Plasmid replication stimulates DNA recombination in Bacillus subtilis

Noirot, Ph.; Petit, Marie‐Agnès; Ehrlich, S. Dusko

doi:10.1016/0022-2836(87)90509-2

Cited by 67 publications

(52 citation statements)

References 21 publications

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“…Double crossover events were also obtained; a strategy was adopted in which shift-down of Campbell-like integrants to 28%C allows plasmid replication and stimulates a second recombination event (12,32,37). The Ts plasmid can be used in this way to introduce and stabilize heterologous or homologous genes of interest.…”

Section: Resultsmentioning

confidence: 99%

New thermosensitive plasmid for gram-positive bacteria

et al. 1992

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We isolated a replication-thermosensitive mutant of the broad-host-range replicon pVWVOl. The mutant pVE6002 is fuly thermosensitive above 35°C in both gram-negative and gram-positive bacteria. Four clustered mutations were identified in the gene encoding the replication protein of pVE6002. The thermosensitive derivative of the related plasmid pE194 carries a mutation in the analogous region but not in the same position. Derivatives of the thermosensitive plasmid convenient for cloning purposes have been constructed. The low shut-off temperature of pVE6002 makes it a useful suicide vector for bacteria which are limited in their own temperature growth range. Using pVE6002 as the delivery vector for a transposon TnlO derivative in BaciUus subtilis, we observed transposition frequencies of about 1%.

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Section: Resultsmentioning

confidence: 99%

New thermosensitive plasmid for gram-positive bacteria

et al. 1992

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“…For small-scale plasmid extraction from C. cellulolyticum, 6 ml of an overnight culture was subjected to the extraction procedure described previously by Williams et al (1990a) for Clostridium beijerinckii NCIMB 8052. Total DNA was extracted using a method adapted from that of Noirot et al (1987).…”

Section: Methodsmentioning

confidence: 99%

Gene transfer to Clostridium cellulolyticum ATCC 35319

et al. 2000

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Although much is known about the bacterial cellulosome and its various protein components, their contributions to bacterial growth on cellulose and the process of cellulolysis in vivo cannot currently be assessed. To remedy this, the authors have developed gene transfer techniques for Clostridium cellulolyticum ATCC 35319. Firstly, transfer of Tn1545 has been obtained using an Enterococcus faecalis donor. Secondly, IncP-mediated conjugative mobilization of plasmids from Escherichia coli donors has also been achieved. The yield of transconjugants in both cases was low and was probably limited by the suboptimal growth conditions that must of necessity be employed for the co-culture of oligotrophic C. cellulolyticum with copiotrophic donors. A restriction endonuclease was detected in crude extracts of C. cellulolyticum. This enzyme, named CceI, is an isoschizomer of MspI (HpaII). Electrotransformation was employed to establish plasmids containing the replication functions of pAMβ1 (En. faecalis), pIM13 (Bacillus subtilis), pCB102 (Clostridium butyricum), pIP404 (Clostridium perfringens) and pWV01 (Lactococcus lactis subsp. cremoris) in C. cellulolyticum. Transformants were only obtained if the DNA was appropriately methylated on the external C of the sequence 5'-CCGG-3' using either BsuFI methylase in vivo or MspI methylase in vitro. Plasmids based on the pAMβ1 and pIM13 replicons were more stably maintained than one based on the pCB102 replicon. Selection of transformants on solid medium led to low apparent transformation efficiencies (approx. 10 2 transformants per µg DNA) which might, in part, reflect the low plating efficiency of the organism. Selection of transformants in liquid medium led to a higher apparent yield of transformants (between 10 5 and 10 7 transformants per µg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo.

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“…Sub-culturing steps and colony counts were repeated until each bacterial population had undergone about 30 generations of growth. The segregation frequency was calculated by dividing the proportion of antibiotic-sensitive colonies by the number of generations of growth (Noirot et d., 1987). This suggests that a small proportion of the incoming p JC4PTA a n d p JC4BK molecules integrated into the chromosome by homologous recombination.…”

Section: Measurement Of the Recombination Frequencymentioning

confidence: 99%

Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824

Green¹,

Boynton²,

Harris³

et al. 1996

Microbiology

190

118

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IL 60208-31 20, USA lntegrational plasmid technology has been used to disrupt metabolic pathways leading to acetate and butyrate formation in CIostridium acetobutylicum ATCC 824. Non-replicative plasmid constructs, containing either clostridial phosphotransacetylase (pta) or butyrate kinase (buk) gene fragments, were integrated into homologous regions on the chromosome. Integration was assumed to occur by a Campbell-like mechanism, inactivating either pta or buk. Inactivation of the pta gene reduced phosphotransacetylase and acetate kinase activity and significantly decreased acetate production. Inactivation of the buk gene reduced butyrate kinase activity, significantly decreased butyrate production and increased butanol production.

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Plasmid replication stimulates DNA recombination in Bacillus subtilis

Cited by 67 publications

References 21 publications

New thermosensitive plasmid for gram-positive bacteria

New thermosensitive plasmid for gram-positive bacteria

Gene transfer to Clostridium cellulolyticum ATCC 35319

Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824

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