A mutation affecting L-serine and energy metabolism in E. coli K12

Summary The effects of a pleiotropic mutation ssd are described. This mutation results in decreased efficiency in the use of glucose and fructose as carbon source, inability to use succinate or to grow anaerobically, an alteration in the activity of enzymes responsible for the synthesis and degradation of L-serine, increased resistance to certain antibiotics, and a deficiency in proline transport. This mutation resembles various previously described mutations throught to affect energy coupling factor and is located in the same region of the chromosome. While the gene product affected by this mutation is still unidentified, it is clear that L-serine metabolism cannot be understood merely in terms of providing L-serine and its derivatives.     

Membrane mutation affecting energy-linked functions inEscherichia coli K 12

A small-colony forming variant ofEscherichia coli with a mutation in thenof gene was analysed. The alternation of the protein composition in the cytoplasmic membrane and the interaction with K and E group colicina indicated a membrane mutation. The effect of this mutation on some membrane-bound processes, the activity of Mg²⁺-activated ATPase, the growth on different carbon sources and the active transport of amino acids, is described. This mutation does not exert any effect on the electron transport system.     

Factors influencing the in vivo stability of L-serine deaminase activity in E. coli K12.

L-Serine deaminase (L-SD) is unstable in intact cells of Escherichia coli K12. The extent of this instability is dependent on the nitrogen content of the medium in which the enzyme is synthesized, and on that in which it is tested. Enzyme activity in cells grown with an inorganic nitrogen source is unstable in the presence of inorganic nitrogen; enzyme activity in cells grown with an organic nitrogen source is unstable in the presence of the amino acids glycine and leucine.     

A relationship between L-serine degradation and methionine biosynthesis in Escherichia coli K12

While wild-type Escherichia coli K12 cannot grow with L-serine as carbon source, two types of mutants with altered methionine metabolism can. The first type, metJ mutants, in which the methionine biosynthetic enzymes are expressed constitutively, are able to grow with L-serine as carbon source. Furthermore, a plasmid carrying the metC gene confers ability to grow on L-serine. These observations suggest that in these mutants, L-serine deamination may be a result of a side-reaction of the metC gene product, cystathionine beta-lyase. The second type is exemplified by two newly isolated strains carrying mutations mapping between 89.6 and 90 min. These mutants use L-serine as carbon source, and also require methionine for growth with glucose at 37 degrees C and above. The phenotypes of the new mutants resemble those of both met and his constitutive mutants in some respects, but have been differentiated from both of them.     

Ter mutation and susceptibility to phi X174 phage in E. coli K12.

The Ter-15 mutant derived from E. coli K12 W2252-11U^? RC^str (wild type I) is found to be sensitive to φx174 phage infection. Lipopolysaccharide extracted from this mutant inactivates the phage, and has core oligosaccharides identical in amounts to those in the lipopolysaccharide from wild type cells.In contrast, the Ter-21 mutant derived from E. coli K12 W2252-11U^? RC^rel (wild type II) is not sensitive to this phage infection, and its lipopolysaccharide does not inactivate the phage. Its lipopolysaccharide sugars are found to be D-glucose and D-ribose, thus differing from the lipopolysaccharide sugars of the wild type cells.     

A role for EIIANtr in controlling fluxes in the central metabolism of E. coli K12

To investigate a possible role of the nitrogen-PTS (PTS^Ntr) in controlling carbon metabolism, we determined the growth of Escherichia coli LJ110 and of isogenic derivatives, mutated in components of the PTS^Ntr, on different carbon sources. The PTS^Ntr is a set of proteins homologous to the PEP-dependent phosphotransferase system (C-PTS) that transfers a phosphate group from PEP over EI^Ntr (encoded by ptsP) and NPr (encoded by ptsO) to EIIA^Ntr (encoded by ptsN). Strains deleted in ptsN were characterized by a high acetate production coupled to slow growth on glycolytic substrates. The ΔptsP and the ΔptsO strain showed the same behavior as the parent strain. As the phosphorylation level of EIIA^Ntr in these mutants differed significantly from that of the parent strain, phosphorylation of EIIA^Ntr obviously is not important for its function. During growth in minimal medium with defined carbon sources, EIIA^Ntr was always completely phosphorylated in LJ110. Significant amounts of dephosphorylated EIIA^Ntr were only visible in strains lacking EI^Ntr or NPr. mRNA expression studies on glucose revealed a downregulation of genes encoding TCA cycle enzymes when EIIA^Ntr was absent. ¹³C-flux analyses confirmed higher fluxes towards acetate and lower fluxes in the TCA cycle in the ptsN mutants but additionally hinted to a slightly but significantly increased flux through the pyruvate dehydrogenase complex (PDH). During growth on succinate the ΔptsN strain accumulated mutations in rpoS, while no rpoS mutants were observed for the ΔptsN-O strain. This hints to an additional function of NPr during growth with succinate.     

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.     

Increased mutation rate of E. coli K12 lambda cultures maintained in continuous logarithmic growth

Continuous logarithmic growth of E. coli K12 lambda in an automatic culture cell resulted in marked increases in the proportion of several mutants. The P1 phage-resistant cells increased 10 to 3000 times, the T2 phage-resistant cells 1 to 1000 times, the neomycin-resistant cells 1 to 10 times, and the virus-producing cells 30 to 70 times. No change occurred in the penicillin-resistant cells. Calculation of the growth curves and direct determination of the mutation rates by the null fraction method showed that the increases in the proportion of mutants were due to increases in the mutation rates.     

Influence of recC and uvrD on ultraviolet-induced mutation to valine resistance in E. coli K12

The production of mutants in E. coli exposed to ultraviolet light is initiated by photochemical reactions, and completed by metabolic processes controlled by recA and other genes. Ultraviolet-induced mutagenesis to valine resistance was measured in cells carrying recC, uvrD, or both recC and uvrD. The spontaneous and UV-induced mutagenesis was slightly greater in those carrying uvrD, as compared to recC or wild-type. At low doses, UV mutagenesis in the recC uvrD double mutant was greater than in either recC or wild-type, and was comparable to that in the uvrD strain, although this double mutant was very UV-sensitive and showed poor survival at doses above 2 J/m².     

Synthesis of paf-acether by E. coli K12

Paf-acether (platelet-activating factor) is one of the most potent mediator of inflammation released from and acting on most cells that participate in inflammatory diseases. Its molecular structure is 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine. Two metabolic steps are involved in its biosynthesis: the action of a phospholipase A2 on choline-containing membrane alkyl-ether lipids results in the production of lyso paf-acether and acetylation of the lyso compound by an acetyltransferase yields the biologically active molecule. Membrane alkyl-ether lipids can therefore be considered as potential precursors of paf-acether and their composition has been studied in various cell types. In this work, we investigated the presence of paf-acether in E. coli. Our results showed that paf-acether can be obtained from E. coli K12 under a variety of bacterial growth conditions. Paf-acether from E. coli exhibited the same physicochemical and biological characteristics as synthetic paf-acether and that from eucaryotic cells. Therefore, it appears that E. coli itself has the ability of producing paf-acether, a result that could be of some importance with respect to the pathogenesis of Enterobacteria and the use of E. coli in the recombinant DNA technology.     

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.     

Trans-dominant mutation affecting restriction and modification in Escherichia coli K-12

Summary We have analysed the mechanism of action of a ts mutation in E. coli, which has an effect on the expression of the restriction and modification phenotype. The frequencies of recombinants obtained in transduction experiments support the idea that the temperature sensitive mutation is located outside the hsd operon in the gene denoted hsd. X. Complementation experiments demonstrated the trans-dominant nature of the temperature sensitive mutation. The possible role of the hsd.X product in the formation of EcoR.K and EcoM.K complexes and their interaction with the recognition site on the DNA is discussed.     

Mutation fruB in the fructose regulon affecting beta-galactosidase synthesis and adenylate cyclase activity of E. coli K12

Escherichia coli mutant devoid of fructosespecific factor III (factor IIIfru) of phosphoenolpyruvate (PEP) carbohydrate phosphotransferase system was isolated. The mutation fruB was localized on 46 min of chromosomal map of Escherichia coli in the fru-operon region. The mutant bacteria are unable to accumulate fructose. PEP-dependent phosphorylation of this carbohydrate in cellular extracts was considerably decreased. The mutational damage of factor IIIfru results in the suppression of beta-galactosidase synthesis. The impaired synthesis of the enzyme was partially resistant to glucose catabolite repression. However, the deficiency was suppressed by addition of exogenous cyclic AMP. The adenylatecyclase activity in fruB mutant was found to be 50% lower as compared with the one in the parent strain.     

A thermosensitivepdxJ mutation affecting vitamin B6 biosynthesis inEscherichia coli K-12

A temperature-sensitive mutation affecting pyridoxine biosynthesis inEscherichia coli has been isolated following nitrosoguanidine mutagenesis. This is the first report of a conditional mutation affecting that pathway. Three-point transductional analysis and an Hfr mating test indicate that the affected locus ispdxJ and that the gene order isglyA-purI-pdxJ-nadB. The data confirm the present genetic map position ofpdxJ, which had not been accurately determined. Cells carrying the mutation exhibit an absolute requirement for 2.5 μM vitamin B₆ when inenbated at temperatures of 38°C or higher.