A novel L-serine deaminase activity in Escherichia coli K-12.

We demonstrate here that Escherichia coli K-12 synthesizes two different L-serine deaminases (L-SD) catalyzing the nonoxidative deamination of L-serine to pyruvate, one coded for by the previously described sdaA gene and a second, hitherto undescribed enzyme which we call L-SD2. A strain carrying a null mutation in sdaA made no detectable L-SD in minimal medium, but had activity in Luria broth. We describe a mutation, sdaX, which affects the regulation of L-SD2 and permits its expression in minimal medium, and an insertion mutation, sdaB, which abolishes L-SD2 activity completely. Both mutations lie near 60.5 min on the E. coli genetic map. The two L-SD enzymes have similar enzyme parameters, and both require posttranslational activation.     

Studies on L-serine deaminase in Escherichia coli K-12

l-Serine deaminase has been studied in toluene-treated cells of Escherichia coli K-12 and shown to be a discrete entity distinct from l-threonine deaminase. Its level in the cell varies as a function of nitrogen nutrition, carbon source, and amino acids (glycine and leucine). The metabolic role of the enzyme remains unclear but may be related to serine toxicity. The enzyme is unstable within the cell in the presence of its inducers, glycine and leucine, but not in their absence.     

A mutation in Escherichia coli K-12 results in a requirement for thiamine and a decrease in L-serine deaminase activity

Mutants of Escherichia coli K-12 deficient in L-serine deaminase (L-SD) activity have been isolated. These strains required thiamine and grew normally when it was provided. The decrease in L-SD activity caused no obvious metabolic deficiency. A study of revertants and transductants showed that a single mutation was responsible for the thiamine requirement and for the decrease in L-SD activity.     

In vitro and in vivo activation of L-serine deaminase in Escherichia coli K-12.

Escherichia coli L-serine deaminase (L-SD) in crude extracts made in glycylglycine could be activated by incubation with iron sulfate and dithiothreitol. This activation could also be demonstrated in vitro in two mutants which were physiologically deficient in L-SD activity in vivo. This suggests that these mutants were deficient not in L-SD but in an enzyme(s) activating L-SD. The suggestion is made that production of a functional L-SD in vivo requires activation of the structural gene product by an enzyme or enzymes that reduce the protein to an active form.     

Two proline porters in Escherichia coli K-12

Escherichia coli mutants defective at putP and putA lack proline transport via proline porter I and proline dehydrogenase activity, respectively. They retain a proline uptake system (proline porter II) that is induced during tryptophan-limited growth and are sensitive to the toxic L-proline analog, 3,4-dehydroproline. 3,4-Dehydroproline-resistant mutants derived from a putP putA mutant lack proline porter II. Auxotrophic derivatives derived from putP+ or putP bacteria can grow if provided with proline at low concentration (25 microM); those derived from the 3,4-dehydroproline-resistant mutants require high proline for growth (2.5 mM). We conclude that E. coli, like Salmonella typhimurium, possesses a second proline porter that is inactivated by mutations at the proP locus.     

A possible mechanism for the in vitro activation of L-serine deaminase activity in Escherichia coli K12

L-Serine deaminase is inactive in crude extracts of Escherichia coli K12, but can be activated by incubation with iron and dithiothreitol. This activation requires oxygen, and is inhibited by free radical scavengers and by diethylene triamine pentaacetic acid, which prevents Fe cycling. We suggest that in vitro activation of L-serine deaminase is catalyzed by an oxidant (perhaps hydroxyl radicals). Also, activation may be accompanied by a decrease in molecular weight and involve both a cleavage of the polypeptide chain and a reversible reduction of the molecule.     

Escherichia coli K-12 mutant forming a temperature-sensitive D-serine deaminase.

A single-site mutant of Escherichia coli K-12 able to grow in minimal medium in the presence of D-serine at 30 C but not at 42 C was isolated. The mutant forms a D-serine deaminase that is much more sensitive to thermal denaturation in vitro at temperatures above but not below 47 C than that of the wild type. No detectable enzyme is formed by the mutant at 42 C, however, and very little is formed at 37 C. The mutant enzyme is probably more sensitive to intracellular inactivation at high temperatures than the wild-type enzyme. The mutation lies in the dsdA region. The mutant also contains a dsdO mutation, which does not permit hyperinduction of D-serine deaminase synthesis.     

rorA mutation of Escherichia coli K-12 affects the recB subunit of exonuclease V.

The ATP-dependent nuclease, exonuclease V, of Escherichia coli plays an important role in repair and recombination. The enzyme is composed of two subunits, one of which is the product of the recB and recC genes. In this communication it is shown by mapping and complementation experiments that the rorA mutation, which results in radiation sensitivity but not the loss of recombination ability, is an allele of the recB gene.     

Dual autogenous regulatory role of threonine deaminase in Escherichia coli K-12.

Summary We describe the regulatory properties of two strains carrying either the ilvA624 or the ilvA625 mutations, located in the structural gene for threonine deaminase. Crude extracts of both these strains possess a threonine deaminase activity migrating on polyacrylamide gels, differently from the wild type enzyme. Growth studies demonstrate that these mutations do not cause a limitation of isoleucine biosynthesis, suggesting normal catalytic activity of deaminase.A regulatory consequence of the ilvA624 allele is a derepression of the isoleucine-valine biosynthetic enzymes, which is recessive to an ilvA ⁺ allele. The ilvA625 mutation causes a derepression which is dominant in an ilvA625/ilvA ⁺ diploid. We interpret these data assuming that threonine deaminase, previously shown to be an autogenous regulator of the ilv genes, lacks a repressor function in the ilvA624 mutant, while in the ilvA625 mutant it is a better activator than wild type threonine deaminase.The data are discussed in terms of a model requiring that threonine deaminase, or a precursor of it, is in equilibrium between two forms, one being an activator of gene expression and the other being a repressor.     

L-Serine deaminase activity is induced by exposure of Escherichia coli K-12 to DNA-damaging agents.

The synthesis of L-serine deaminase in Escherichia coli K-12 was induced after exposure of cells to a variety of DNA-damaging agents, including UV irradiation, nalidixic acid, and mitomycin C. Synthesis was also induced during growth at high temperature. A mutant constitutive for SOS functions showed an elevated level of L-serine deaminase activity. The response to DNA-damaging agents thus may be mediated via the SOS system.     

Identification of a mutation affecting an alanine-alpha-ketoisovalerate transaminase activity in Escherichia coli K-12.

Summary A mutation affecting alanine--ketoisovalerate transaminase activity has been shown to be cotransducibe with the ilv gene cluster. The transaminase deficiency results in conditional isoleucine auxotrophy in the presence of alanine.     

Acetohydroxy acid synthase activity from a mutation at ilvF in Escherichia coli K-12.

Examination of the ilvF locus at 54 min on the Escherichia coli K-12 chromosome revealed that it is a cryptic gene for expression of a valine-resistant acetohydroxy acid synthase (acetolactate synthase; EC 4.1.3.18) distinct from previously reported isozymes. A spontaneous mutation, ilvF663, yielded IlvF+ enzyme activity that was multivalently repressed by all three branched-chain amino acids, was completely insensitive to feedback inhibition, was highly stable at elevated temperatures, and expressed optimal activity at 50 degrees C. The IlvF+ enzyme activity was expressed in strains in which isozyme II was inactive because of the ilvG frameshift in the wild-type strain K-12 and isozymes I and III were inactivated by point mutations or deletions. Tn5 insertional mutagenesis yielded two IlvF- mutants, with the insertion in ilvF663 in each case. These observations suggest that the ilvF663 locus may be a coding region for a unique acetohydroxy acid synthase activity.     

L-serine degradation in Escherichia coli K-12: cloning and sequencing of the sdaA gene.

A new mutant of Escherichia coli K-12 unable to grow with L-serine, glycine, and L-leucine has been isolated by lambda plac Mu insertion and shown to be deficient in L-serine deaminase activity. The corresponding gene, sdaA, has been cloned from a prototrophic strain, and the clone has been characterized and sequenced. The evidence is consistent with the hypothesis that sdaA is the structural gene for L-serine deaminase. However, other possibilities are also considered. No significant homology with previously reported DNA or protein sequences was detected.     

formation for phenylethylamine oxidase expressed in Escherichia coli K-12

Abstract Arthrobacter globiformis amine oxidase produced by Escherichia coli cells grown in copper-depleted media was reported to undergo activation due to formation of its topaquinone cofactor in a copper-dependent autocatalytic reaction. Likewise, a mutated E. coli amine oxidase located in the cytoplasm was reported to form topaquinone autocatalytically in an EDTA-sensitive reaction. Here we show unequivocally that formation of an amine oxidase lacking topaquinone is primarily a consequence of the location of the enzyme in the cytoplasm rather than the level of copper in the growth medium. For E. coli , insertion of copper into apoamine oxidase and subsequent topaquinone formation occur after export of the apoenzyme into the periplasm.     

L-serine degradation in Escherichia coli K-12: a combination of L-serine, glycine, and leucine used as a source of carbon.

Escherichia coli K-12 strain CU1008 cannot use L-serine as the sole carbon source, but it could use L-serine as an auxiliary carbon source with glucose, L-alanine, or pyruvate and could derive energy from L-serine to support oxygen uptake. CU1008 grew with L-serine if it was also provided with glycine and leucine. These may act by increasing the available activity of L-serine deaminase; other explanations are also explored.     

Chromosomal mutation for citrate utilization by Escherichia coli K-12.

A mutant strain of Escherichia coli K-12 that utilizes citrate as a sole source of carbon and energy was isolated. Citrate utilization arose as the consequence of two mutations in genes citA and citB, which are linked to the gal operon. The mutant strain expresses a semiconstitutive citrate transport system, and it utilizes both citrate and isocitrate as carbon and energy sources. It is capable of utilizing cis- and trans-aconitate, but only if it is preinduced by growth on citrate.     

Map location of the ssd mutation in Escherichia coli K-12.

A pleiotropic mutation at the ssd locus was mapped at 86 min near rha. A mutation at the ssd locus resulted in elevated L-serine deaminase activity, inability to grow with succinate as the carbon source, and inability to grow anaerobic conditions.