Effect of growth conditions on the synthesis of nitrate reductase components in chlorate resistant mutants of Escherichia coli K 12.

The chlorate resistant mutants of $\text{Escherichia coli}$ synthetize, in variable quantities, proteins which give immunocomplex with specific nitrate reductase antiserum. The biosynthesis of these cross reacting materials presents the same type of regulation as nitrate reductase of the wild type. C.R.M. biosynthesis is repressed by oxygen and even in the presence of nitrate, the oxygen inhibition is not reversed with chlorate mutants and wild type. With anaerobically grown cells, nitrate acts as an inducer and increases the amount of antibody-precipitable material, three times in mutants and even four times with Chl-E.     

Nitrate reductase and the membrane composition of pleiotropic chlorate resistant mutants of Escherichia coli K-12.

Attachment of rat hepatocytes to collagen but not to fibronectin substrata was efficiently inhibited by antibodies against the hepatocyte surface. Further analyses of this inhibition suggested that hepatocyte attachment to collagen involves cell surface antigens which are not identical to membrane bound fibronectin or collagen.Rabbit antibodies against rat fibronectin inhibited hepatocyte attachment to rat fibronectin but not to collagen or rabbit fibronectin. After plasmin digestion of fibronectin, peptides were isolated that lacked affinity for collagen but could serve as a substratum for hepatocyte attachment. These results suggested that attachment to fibronectin does not involve membrane bound fibronectin or collagen.     

Altered dihydrofolate reductase in fol regulatroy mutants of Escherichia coli K12.

Summary Culture of the thymine-dependent mutant of Rhizobium trifolii T37 was synchronized with phenethanol. During bacterial synchronous growth with synchronized replication of DNA, cells were differentially labeled using subsequently ³H-thymidine of low and high specific activity. The grain tracks produced in autoradiographs of chromosomes were denser on both ends than in the middle. In control experiments with bacteria labeled only with ³H-thymidine of low specific activity, the grain density was uniform throughout the grain track. The results constitute clear evidence of bidirectional replication of R. trifolli chromosome.     

Kinetic analysis of respiratory nitrate reductase from Escherichia coli K12

Purified respiratory nitrate reductase from Escherichia coli is able to use either reduced viologen dyes or quinols as the electron donor and nitrate, chlorate, or bromate as the electron acceptor. When reduced viologen dyes act as the electron donor, the enzyme follows a compulsory-order, "Theorell-Chance" mechanism, in which it is an enzyme-nitrate complex that is reduced rather than the free enzyme. In contrast, if quinols are used as the electron donor, then the enzyme operates by a two-site, enzyme-substitution mechanism. Partial proteolysis of the cytochrome b containing holoenzyme by trypsin results in loss of cytochrome b and in cleavage of one of the enzyme's subunits. The cytochrome-free derivative exhibits a viologen dye dependent activity that is indistinguishable from that of the holoenzyme, but it is incapable of catalyzing the quinol-dependent reaction. The quinol-dependent, but not the viologen dye dependent, activity is inhibited irreversibly by exposure to diethyl pyrocarbonate and reversibly by treatment with 2-n-heptyl-4-hydroxyquinoline N-oxide. We conclude that the holoenzyme has two independent and spatially distinct active sites, one for quinol oxidation and the other for nitrate reduction.     

Electron-paramagnetic-resonance studies on the molybdenum of nitrate reductase from Escherichia coli K12.

Studies on the respiratory nitrate reductase (EC 1.7.99.4) from Escherichia coli K12 by electron-paramagnetic-resonance spectroscopy indicate that its molybdenum centre is comparable with that in other molybdenum-containing enzymes. Two Mo(V) signals may be observed; one shows interaction of Mo(V) with a proton exchangeable with the solvent and has: A (1H) 0.9-1.2mT; g1 = 1.999; g2=1.985; g3 = 1.964; gav. = 1.983. Molybdenum of both signal-giving species may be reduced with dithionite and reoxidized with nitrate.     

Complementation in vitro between guaB mutants of Escherichia coli K12

Guanine auxotrophs of Escherichia coli were isolated following mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine or ethyl methanesulphonate. The mutants were classified according to growth properties and absence of IMP dehydrogenase or GMP synthetase activity. Mutations in guaB (IMP dehydrogenase-less) were analysed by reversion and suppression tests; all were of the base substitution missense type except for one possible frameshift and one polar nonsense mutation. GuaB mutants were examined for protein (CRM) that cross-reacts with monospecific antibodies to IMP dehydrogenase; approximately half were CRM+. Enzyme complementation in vitro was detected in mixed denatured and renatured cell-free extracts of any CRM+ guaB mutant and PL1138 (guaB105, CRM+); CRM- mutants did not complement. GuaB105 maps distal to all other guaB mutations except guaB86 (CRM-). Two hybrid enzymes produced by complementation were less stable to heat than native IMP dehydrogenase, although kinetic constants were similar. These observations indicate interallelic complementation between guaB mutants and are consistent with the demonstration of identical subunits for IMP dehydrogenase (Gilbert et al., 1979). Only the subunits supplied by PL1138 are catalytically active in the hybrid enzymes suggesting that this mutant may produce a repairable polypeptide whereas the enzymes of complementing mutants may be defective at the active site.     

Synthesis of nitrate reductase components in chlorate-resistant mutants of Escherichia coli.

Specific antibody to purified nitrate reductase from Escherichia coli was used to identify enzyme components present in mutants which lack functional nitrate reductase. chlA and B mutants contained all three subunits present in the wild-type enzyme. Different peptides with a broad range of molecular weights could be precipitated from chlCmutants, and chlE mutants contained either slightly degraded enzyme subunits or no precipitable protein. No mutants produced significant amounts of cytoplasmic enzyme. The chlA and B loci are suggested to function in the synthesis and attachment of a molybdenum-containing factor. The chlC locus is suggested to be the structural gene for nitrate reductase subunit A and chlE is suggested to be involved in the synthesis of the cytochrome b1 apoprotein.     

Phenotypic restoration by molybdate of nitrate reductase activity in chlD mutants of Escherichia coli

ChlD mutants of Escherichia coli are pleiotropic, lacking formate-nitrate reductase activity as well as formate-hydrogenlyase activity. Whole-chain formate-nitrate reductase activity, assayed with formate as the electron donor and measuring the amount of nitrite produced, was restored to wild-type levels in the mutants by addition of 10(-4)m molybdate to the growth medium. Under these conditions, the activity of each of the components of the membrane-bound nitrate reductase chain increased after molybdate supplementation. In the absence of nitrate, the activities of the formate-hydrogenlyase system were also restored by molybdate. Strains deleted for the chlD gene responded in a similar way to molybdate supplementation. The concentration of molybdenum in the chlD mutant cells did not differ significantly from that in the wild-type cells at either low or high concentrations of molybdate in the medium. However, the distribution of molybdenum between the soluble protein and membrane fractions differed significantly from wild type. We conclude that the chlD gene product cannot be a structural component of the formate-hydrogenlyase pathway or the formate-nitrate reductase pathway, but that it must have an indirect role in processing molybdate to a form necessary for both electron transport systems.     

Enzymic properties of a mutant of Escherichia coli K 12 lacking nitrate reductase

Summary A pleiotropic mutant of Escherichia coli K ₁₂ lacking reduced NAD: nitrate oxidoreductase, soluble formate dehydrogenase and membrane-bound formate:ferricytochrome b₁ oxidoreductase is described. Levels of several other enzymes and cytochromes have been measured and found to differ little from those normally present in the wild type with the exceptions of cytochrome c₅₂₂, reduced NAD:cytochrome c oxidoreductase and reduced NAD:nitrite oxidoreductase which are very high. Although the affected gene maps in a different position from that reported for chl A by other workers it seems likely that the two loci are identical.     

Antibiotic resistant mutants of Escherichia coli K12 show increases in heterologous gene expression

Using a variety of antibiotics, it was found that nine separate isolates of spontaneous antibiotic resistant mutants of Escherichia coli K12 pPSX-vioABCDE overproduce the anti-tumour antibiotic violacein. Subsequent analysis showed that seven of these mutations occurred on the plasmid pPSX-vioABCDE. The other two overproducing strains carried spontaneous chromosomal mutations to lincomycin and kanamycin. The kanamycin resistant mutant of E. coli K12 DH10B (AA23) and a lincomycin resistant mutant of E. coli K12 LE392 (AA24) increased the synthesis of violacein. The plasmid pPSX-vioABCDE opv-1 contains a violacein over-production (opv-1) mutation which when introduced into either E. coli K12 AA23 or AA24, resulted in a hyper-production of violacein. Remarkably, E. coli K12 AA23 pPSX-vioABCDE opv-1 produced 41 times the normal level of violacein. In addition, both E. coli K12 AA23 and E. coli K12 AA24 demonstrated an increase in expression of an alpha amylase gene from Streptomyces lividans and the urease gene cluster from Klebsiella oxytoca. These results suggest that selection of antibiotic resistant mutants can increase heterologous gene expression in E. coli K12. Additionally, the increased expression is a general effect applicable to genes and gene clusters cloned into E. coli K12 from both Gram-positive and Gram-negative bacteria.     

Purification and further characterization of the second nitrate reductase of Escherichia coli K12

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr-230,000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.     

Properties of cysK mutants of Escherichia coli K12.

Triazole and azaserine resistant mutants of E. coli K12 affecting cysK gene coding for O-acetylserine sulphydrylase were isolated. The cysK gene in E. coli is located in the same region of chromosome as the cycK gene in Salmonella typhimurium. All azaserine and some triazole resistant mutants require cysteine for growth at a normal rate. The cysK mutants have reduced sulphate uptake. Stability and transfer by conjugation of triazole resistant phenotype were checked. Differences in sulphate metabolism between closely related organisms E. coli and S. typhimurium are discussed.