophilus

Respiratory reduction of nitrate and nitrite is encoded in Thermus thermophilus by the respective transferable gene clusters. Nitrate is reduced by a heterotetrameric nitrate reductase (Nar) encoded along transporters and regulatory signal transduction systems within the nitrate respiration conjugative element (NCE). The nitrite respiration cluster (nic) encodes homologues of nitrite reductase (Nir) and nitric oxide reductase (Nor). The expression and role of the nirSJM genes in nitrite respiration were analyzed. The three genes are expressed from two promoters, one (nirSp) producing a tricistronic mRNA under aerobic and anaerobic conditions and the other (nirJp) producing a bicistronic mRNA only under conditions of anoxia plus a nitrogen oxide. As for its nitrite reductase homologues, NirS is expressed in the periplasm, has a covalently bound heme c, and conserves the heme d₁ binding pocket. NirJ is a cytoplasmic protein likely required for heme d₁ synthesis and NirS maturation. NirM is a soluble periplasmic homologue of cytochrome c₅₅₂. Mutants defective in nirS show normal anaerobic growth with nitrite and nitrate, supporting the existence of an alternative Nir in the cells. Gene knockout analysis of different candidate genes did not allow us to identify this alternative Nir protein but revealed the requirement for Nar in NirS-dependent and NirS-independent nitrite reduction. As the likely role for Nar in the process is in electron transport through its additional cytochrome c periplasmic subunit (NarC), we concluded all the Nir activity takes place in the periplasm by parallel pathways.     

Light and Dark Controls of Nitrate Reduction in Wheat (Triticum aestivum L.) Protoplasts

Protoplasts were isolated from the leaves of nitrate-cultured wheat (Triticum aestivum L. var. Frederick) seedlings. When incubated in the dark, protoplasts accumulated nitrite under anaerobic, but not under aerobic, conditions. The assimilation of [¹⁵N]nitrite by protoplasts was strictly light-dependent, and no loss of nitrite from the assay medium was observed under dark aerobic conditions. Therefore, the absence of nitrite accumulation under dark aerobic conditions was the result of an O₂ inhibition of nitrate reduction and not a stimulation of nitrite reduction. In the presence of antimycin A, protoplasts accumulated nitrite under dark aerobic conditions. The oxygen inhibition of nitrate reduction was apparently due to a competition between nitrate reduction and dark respiration for cytoplasmic-reducing equivalents.