Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans

Jäger, Wolfgang; Schäfer, Andreas; Pühler, Alfred; Labes, G.; Wohlleben, Wolfgang

doi:10.1128/jb.174.16.5462-5465.1992

Cited by 159 publications

(83 citation statements)

References 16 publications

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“…This plasmid is mobilizable from E. coli to both Gram positive and Gram negative bacteria including Agrobacterium tumefaciens (Schäfer et al, 1994). Furthermore, its sacB gene produces a substance toxic to the host under high sucrose conditions (Steinmetz et al, 1983;Gay et al, 1985;Jäger et al, 1992). This gene is useful for counter-selection as well as for selection of cells lacking it.…”

Section: Resultsmentioning

confidence: 99%

A novel plasmid curing method using incompatibility of plant pathogenic Ti plasmids in Agrobacterium tumefaciens.

2002

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Ti (Tumor inducing) plasmids in Agrobacterium tumefaciens can transfer their T-DNA region into dicotyledonous plants, in which the expression of T-DNA genes causes plant tumors and the production of bacterial nutrients, e.g., opines such as nopaline. Naturally occurring Ti plasmids (pTi) are difficult to cure by conventional curing methods because of their high stability. Here, we developed a novel curing method based on plasmid incompatibility. For this, a curing plasmid, pMGTrep1, was newly constructed and subsequently introduced into A. tumefaciens strains harboring pTi by conjugation with Escherichia coli harboring pMGTrep1. The conjugation yielded 32-99% nopaline non-utilizing agrobacterial transconjugants in which pMGTrep1 replaced pTi due to incompatibility. Then, pMGTrep1-less derivatives of the transconjugants are easily selected in the presence of sucrose because pMGTrep1 contains a sucrose-sensitive sacB gene. This efficient method is directly applicable for curing plasmids with the same incompatibility group and shoud also applicable to other types of plasmids in Agrobacterium groups, including A. rhizogenes, by replacing the rep gene region of the curing plasmid with that of the corresponding incompatibility.

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Section: Resultsmentioning

confidence: 99%

A novel plasmid curing method using incompatibility of plant pathogenic Ti plasmids in Agrobacterium tumefaciens.

2002

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“…30 copies per cell) derived from the endogenous plasmids pBL1 (Santamaria et al, 1984) and pCG1 (Jager et al, 1992) respectively. Therefore, the high level of FtsZ produced might be responsible for the lethality of FtsZ as described previously .…”

Section: Resultsmentioning

confidence: 99%

“…To express ftsZ Cg in C. glutamicum, a 2?1 kb BglII fragment from the C. glutamicum chromosome (obtained from plasmid pPHEZQ1; see Table 1) containing the whole ftsZ gene and upstream (358 nt) and downstream (433 nt) sequences was cloned into the unique BglII site of plasmid pUL880M (Adham et al, 2001b) or into the high-copy-number conjugative bifunctional plasmid pECM2 (Jager et al, 1992), creating plasmids pBZ81 and pECZ1 respectively (Table 1). These plasmids were constructed in E. coli and transferred to C. glutamicum by electroporation or conjugation respectively.…”

Section: Methodsmentioning

confidence: 99%

Altered morphology produced by ftsZ expression in Corynebacterium glutamicum ATCC 13869

et al. 2005

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Corynebacterium glutamicum is a Gram-positive bacterium that lacks the cell division FtsA protein and actin-like MreB proteins responsible for determining cylindrical cell shape. When the cell division ftsZ gene from C. glutamicum (ftsZ Cg ) was cloned in different multicopy plasmids, the resulting constructions could not be introduced into C. glutamicum; it was assumed that elevated levels of FtsZ Cg result in lethality. The presence of a truncated ftsZ Cg and a complete ftsZ Cg under the control of Plac led to a fourfold reduction in the intracellular levels of FtsZ, generating aberrant cells displaying buds, branches and knots, but no filaments. A 20-fold reduction of the FtsZ level by transformation with a plasmid carrying the Escherichia coli lacI gene dramatically reduced the growth rate of C. glutamicum, and the cells were larger and club-shaped. Immunofluorescence microscopy of FtsZ Cg or visualization of FtsZ Cg -GFP in C. glutamicum revealed that most cells showed one fluorescent band, most likely a ring, at the mid-cell, and some cells showed two fluorescent bands (septa of future daughter cells). When FtsZ Cg -GFP was expressed from Plac, FtsZ rings at mid-cell, or spirals, were also clearly visible in the aberrant cells; however, this morphology was not entirely due to GFP but also to the reduced levels of FtsZ expressed from Plac. Localization of FtsZ at the septum is not negatively regulated by the nucleoid, and therefore the well-known occlusion mechanism seems not to operate in C. glutamicum.

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“…Next, each kanamycin-resistant integrant was grown without kanamycin for 1 d to allow for a second recombination event to take place, and an appropriate dilution (10 4 -10 5 cells) of cells was spread on BY plates containing 10% sucrose, yielding about 10 2 colonies. The sacB positive selection system 17) was used for this purpose. Since expression of integrated plasmid-bearing sacB in the presence of sucrose is lethal to C. glutamicum, only cells in which sacB was deleted as a consequence of the second homologous recombination grew in selective plates.…”

Section: Cadaverinementioning

confidence: 99%

Metabolic Engineering ofCorynebacterium glutamicumfor Cadaverine Fermentation

Mimitsuka

Sawai

Hatsu

et al. 2007

Bioscience, Biotechnology, and Biochemistry

210

119

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Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans

Cited by 159 publications

References 16 publications

A novel plasmid curing method using incompatibility of plant pathogenic Ti plasmids in Agrobacterium tumefaciens.

A novel plasmid curing method using incompatibility of plant pathogenic Ti plasmids in Agrobacterium tumefaciens.

Altered morphology produced by ftsZ expression in Corynebacterium glutamicum ATCC 13869

Metabolic Engineering ofCorynebacterium glutamicumfor Cadaverine Fermentation

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