Proline Auxotroph Research Articles

Spontaneous deletion formation in several classes of Escherichia coli mutants deficient in recombination ability

The frequency of spontaneous chromosomal deletions in 2 separate regions of the Escherichia coli chromosome has been measured in rec +. recA, recC, and rep strains. When spontaneous T7 r mutants were selected from cultures of these strains, 5–50% of the T7 r mutants were found to have become proline auxotrophs as well. Although the proportion of T7 r-Pro − deletions among T7 r mutants and the rate of T7 r formation varied according to the strain line, the state of the rec or rep alleles did not affect either of these properties within a given strain line. The rate of colB r-colV r deletion formation was also found to be independent of the state of the rec alleles and dependent upon the strain line. Thus, the products of the recA, recB, and recC loci, which are necessary for efficient recombination, and the product of the rep locus, whose function is unknown, are not required for the production of chromosomal deletions.

Proline uptake by disrupted membrane preparations from Escherichia coli

Isolated membrane preparations from E. coli W6, a proline auxotroph, continue to catalyze energy-dependent proline uptake after disruption by sonication or passage through a French pressure cell. Membranes from E. coli W157, a mutant deficient in proline uptake and exchange capacity, do not catalyze proline uptake before or after disruption. The proline uptake by the sonicated W6 preparations is not due to undisrupted membranes and the proline taken up is loosely associated with a particulate, sedimentable fraction. This uptake system is inhibited by the same metabolic inhibitors that inhibit proline uptake by intact membranes, has the same temperature optimum as intact membranes, and displays similar responses to osmolarity changes as intact membranes. Furthermore, in electron micrographs, sonicated membranes appear to be identical to intact membranes. With the exception of size, both preparations consist of grossly intact vesicles. Finally, it is demonstrated by light scattering techniques that sonicated membrane vesicles respond to changes in the concentrations of either sucrose or potassium phosphate in the same manner as undisrupted membranes.

Genetical analysis of proline mutants and their suppressors in Aspergillus nidulans.

1.Complementation between thirteen proline auxotrophs inA. nidulanswas studied. Two groups of mutants with different complementation pattern were found. These two groups could also be distinguished on the basis of recombination tests.2. The spontaneous reversion rate of proline mutants was established. In all cases studied the reversions were due to suppressor mutations. Dominant, semi-dominant and recessive suppressors were distinguished.3. Complementation between recessive suppressors was studied. Only a few of the suppressors obtained could be located in different complementation groups.4. Three suppressor loci were mapped, two of them,su-2andsu-6in chromosome III linked to thephen-2locus, respectively 22 and 26 map units distant, and the third in chromosome I, linked to thead-9locus (1·9 map units).su-2is a mutant at theSu-4 prolocus already identified by Forbes (1956).5. The action of these suppressors is thought to consist in affecting the pathway of arginine synthesis by one of three mechanisms: (1) accumulation of an inter mediate (ornithine); (2) increased activity of ornithine δ-transaminase; and (3) a third, as yet, unclear process possibly involving feed-back regulation of arginine synthesis or the regulation of arginine breakdown to ornithine.

Observations on the Conversion of N-Acetylglutamate to Proline in Extracts of Escherichia coli

Abstract An extract of Escherichia coli was found capable of synthesizing proline from N-acetylglutamate. This synthesis was shown to proceed by transformation of the 5 carbon skeleton of acetylglutamate into ornithine by way of the familiar reactions of arginine biosynthesis. This ornithine then undergoes a transamination of its δ-amino group with α-ketoglutarate, yielding Δ'-pyrroline-5-carboxylic acid. The synthesis of the latter compound from acetylglutamate requires both an adenosine triphosphate- and a reduced triphosphopyridine nucleotide-generating system and is stimulated by l-glutamate. Evidence is presented, however, that the synthesis of proline in vivo does not proceed by this pathway. (a) Exogenous acetylglutamate is incorporated preferentially into arginine, rather than into proline, when compared with exogenous glutamate. (b) Cells grown in the presence of arginine yield extracts devoid of activity for synthesizing proline from acetylglutamate, yet such cells do not require exogenous proline for growth. (c) A proline auxotroph, strain 55–25, blocked between glutamate and Δ'-pyrroline-5-carboxylic acid does not appear to be blocked either between glutamate and ornithine or between ornithine and Δ'-pyrroline-5-carboxylic acid. (d) Proline does not inhibit the transamination of ornithine to yield Δ'-pyrroline-5-carboxylic acid.

Proline metabolism in Escherichia coli: III. The proline catabolic pathway

Proline was oxidized by a proline auxotroph to glutamate via the intermediate, Δ 1-pyrroline-5-carboxylate (PCA). The reactions, like those in animal tissues, involve the successive action of an oxidase and a pyridine nucleotide dehydrogenase. Proline oxidase and the bioxynthetic PCA reductase were unrelated, as shown by imposing a second mutational loss in the auxotroph which removed PCA reductase but not proline oxidase activity.

PROLINE METABOLISM IN ESCHERICHIA COLI. II. REGULATION OF TOTAL GROWTH OF A PROLINE AUXOTROPH BY A PROLINE-OXIDIZING SYSTEM.

Frank, Leonard (The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Md.). Proline metabolism in Escherichia coli. II. Regulation of total growth of a proline auxotroph by a proline-oxidizing system. J. Bacteriol. 86:781-784. 1963.-With a simple kinetic model, it was shown that a proline-requiring mutant of Escherichia coli was subject to growth limitation on proline by a proline-oxidizing system active in the growing cells.

Amino acid starvation in an Escherichia coli auxotroph : III. Incorporation of 32P into ribonucleic acid and other cell components

During amino acid starvation in leucine and proline auxotrophs of E. coli, although net protein and RNA synthesis have ceased, there is continuing incorporation of 32P into RNA nucleotides. The turnover rate approximates 10% per hour initially and is nearly equal in the debris fraction, the ribosomes, and the soluble supernatant. Both early and late in starvation and in each centrifugal fraction the specific radioactivities of the four RNA nucleotides are approximately equal. These findings are discussed with reference to the interrelation of RNA and protein synthesis and the cessation of both during amino acid starvation.