Regulation of the nitrate reductase operon: Effect of mutations in chl A, B, D and E genes

Summary Introduction of chlA, B or E mutant alleles into strains carrying fusions between the lac structural genes and the promoter of the nitrate reductase operon led to the partial or total constitutive expression of the fusion. Presence of chlD mutated alleles in the same strains did not result in constitutive expression of the fusion and allowed full induction by nitrate only in the presence of molybdenum. It is proposed that the molybdenum cofactor, Mo-X, of the nitrate reductase is also corepressor of the operon. The chlA, B and E genes would be involved in the biosynthesis of the X-moity. Mutations in these genes would give an altered X-moity which still binds to molybdenum but leads to a less effcient repressor complex; chlD gene would code for an enzyme inserting molybdenum in the X-moity of the cofactor. Mutations in chlD give an empty cofactor leading to a complex which permanently represses the operon unless molybdenum is added.     

Regulation of the trimethylamine N-oxide (TMAO) reductase in Escherichia coli: analysis of tor::Mud1 operon fusion

Summary Mud1 insertion mutants of Escherichia coli were obtained in which the lac structural genes were fused to the promoter of torA, a gene encoding the trimethylamine N-oxide (TMAO) reductase. Expression of the fusion is induced by TMAO and repressed by oxygen. However, in contrast to the nar operon which codes for the nitrate reductase structural genes, the tor::Mud1 fusion was found to be independent of the positive control exerted by the nirR gene product and not repressed by the molybdenum cofactor. The torA gene which is strongly linked to pyrF (28.3 U) is different from any tor gene already described in E. coli or in Salmonella typhimurium.     

Reversion and immobilization of phage Mud1 cts (Apr lac) insertion mutations in Salmonella typhimurium

Summary Phage Mud1 cts (Ap^r lac), or Mud1, insertion mutations may be accompanied by adjacent deletion formation which can complicate use of lac fusions generated with this phage for gene regulatory studies. As for phage Mu insertion mutations, phage Mud1 insertions fail to revert at significant frequency (whether or not accompanied by an adjacent deletion). We describe isolation of revertible (X mutant) derivatives of phage Mud1 in Salmonella typhimurium. The X mutant derivatives allow use of reversion as a simple test to determine whether a Mud1 insertion has occurred precisely without an adjacent deletion that may have fused the lac genes to a promoter outside of the gene of interest. In addition, a simple method for stabilizing Mud1 generated lac fusions against subsequent transposition is described.     

Operon fusions in the nitrate reductase operon and study of the control gene nir R in Escherichia coli

Summary Strains carrying operon fusions between the promotor of the chl I gene and the lac structural genes were constructed. From these strains in which the expression of the lac genes is under the control of both nitrate and oxygen, spontaneous regulatory mutants were selected: (i) mutants which synthesize -galactosidase constitutively in anaerobiosis; (ii) mutants in which -galactosidase synthesis is no longer repressed by oxygen.Introduction of the nir R mutated allele into strains carrying these fusions resulted in the total loss of -galactosidase synthesis, confirming that nir R is a regulatory gene controlling the expression of the biosynthesis of the nitrate reductase.     

Damage to DNA induces expression of the ruv gene of Escherichia coli

Summary Regulation of the ruv gene of E. coli was studied using phage Mud (Ap lac) to obtain a fusion of the lac genes to the ruv promoter. -galactosidase synthesis in the ruv-lac fusion strain was induced by mitomycin C and other agents that damage DNA. The induction of -galactosidase could be altered by mutations either in lexA or recA from which it is concluded that ruv is regulated by lexA repressor. A possible function of ruv in promoting cell recovery following damage to DNA is discussed.     

Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli

The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl₂, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over-expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.     

Use of lac operon fusions to isolate Escherichia coli mutants with altered expression of the fumarate reductase system in response to substrate and respiratory controls.

Strains of $\text{E}\text{.}\text{coli}$ with fusions between the $\text{lac}$ structural genes and the promoter region of the fumarate reductase system were constructed from a parental strain deleted in the native $\text{lac}$ operon. Like fumarate reductase in wild-type cells, β-galactosidase in these fusion strains is inducible by fumarate, but only under anaerobic conditions. From one of these strains, three classes of mutants altered in the expression of the hybrid operon were isolated. By anaerobic selection for growth on lactose in the absence of fumarate, mutants that synthesize β-galactosidase constitutively both aerobically and anaerobically were obtained. By aerobic selection for growth on lactose in the presence of fumarate, mutants that are inducible in the enzyme both aerobically and anaerobically and mutants that are inducible in the enzyme only aerobically were obtained. The regulatory behaviors of the mutants studied suggest that substrate and respiratory control of the expression of the fumarate reductase complex are mechanistically connected.     

Alteration by mutation of the control by oxygen of the nar operon in Escherichia coli

Summary A nar-lac operon fusion was used to isolate a mutant in which the expression of the nar operon was no longer repressed by oxygen. The nar ^d mutation, located upstream of the nar structural genes, was found to be cis dominant; it led to independence from the Fnr protein which, in the wild-type strain, exerts a strict positive control on the nar operon. Both other known controls, nitrate induction and autoregulation, were unaffected. It is proposed that molecular oxygen controls the expression of nar via Fnr and that the nar ^d mutation affects the Fnr binding site of the narGHI control region.     

Nitrate reductase and cytochrome bnitrate reductase structural genes as parts of the nitrate reductase operon

Summary The existence of a nitrate-reductase operon in the tryptophane region was deduced from the effects of prophage insertion in each of chl I and chl C genes and from transposition of the Mu-mediated host DNA fragments on F-prime. This operon appears to be polarized from chlC to chlI and the gene order in the region is trp-chlI-chlC-purB.     

Interaction between the nitrate respiratory system of Escherichia coli K12 and the nitrogen fixation genes of Klebsiella pneumoniae.

Hybrids were constructed between $\text{E. coli}$ K12 $\text{chl}$^? mutants defective in nitrate respiration and an F′ plasmid carrying nitrogen fixation genes from $\text{K. pneumoniae}$. Examination of these hybrids showed that expression of $\text{nif}$_Kp⁺ genes does not require a functional nitrate respiratory system, but that nitrate reductase and nitrogenase do share some Mo-processing functions. For nitrate repression of nitrogenase activity, reduction of nitrate to nitrite is not necessary, but the Mo-X cofactor encoded by $\text{chl}$ genes is essential. Nitrate probably inhibits nitrogen fixation by affecting the membrane relationship of the nitrate and fumarate reduction systems such that the membrane cannot be energized for nitrogenase activity.     

Mapping of chlorate-resistant mutants of Citrobacter freundii by deletion and complementation analysis

Summary From Citrobacter freundii mutants have been isolated, with deletions extending chl genes. 53% of these mutants mapped in the gal-bio region of the chromosome. Genetic mapping by three methods—deletion analysis, linkage analysis in crosses with C. freundii Hfr donors and complementation with Escherichia coli F factors—establishes the gene order: chl H-gal-chl D-hut-bio-uvr B-chl A-chl I-chl E In an other segment a gene order ilv-chl-pdx was found. Furthermore chl loci were found adjacent to 7 different chromosomal markers. C. freundii can form nitrate reductase A, thiosulfate reductase, tetrathionate reductase and formic dehydrogenase. These enzymes are not formed in most of the chlorate-resistant mutants. In some of these mutants the enzyme activities can be restored by complementation with F factors of E. coli.     

Catabolite-repressor-like protein regulates the expression of a gene under the control of the Escherichia coli lac promoter in the plant pathogen Xanthomonas campestris pv. campestris

  Xanthomonas campestris pv. campestris, the causal agent of black-rot disease of cruciferous plants, and an important industrial microbe, was able to express the Escherichia coliβ-glucuronidase reporter gene (uidA) when fused to the E. coli lactose operon promoter on a wide-host-range plasmid vector. The gene fusion is expressed constitutively at high levels in both complex and defined media using a wide range of carbon sources, and is not repressible by glucose or inducible by the gratuitous lac inducer isopropyl β-d-thiogalactoside. An X. campestris campestris strain with a lesion in the clp (catabolite-repressor-like protein) locus, and containing the plac/uidA fusion, was tested for β-glucuronidase activity. We found that the expression of the plac/uidA fusion gene is dependent on the presence of catabolite-repressor-like protein, with an approximately 75% reduction of expression in the clp -deficient mutant. Received: 1 April 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996     

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     

glnF-lacZ fusions in Escherichia coli: studies on glnF expression and its chromosomal orientation

Summary The regulatory gene, glnF, of Escherichia coli was fused to the structural genes of the lac operon by use of the hybrid Mu phage derivative Mudl (Ap lac). Analysis of two of these fusions showed that the glnF gene is expressed constitutively, i.e., independent of either the nitrogen source in the growth medium or the availability of the glnA, glnL, glnG or glnF functional gene products. The orientation of the Mud1 (Ap lac) insertions was determined by chromosome mobilization in F-merogenotes carrying either of the two glnF:: Mud1 chromosomal insertions isolated, and either one of a pair of F'lacZ:: Mucts62 episomes; the two episomes differing in that their Mucts62 insertions are located in opposite orientations with regard to lacZ. The direction of chromosome mobilization by the Hfrs that were probably formed via Mu homology demonstrated that orientation of the glnF gene is clockwise relative to that of the chromosome.     

A genetical and biochemical study of chlorate-resistant mutants ofSalmonella typhimurium

S.typhimurium can form nitrate reductase A, chlorate reductase C, thiosulfate reductase, tetrathionate reductase and formic dehydrogenase. None of these enzymes are formed in chlorate-resistant mutants. Conjugation experiments showed the presence of a strong linkage between thechl andgal markers of the bacterial chromosome. By deletion mapping the gene ordernic A aro G gal bio chl D uvr B chl A was found. Strains with deletions terminating betweenbio anduvr B or betweenuvr B andchl A have a number of aberrant properties. Though resistant against chlorate they reduce nitrate and form gas. After growth with nitrate they form less nitrate reductase than the wild type which may explain the resistance against chlorate. After growth with thiosulfate they form small amounts of thiosulfate reductase and chlorate reductase C. In crosses between anE.coli Hfrchl ⁺ strain and aS.typhimurium chl A strain recombinants were obtained, forming nitrate reductase A and chlorate reductase C. These recombinants do not form gas, which indicates that thechl ⁺ gene fromE.coli does not function normally inS.typhimurium.The author is very gratefull to Miss C. W. Bettenhaussen, Miss W. M. C. Kapteijn and Mr. K. Pietersma for technical assistance. Helpfull suggestions of Dr. P. van de Putte (Medical Biological Laboratory of the National Defence Organization TNO, Rijswijk) are gratefully acknowledged.     

A genetic assay for transversion mutations in bacteriophage T4

Summary Seventy-two mutants deficient in formate-nitrate reductase activity were selected in Escherichia coli strain PK 27, by two different procedures. Forty-five strains were selected on the basis of chlorate resistance and 27 strains were selected by their inability to reduce nitrate with formate as an electron donor. Genetic analysis of these strains showed that the two techniques yield distinctly different distributions of mutants among the various controlling genetic loci. Chlorate resistance appears to select for severe alterations in the nitrate reductase system; 98% of these mutants fell into the pleiotropic chl A, B, D and E classes and are deficient in all the activities of the formate-hydrogenlyase pathway as well as formate-nitrate reductase pathway. In contrast, 48% of the mutants selected for their inability to reduce nitrate with formate as the electron donor were of the chl C class and two new classes were identified among mutants selected by this procedure. Chl F mutants are linked to tryptophan and the chl C locus. Chl G mutants map at zero minutes on the E. coli genetic map.     

The narA Locus of Synechococcus sp. Strain PCC 7942 Consists of a Cluster of Molybdopterin Biosynthesis Genes

The narA locus required for nitrate reduction in Synechococcus sp. strain PCC 7942 is shown to consist of a cluster of genes, namely, moeA, moaC, moaD, moaE, and moaA, involved in molybdenum cofactor biosynthesis. The product of the moaC gene of strain PCC 7942 shows homology in its N-terminal half to MoaC from Escherichia coli and in its C-terminal half to MoaB or Mog. Overexpression of the Synechococcus moaC gene in E. coli resulted in the synthesis of a polypeptide of 36 kDa, a size that would conform to a protein resembling a fusion of the MoaC and MoaB or Mog polypeptides of E. coli. Insertional inactivation of the moeA, moaC, moaE, and moaA genes showed that the moeA-moa gene cluster is required for growth on nitrate and expression of nitrate reductase activity in strain PCC 7942. The moaCDEA genes constitute an operon which is transcribed divergently from the moeA gene. Expression of the moeA gene and the moa operon was little affected by the nitrogen source present in the culture medium.     

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.     

Regulation of ferric iron transport in Escherichia coli K12: Isolation of a constitutive mutant

Summary The lac genes were inserted with phage Mu(Ap, lac) into the fhuA, fepA, cir and tonB genes which specify components of iron uptake systems. The expression of lac in all these operon fusions was controlled by the availability of iron to the cells, thereby facilitating a quick and simple measurement of the expression of the genes listed above. In an iron rich medium under anaerobic conditions all systems were strongly repressed. fhuA was depressed at higher iron concentration than was fepA or cir, and tonB was repressed only under anaerobic conditions and could be induced by iron limitation.Mutants constitutive for the expression of -galactosidase were selected in a fhuA-lac fusion strain. The outer membrane proteins Cir, FhuA, FecA, 76K and 83K were made constitutively in such mutant strains. Therefore, they were termed fur mutants. In these fur mutant strains, the synthesis of a 19K protein was reduced. Furthermore, it was found that transport of ferric enterochelin and ferrichrome was also constitutive in the fur mutant cells, and that ferric citrate uptake could be induced by only 10 M citrate in the growth medium in contrast to wild-type cells in which at least 100 M citrate was necessary. The fepA gene was concluded to be under an additional control, because it was not fully derepressed by the fur mutation.