A mutation in Escherichia coli K-12 results in a requirement for thiamine and a decrease in L-serine deaminase activity

Newman, E. B.; Miller, B; Colebrook, Lawrence D.; Walker, C

doi:10.1128/jb.161.1.272-276.1985

Cited by 21 publications

(8 citation statements)

References 23 publications

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“…The plasmids used were pBR322 (3) and pBAD22 (10). Minimal medium, LB, and growth conditions were as previously described (31,34). Carbon sources were added at 0.2%.…”

Section: Methodsmentioning

confidence: 99%

Lack of S -Adenosylmethionine Results in a Cell Division Defect in Escherichia coli

Newman¹,

Budman²,

Chan

et al. 1998

J Bacteriol

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The enzyme S-adenosylmethionine (SAM) synthetase, theEscherichia coli metK gene product, produces SAM, the cell’s major methyl donor. We show here that SAM synthetase activity is induced by leucine and repressed by Lrp, the leucine-responsive regulatory protein. When SAM synthetase activity falls below a certain critical threshold, the cells produce long filaments with regularly distributed nucleoids. Expression of a plasmid-carried metKgene prevents filamentation and restores normal growth to themetK mutant. This indicates that lack of SAM results in a division defect.

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“…The plasmids used were pBR322 (3) and pBAD22 (10). Minimal medium, LB, and growth conditions were as previously described (31,34). Carbon sources were added at 0.2%.…”

Section: Methodsmentioning

confidence: 99%

Lack of S -Adenosylmethionine Results in a Cell Division Defect in Escherichia coli

Newman¹,

Budman²,

Chan

et al. 1998

J Bacteriol

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“…We report here that the enzyme prepared in glycylglycine is quite stable and is subject to an elaborate activating system which is described in this paper. This activating system appears to be defective in two mutants previously described as being defective in L-SD (23).…”

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confidence: 82%

“…Many factors which influence the synthesis of this activity are known (20). Mutants with more activity than the parent have been described previously (22), but other mutants showed very little activity in the permeabilized cell assay and were unable to deaminate L-serine in vivo (23).…”

mentioning

confidence: 96%

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In vitro and in vivo activation of L-serine deaminase in Escherichia coli K-12

Newman¹,

Dumont²,

Walker³

1985

J Bacteriol

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Escherichia coli L-serine deaminase (L-SD) in crude extracts made in glycylglycine could be activated by incubation with iron sulfate and dithiothreitol. This activation could also be demonstrated in vitro in two mutants which were physiologically deficient in L-SD activity in vivo. This suggests that these mutants were deficient not in L-SD but in an enzyme(s) activating L-SD. The suggestion is made that production of a functional L-SD in vivo requires activation of the structural gene product by an enzyme or enzymes that reduce the protein to an active form.

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“…A viable alternative to antibiotics is the use of auxotrophic selective markers which require a strain engineered to have a loss of function mutation in a key enzyme of an essential biosynthetic pathway. Examples of commonly used auxotrophic strains in industrial and academic labs include uracil, histidine, and tryptophan auxotrophs [18][19][20] . Two approaches have been taken to generate P. tricornutum auxotrophs.…”

Section: Introductionmentioning

confidence: 99%

Cas9-generated auxotrophs ofPhaeodactylum tricornutumare characterized by small and large deletions that can be complemented by plasmid-based genes

Slattery

Wang

Kocsis

et al. 2020

Preprint

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The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Editing events are characterized by loss of heterozygosity and by the occurrence of large deletions of up to ∼2.7-kb centred on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-FOA and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyl transferase activity in knockout strains. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains. pathways of P. tricornutum and show for the first time that plasmid-based copies of the intact PtUMPS and PtPRA-PH/CH genes can complement the uracil-and histidine-requiring phenotypes, respectively. Individual auxotroph strains are characterized by loss of heterozygosity at the edited alleles, and Nanopore sequencing of the edited population reveals large, heterogeneous deletions up to ∼ 2.7 kb. The uracil and histidine auxotrophs and their respective complementation markers are a potential alternative to antibiotic-based selection of plasmids in P. tricornutum. Our results also suggest a simple methodology to cure plasmids from uracil auxotrophs to enable strain and genome engineering.

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A mutation in Escherichia coli K-12 results in a requirement for thiamine and a decrease in L-serine deaminase activity

Cited by 21 publications

References 23 publications

Lack of S -Adenosylmethionine Results in a Cell Division Defect in Escherichia coli

Lack of S -Adenosylmethionine Results in a Cell Division Defect in Escherichia coli

In vitro and in vivo activation of L-serine deaminase in Escherichia coli K-12

Cas9-generated auxotrophs ofPhaeodactylum tricornutumare characterized by small and large deletions that can be complemented by plasmid-based genes

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