Coumermycin A1: a preferential inhibitor of replicative DNA synthesis in Escherichia coli. I. In vivo characterization

Biochemistry ◽  
1976 ◽  
Vol 15 (17) ◽  
pp. 3769-3777 ◽  
Author(s):  
Michael J. Ryan
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
In Vivo Characterization

scholarly journals In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Fabien Le Chevalier ◽  
Isabelle Correia ◽  
Lucrèce Matheron ◽  
Morgan Babin ◽  
Mireille Moutiez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biological Activities ◽  
Gene Clusters ◽  
Chemical Diversity ◽  
E Coli ◽  
Chemical Structures ◽  
In Vivo Characterization ◽  
Culture Supernatants

Abstract Background Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing Cα-Cβ dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines. Results We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23. Conclusions Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.

scholarly journals Escherichia coli DNA polymerase II catalyzes chromosomal and episomal DNA synthesis in vivo

1997 ◽  
Vol 94 (3) ◽  
pp. 946-951 ◽  
Author(s):  
S. Rangarajan ◽  
G. Gudmundsson ◽  
Z. Qiu ◽  
P. L. Foster ◽  
M. F. Goodman
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerase Ii ◽  
Episomal Dna

scholarly journals In vivo characterization of the dTDP-d-desosamine pathway of the megalomicin gene cluster from Micromonospora megalomicea

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 667-673 ◽  
Author(s):  
Eduardo Rodríguez ◽  
Salvador Peirú ◽  
John R. Carney ◽  
Hugo Gramajo
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Ms Analysis ◽  
In Vivo Characterization

In vivo reconstitution of the dTDP-d-desosamine pathway of the megalomicin gene cluster from Micromonospora megalomicea was achieved by expression of the genes in Escherichia coli. LC/MS/MS analysis of the dTDP-sugar intermediates produced by operons containing different sets of genes showed that production of dTDP-d-desosamine from dtdp-4-keto-6-deoxy-d-glucose requires only four biosynthetic steps, catalysed by MegCIV, MegCV, MegDII and MegDIII, and that MegCII is not involved. Instead, bioconversion studies demonstrated that MegCII is needed together with MegCIII to catalyse transfer of d-desosamine to 3-α-mycarosylerythronolide B.

DNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light

1987 ◽  
Vol 162 (2) ◽  
pp. 305-309
Author(s):  
Gaspar BANFALVI ◽  
Viera SLEZARIKOVA ◽  
Milena SEDLIAKOVA ◽  
Ferenc ANTONI
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Ultraviolet Light

scholarly journals DNA polymerase II of Escherichia coli in the bypass of abasic sites in vivo.

Genetics ◽  
1994 ◽  
Vol 136 (2) ◽  
pp. 439-448 ◽  
Author(s):  
I Tessman ◽  
M A Kennedy
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Quantitative Difference ◽  
Spontaneous Mutations ◽  
Pol Ii ◽  
Abasic Sites ◽  
Dna Polymerase Ii

Abstract The function of DNA polymerase II of Escherichia coli is an old question. Any phenotypic character that Pol II may confer upon the cell has escaped detection since the polymerase was discovered 24 yr ago. Although it has been shown that Pol II enables DNA synthesis to proceed past abasic sites in vitro, no role is known for it in the bypass of those lesions in vivo. From a study of phage S13 single-stranded DNA, we now report SOS conditions under which Pol II is needed for DNA synthesis to proceed past abasic sites with 100% efficiency in vivo. Overproduction of the GroES+L+ heat shock proteins, which are members of a ubiquitous family of molecular chaperones, eliminated this requirement for Pol II, which may explain why the role of Pol II in SOS repair had eluded discovery. Mutagenesis accompanied SOS bypass of abasic sites when the original occupant had been cytosine but not when it had been thymine; the quantitative difference is shown to imply that adenine was inserted opposite the abasic sites at least 99.7% of the time, which is an especially strict application of the A-rule. Most, but not all, spontaneous mutations from Rifs to Rifr, whether in a recA+ or a recA(Prtc) cell, require Pol II; while this suggests that cryptic abasic lesions are a likely source of spontaneous mutations, it also shows that such lesions cannot be the exclusive source.

Deoxyribonucleotide Synthesis in an Escherichia coli Mutant (H 1491) which Lacks Ribonucleotide Reductase Subunit B2

1989 ◽  
Vol 44 (7-8) ◽  
pp. 715-718 ◽  
Author(s):  
Jens Harder ◽  
Hartmut Follmann ◽  
Klaus Hantke
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Strictly Anaerobic ◽  
Anaerobic Conditions ◽  
Sensitive Mutant ◽  
E Coli ◽  
Key Enzyme ◽  
Minimal Media

An iron-sensitive mutant of E. coli with a Mudl phage insertion in the nrdB gene lacks subunit B2 of the key enzyme of DNA synthesis, ribonucleotide reductase. Nevertheless, these cells are capable of growing in minimal media under anaerobic conditions, indicating a second enzyme or pathway for deoxyribonucleotide synthesis. We here show that ribonucleotide reduction cannot be unambiguously measured in bacterial extracts whereas phosphorylase- catalyzed deoxyribosyl transfer does occur; however these salvage reactions could not function in vivo in the absence of deoxyribosides. It is suggested that the cells possess a specific, anaerobic ribonucleotide reductase which escapes detection under aerobic standard conditions, similar to the situation found in strictly anaerobic methanogens.

Inhibitors of DNA synthesis cause excessive DNA synthesis in dnaA mutants of Escherichia coli K12

1978 ◽  
Vol 24 (10) ◽  
pp. 1250-1252
Author(s):  
Jukka Heinonen ◽  
Marja-Leena Pekola
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Relative Rate ◽  
Cell Mass ◽  
Chromosomal Dna ◽  
E Coli ◽  
Escherichia Coli K12 ◽  
Low Concentrations ◽  
Dna Synthesis Inhibitors

The relative rate of net DNA synthesis was stimulated when cells of dnaA mutants of Escherichia coli K12 were grown in the presence of low concentrations of DNA synthesis inhibitors. This led to a supernormal DNA/cell mass ratio. The excessive DNA was similar to the normal chromosomal DNA in size and stability in vivo. However, the cells did not divide but turned into long filaments. Excessive DNA synthesis in the presence of inhibitors of DNA synthesis was observed in the cultures of two independent dnaA mutants of E. coli, but dnaB and dnaC mutants behaved like the wild type in this respect.

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