Inhibition of protein biosynthesis in Escherichia coli B by tri-L-ornithine.
TL;DR: The treatment of E. coli B cells with triornithine impaired the protein biosynthetic system of the treated cells in vitro, and the biological activity of the ribosomal fraction is impaired within the bacterial cell.
Abstract: 1
Triornithine completely inhibited growth of Escherichia coli B in the minimal liquid medium M63. The di-, tetra- and pentapeptides showed no effect on the growth of the bacterium.
2
Triornithine, when added to a bacterial culture, inhibited the incorporation of 14C-labeled amino acids into the protein fraction, but did not suppress the incorporation of [3H]uridine and [3H]thymidine.
3
The tripeptide inhibited translation of the induced β-galactosidase mRNA and also inhibited the synthesis of alkaline phosphatase initiated by derepression.
4
Triornithine had no effect on the reaction in vitro of T4-DNA-directed RNA synthesis.
5
Incubation of E. coli B cells with triornithine impaired the protein biosynthetic system of the treated cells in vitro. By this treatment the biological activity of the ribosomal fraction is impaired within the bacterial cell.
6
Triornithine, even at a high concentration, did not inhibit in vitro the polypeptide synthesis in a cell-free system.
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TL;DR: It is hoped that studies on the structural requirements of microbial peptide permeases and peptidases, and of the effects of peptides on microbial growth, may be useful not only in their immediate context, but they may also act as a model system to provide information relevant to peptide interactions in more complex biological systems of free amino acids.
Abstract: Publisher Summary This chapter focuses on the current assessment of peptide utilization by microorganisms from the standpoints of cleavage and transport. It also lays emphasis on the effects of peptides on other aspects of microbial physiology. It is clear that prior to their nutritional utilization peptides must be hydrolyzed to yield free amino acids, and that this cleavage may occur before or after absorption. Variations in these two essential features of cleavage and transport produce circumstances in which particular peptides may give a growth response that is greater than, equal to, or less than an equivalent mixture. Simple peptides offer a vast reservoir of biologically active compounds and techniques exist for the synthesis of any desired sequence. It is hoped that studies on the structural requirements of microbial peptide permeases and peptidases, and of the effects of peptides on microbial growth, may be useful not only in their immediate context, but they may also act as a model system to provide information relevant to peptide interactions in more complex biological systems of free amino acids.
82 citations
TL;DR: A direct study of peptide uptake by E. coli showed that peptidase-resistant substrates, e.g. triornithine and glycylsarcosine, which can be similarly estimated in cell extracts, were accumulated about 1,000-fold.
Abstract: A direct study of peptide uptake by Escherichia coli was made using a fluorescent procedure. After incubation with the bacteria, peptides remaining in the medium were dansylated, separated chromatographically, and quantitated from their fluorescent intensities and/or from their incorporated radioactivity when tritiated dansyl derivatives were prepared. Peptide uptake was apparently not regulated and proceeded continuously until complete, with the absorbed peptides undergoing rapid intracellular hydrolysis and the excess amino acid residues leaving the cell. Thus, peptide uptake and amino acid exodus occur concurrently. However, peptidase-resistant substrates, e.g. triornithine and glycylsarcosine, which can be similarly estimated in cell extracts, were accumulated about 1,000-fold. The influence of amino acid composition and chain length on rates of transport was assessed. Different strains of E. coli showed variability in their rates of di- and oligopeptide transport. With respect to energy coupling, both the di- and oligopeptide permeases behaved like shock-sensitive transport systems.
60 citations
TL;DR: Triornithine-resistant (TOR) mutants were isolated from several strains of Escherichia coli K-12 and found to be oligopeptide transport-deficient (Opp-), finding the opp gene is the closest marker to the trp operon.
53 citations
TL;DR: Mutants harboring defects in both peptide transport loci defined in this study would not grow on nutritional peptides except for tri-L-methionine, were totally resistant to toxic peptides, and would not actively transport L-[U-14C]alanyl-L alanyl-lanyl- L-alanine.
Abstract: The composition of the outer membrane channels formed by the OmpF and OmpC porins is important in peptide permeation, and elimination of these proteins from the Escherichia coli outer membrane results in a cell in which the primary means for peptide permeation through this cell structure has been lost. E. coli peptide transport mutants which harbor defects in genes other than the ompF/ompC genes have been isolated on the basis of their resistance to toxic tripeptides. The genetic defects carried by these oligopeptide permease-negative (Opp-) strains were found to map in two distinct chromosomal locations. One opp locus was trp linked and mapped to the interval between att phi 80 and galU. Complementation studies with F'123 opp derivatives indicated that this peptide transport locus resembles that characterized in Salmonella typhimurium as a tetracistronic operon (B. G. Hogarth and C. F. Higgins, J. Bacteriol. 153:1548-1551, 1983). The second opp locus, which we have designated oppE, was mapped to the interval between dnaC and hsd at 98.5 min on the E. coli chromosome. The differences in peptide utilization, sensitivity and resistance to toxic peptides, and the L-[U-14C]alanyl-L-alanyl-L-alanine transport properties observed with these Opp-E. coli strains demonstrated that the transport systems encoded by the trp-linked opp genes and by the oppE gene(s) have different substrate preferences. Mutants harboring defects in both peptide transport loci defined in this study would not grow on nutritional peptides except for tri-L-methionine, were totally resistant to toxic peptides, and would not actively transport L-[U-14C]alanyl-L-alanyl-L-alanine.
46 citations
Cites background from "Inhibition of protein biosynthesis ..."
...Mutants with defects in the general transport system have most commonly been isolated by resistance to the toxic tripeptide, tri-L-ornithine (Orn3), (7, 14)....
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...Orn3 is an inhibitor of protein synthesis (7, 14), Ala-Ala-Ala-P and L-alanyl-aminoethylphosphonic acid (AlaAla-P) are inhibitors of D-alanine racemase (1-3), and leucine-...
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TL;DR: It is concluded that trimethionine is recognized by the trileucine-trithreonine transport system, as they all relieve triornithine toxicity towards E. coli W and compete with tetralysine utilization as lysine source for growth of aLysine auxotroph of this strain.
Abstract: Trileucine is utilized as a source of leucine for growth of strains of Escherichia coli K-12 that are deficient in the oligopeptide transport system (Opp). Trithreonine is toxic to E. coli K-12. Opp- mutants of E. coli K-12 retain complete sensitivity to this tripeptide. Moreover, E. coli W, which is resistant to trithreonine, can utlize this tripeptide as a threonine source and this capability is fully maintained in E. coli W (Opp-). A spontaneous trithreonine-resistant mutant of E. coli K-12 (Opp-) has been isolated that has an impaired growth response to trileucine and is resistant to trithreonine. Trileucine competes with the uptake of trithreonine as measured by its ability to relieve trithreonine toxicity in E. coli K-12. It is concluded that trileucine as well as trithreonine are transported into E. coli K-12 or W by a common uptake system that is distinct from the Opp system. Trimethionine can act as a competitor of trileucine or trithreonine-supported growth and as an antagonist of trithreonine toxicity in Opp- mutants. It is concluded that trimethionine is recognized by the trileucine-trithreonine transport system. Trithreonine, trimethionine, and trileucine are also transported by the Opp system, as they all relieve triornithine toxicity towards E. coli W and compete with tetralysine utilization as lysine source for growth of a lysine auxotroph of this strain.
36 citations
References
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TL;DR: A stable cell-free system has been obtained from E. coli which incorporates C14-valine into protein at a rapid rate as mentioned in this paper, which was shown that this apparent protein synthesis was energy-dependent, was stimulated by a mixture of L-amino acids, and was markedly inhibited by RNAase, puromycin, and chloramphenicol.
Abstract: A stable cell-free system has been obtained from E. coli which incorporates C14-valine into protein at a rapid rate. It was shown that this apparent protein synthesis was energy-dependent, was stimulated by a mixture of L-amino acids, and was markedly inhibited by RNAase, puromycin, and chloramphenicol.1 The present communication describes a novel characteristic of the system, that is, a requirement for template RNA, needed for amino acid incorporation even in the presence of soluble RNA and ribosomes. It will also be shown that the amino acid incorporation stimulated by the addition of template RNA has many properties expected of de novo protein synthesis. Naturally occurring RNA as well as a synthetic polynucleotide were active in this system. The synthetic polynucleotide appears to contain the code for the synthesis of a “protein” containing only one amino acid. Part of these data have been presented in preliminary reports.2, 3
1,304 citations
TL;DR: The study of galactosidase synthesis in heteromerozygotes of E. coli indicates that the z and i mutations belong to different cistrons, and the kinetics of expression of the i + (inducible) character suggest that the i gene controls the synthesis of a specific substance which represses the synthesis.
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TL;DR: It has been shown that alkaline phosphatase in measurable amount is only formed when Pi becomes limiting in the medium, at which point the enzyme is formed in substantial amount at a maximum rate.
754 citations