Incorporation of monofluorotryptophans into protein during the growth of Escherichia coli

Browne, Douglas T.; Kenyon, George L.; Hegeman, George D.

doi:10.1016/0006-291x(70)90750-3

Cited by 46 publications

(26 citation statements)

References 6 publications

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“…2). The cessation of logarithmic growth of HR15 in Trp resembled the behavior of Escherichia coli in 4-FTrp (22,23 (Fig. 3).…”

Section: Growthmentioning

confidence: 99%

Membership mutation of the genetic code: loss of fitness by tryptophan.

Wong¹

1983

Proc. Natl. Acad. Sci. U.S.A.

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BaciUus subtilis strain QB928, a tryptophanauxotroph, was serially mutated to yield strain HR15. For QB928, tryptophan functioned as a competent amino acid and 4-fluorotryptophan as merely an inferior analogue. For HR15, these roles were reversed. The tryptophan/4-fluorotryptophan growth ratio decreased by a factor of 2 x 104 in the transition from QB928 to HR15.The genetic code distributes 64 triplet codons to 20 amino acids and three termination signals for the construction of protein molecules. In the evolution of the code, membership in the ensemble of encoded amino acids may undergo addition, deletion, or replacement; without changing membership, codons also may be redistributed among the amino acids. Purely codonic changes are implicated in the departure of mitochondrial codes from the universal, or mainstream, code (1-3) and occur to a limited extent in missense and nonsense suppressions (4, 5).Whether or not any membership change has ever occurred in the course of biological evolution is more difficult to decide. Constituents of polysaccharides and lipids are varied. Even with nucleic acids, the use of hydroxymethyl-dCTP as substrate for T4 bacteriophage DNA synthesis at least exemplifies a variation. However, prokaryotic, eukaryotic, mitochondrial (6), and chloroplast (7) proteins uniformly utilize the same 20 proteinous amino acids. Thus there is no evidence from extant organisms that the encoded amino acids have ever changed during three billion years of biological evolution, in spite of the selective advantage to be gained from increased variety in amino acid side chains, as witnessed by as many as 120 kinds of posttranslational modifications of proteins (8), and the availability of over 200 kinds of novel amino acids from cellular metabolism (9) for entry into the code. The invariance is also largely not due to protection by the specificity of aminoacyl-tRNA synthetases; these enzymes distinguish among the 20 incumbent amino acids with extreme fidelity but they permit the incorporation of numerous amino acid analogues into proteins either partially or, in the case of selenomethionine (10) and trifluoroleucine (11), completely in place of an incumbent amino acid. Instead, what appears to be the most powerful barrier stabilizing the code has been the continual optimization of protein sequences on the basis of the incumbent amino acids, such that the replacement of any of them nowadays by an analogue typically brings a steep decline in biological fitness, as measured by cell growth (12,13). This fitness edge has to be not only removed but also reversed in order to achieve a membership change in the code.The coevolution theory of the genetic code proposes that the development of the universal code was inseparably linked to that of pathways for amino acid biosynthesis (14-18). New amino acids formed by primitive catalytic pathways received part or all of the codons of precursor amino acids through pretranslational modification of, or competition against, the precursors. This explains the entry o...

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“…2). The cessation of logarithmic growth of HR15 in Trp resembled the behavior of Escherichia coli in 4-FTrp (22,23 (Fig. 3).…”

Section: Growthmentioning

confidence: 99%

Membership mutation of the genetic code: loss of fitness by tryptophan.

Wong¹

1983

Proc. Natl. Acad. Sci. U.S.A.

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“…2). However, while E. coli tolerates 4fW, it cannot use 4fW as the sole source of tryptophan (5,29). Attempts to grow C600p in higher proportions of 4fW (Ͼ99% 4fW) inevitably resulted in cell death after one or two serial transfers.…”

Section: Resultsmentioning

confidence: 99%

“…Given that the evolved strain could grow in the presence of 99.97% 4fW while the starting strain could not, the evolved strain had likely accumulated mutations that altered proteins adversely affected by 4fW incorporation. For example, previous experiments with proteins involved in lactose utilization revealed that 4fW had deleterious effects on a number of enzyme activities (5,29). In order to identify what types of mutations contributed to the ability to grow on 4fW, a number of genes from clones B7-3 and B7-4 were isolated and compared with the same genes from the parental strain.…”

Section: Figmentioning

confidence: 99%

“…For example, although 4-fluorotryptophan (4fW) is eventually toxic to cells, bacteria can grow for several generations in its presence and proteins that contain high levels of this analogue can be isolated (5,29). Similarly, p-Cl-phenylalanine has been shown to replace phenylalanine in vivo, but it drastically decreases luciferase activity (14).…”

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confidence: 99%

“…Such attempts to develop unnatural organisms should enhance our understanding of the extent to which even the most basic biochemistry is evolvable or optimal and may suggest metabolic alternatives for engineered organisms. In order to explore these possibilities, we attempted to evolve Escherichia coli variants which could survive on 4fW, which is otherwise toxic (5,29).…”

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Selection and Characterization of Escherichia coli Variants Capable of Growth on an Otherwise Toxic Tryptophan Analogue

2001

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Escherichia coli isolates that were tolerant of incorporation of high proportions of 4-fluorotryptophan were evolved by serial growth. The resultant strain still preferred tryptophan for growth but showed improved growth relative to the parental strain on other tryptophan analogues. Evolved clones fully substituted fluorotryptophan for tryptophan in their proteomes within the limits of mass spectral and amino acid analyses. Of the genes sequenced, many genes were found to be unaltered in the evolved strain; however, three genes encoding enzymes involved in tryptophan uptake and utilization were altered: the aromatic amino acid permease (aroP) and tryptophanyl-tRNA synthetase (trpS) contained several amino acid substitutions, and the tyrosine repressor (tyrR) had a nonsense mutation. While kinetic analysis of the tryptophanyl-tRNA synthetase suggests discrimination against 4-fluorotryptophan, an analysis of the incorporation and growth patterns of the evolved bacteria suggest that other mutations also aid in the adaptation to the tryptophan analogue. These results suggest that the incorporation of unnatural amino acids into organismal proteomes may be possible but that extensive evolution may be required to reoptimize proteins and metabolism to accommodate such analogues.Cellular growth in the presence of unnatural amino acids often results in at least the partial incorporation of the analogues into proteins. For example, although 4-fluorotryptophan (4fW) is eventually toxic to cells, bacteria can grow for several generations in its presence and proteins that contain high levels of this analogue can be isolated (5, 29). Similarly, p-Cl-phenylalanine has been shown to replace phenylalanine in vivo, but it drastically decreases luciferase activity (14). Azaleucine can also be partially incorporated in place of arginine and has been shown to suppress an otherwise lethal mutation in thymidylate synthase (20). It has recently been shown that a tRNA synthetase with compromised editing function will allow the replacement of valine with cysteine, threonine, or ␣-aminobutyrate (9). Similarly, a tyrosyl-tRNA synthetase mutant has been isolated which discriminates against azatyrosine less readily than the wild type (13). All of these examples demonstrate that the partial substitution of one amino acid for another is possible.Current methods for unnatural amino acid incorporation in vivo involve either the site-specific incorporation of an unnatural amino acid into a few proteins (11) or the partial incorporation into many proteins. In contrast, we and Wong (37) have attempted to incorporate unnatural amino acids throughout an organismal proteome. Such attempts to develop unnatural organisms should enhance our understanding of the extent to which even the most basic biochemistry is evolvable or optimal and may suggest metabolic alternatives for engineered organisms. In order to explore these possibilities, we attempted to evolve Escherichia coli variants which could survive on 4fW, which is otherwise toxic (5, 29).Evolutiona...

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The Use of Microorganisms in the Study of Fluorinated Compounds

Goldman

1972

Ciba Foundation Symposium 2 ‐ Carbon‐Fluorine Compounds; Chemistry, Biochemistry and Biological Activities

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Incorporation of monofluorotryptophans into protein during the growth of Escherichia coli

Cited by 46 publications

References 6 publications

Membership mutation of the genetic code: loss of fitness by tryptophan.

Membership mutation of the genetic code: loss of fitness by tryptophan.

Selection and Characterization of Escherichia coli Variants Capable of Growth on an Otherwise Toxic Tryptophan Analogue

The Use of Microorganisms in the Study of Fluorinated Compounds

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