Localization of the replication origin of plasmid pE194

Dempsey, Laurie A; Dubnau, David

doi:10.1128/jb.171.5.2866-2869.1989

Cited by 16 publications

(9 citation statements)

References 14 publications

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“…In a control e~periment again using the MalE/PdxB protein no vitamins c}uld be detected. Thus, in agreement with former results [6], phosphoserine transaminase is a pyridoxal phosphate containi~'~g enzyme. It is striking that PLP appears to be involved in i s own biosynthesis.…”

Section: -O-phospho-l-serine :2-oxoglutarate Aminotransferasesupporting

confidence: 92%

4‐O‐Phosphoryl‐l‐threonine, a substrate of the pdxC(serC) gene product involved in vitamin B₆ biosynthesis

et al. 1996

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The Escherichia coli pdxC(serC) gene codes for a transaminase (EC 2.6.1.52). The gene is involved in both pyridoxine (vitamin B6) and serine biosynthesis and was overexpressed as a MalEIPdxC(SerC) fusion protein. The fusion protein was purified by aff'mity chromatography on an amylose resin and hydrolyzed in the presence of protease factor Xa. Both the fusion protein and the PdxC(SerC) protein were characteri~ed (KM value, turnover number, optimum pH). Both enzymes used 4-O-phosphoryl-L-threonine rather than 4-hydroxy-L-threonine as a substrate indicating that the phosphorylated rather than the non-phosphorylated amino acid is involved in pyridoxine biosynthesis. Pyridoxal phosphate was shown to be the cofactor for both enzymes and therefore seems to be involved in its own biosynthesis.

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Section: -O-phospho-l-serine :2-oxoglutarate Aminotransferasesupporting

confidence: 92%

4‐O‐Phosphoryl‐l‐threonine, a substrate of the pdxC(serC) gene product involved in vitamin B₆ biosynthesis

et al. 1996

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“…The culture was incubated at 37 °C, and then streaked onto LB plates that were subsequently incubated overnight at 51 °C52. The colonies cured of knockout plasmid were confirmed by streaking them onto LB plates containing tetracycline (20 mg/L); colonies cured of plasmid fail to grow at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system

Zhang

Duan

2016

Sci Rep

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Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 (ΔsrfC, ΔspoIIAC, ΔnprE, ΔaprE, ΔamyE), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain.

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“…Pspac-cre expression cassette (without lacI) was excised from pDGC with EcoRI/SphI and ligated into pTS, also cut with EcoRI/SphI, yielding plasmid pTSC. For the elimination of pTSC from B. subtilis, a transformant was patched on antibioticfree LB agar and incubated at 51°C (13): the resulting colonies all lost pTSC.…”

Section: Methodsmentioning

confidence: 99%

Cre/ lox System and PCR-Based Genome Engineering in Bacillus subtilis

Yan

Hong

et al. 2008

Appl Environ Microbiol

202

159

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We have developed a fast and accurate method to engineer the Bacillus subtilis genome that involves fusing by PCR two flanking homology regions with an antibiotic resistance gene cassette bordered by two mutant lox sites (lox71 and lox66). The resulting PCR products were used directly to transform B. subtilis, and then transient Cre recombinase expression in the transformants was used to recombine lox71 and lox66 into a double-mutant lox72 site, thereby excising the marker gene. The mutation process could also be accomplished in 2 days by using a strain containing a cre isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible expression cassette in the chromosome as the recipient or using the lox site-flanked cassette containing both the cre IPTG-inducible expression cassette and resistance marker. The in vivo recombination efficiencies of different lox pairs were compared; the lox72 site that remains in the chromosome after Cre recombination had a low affinity for Cre and did not interfere with subsequent rounds of Cre/lox mutagenesis. We used this method to inactivate a specific gene, to delete a long fragment, to realize the in-frame deletion of a target gene, to introduce a gene of interest, and to carry out multiple manipulations in the same background. Furthermore, it should also be applicable to large genome rearrangement.Bacillus subtilis is the best-characterized gram-positive bacterial organism: its biochemistry, physiology and genetics have been studied intensely for more than 50 years. B. subtilis and the closely related Bacillus species are nonpathogenic and free of endotoxins, and their fermentation technology has been well characterized, making them important cell factories for industrial enzymes, fine biochemicals, antibiotics, and insecticides (8, 31). The completion of the sequencing and annotation of the B. subtilis 168 genome supplies a complete view of the B. subtilis protein machinery, inspiring new approaches for analyzing biochemical pathways (23,25). Postgenomic studies require simple and highly efficient tools to enable genetic manipulation. Classically, these chromosomal modifications have been achieved by a method that uses a positive selection marker, usually an antibiotic resistance marker, which is generated by insertion of the marker gene into the chromosome. When introducing multiple modifications into the same background, it is better to evict the selection marker gene, usually through a single-crossover event. Selection of the strain that has lost marker gene is tedious without the aid of counterselectable markers that, under appropriate growth conditions, can promote the death of the microorganisms that harbor them. Until now, four counterselectable markers have been described (6,12,17,38) that improve the genetic manipulations of B. subtilis by allowing the subsequent excision of the selection marker, coupled with positive selection. The genetic manipulations described above, however, are mainly based on restriction enzyme and DNA ligase-dependent vector construction, which requi...

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Localization of the replication origin of plasmid pE194

Cited by 16 publications

References 14 publications

4‐O‐Phosphoryl‐l‐threonine, a substrate of the pdxC(serC) gene product involved in vitamin B₆ biosynthesis

4‐O‐Phosphoryl‐l‐threonine, a substrate of the pdxC(serC) gene product involved in vitamin B₆ biosynthesis

Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system

Cre/ lox System and PCR-Based Genome Engineering in Bacillus subtilis

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Localization of the replication origin of plasmid pE194

Cited by 16 publications

References 14 publications

4‐O‐Phosphoryl‐l‐threonine, a substrate of the pdxC(serC) gene product involved in vitamin B6 biosynthesis

4‐O‐Phosphoryl‐l‐threonine, a substrate of the pdxC(serC) gene product involved in vitamin B6 biosynthesis

Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system

Cre/ lox System and PCR-Based Genome Engineering in Bacillus subtilis

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Product

Resources

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4‐O‐Phosphoryl‐l‐threonine, a substrate of the pdxC(serC) gene product involved in vitamin B₆ biosynthesis

4‐O‐Phosphoryl‐l‐threonine, a substrate of the pdxC(serC) gene product involved in vitamin B₆ biosynthesis