The Mode of Replication Is a Major Factor in Segregational Plasmid Instability in
            <i>Lactococcus lactis</i>

Kiewiet, Rense; Kok, Jan; Seegers, Jos F. M. L.; Venema, Gerard; Bron, Sierd

doi:10.1128/aem.59.2.358-364.1993

Cited by 100 publications

(37 citation statements)

References 36 publications

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“…For overexpression of ␣-amylase, integrant strains harboured plasmid pKTH10 (Table 1). The integration constructs used and the resulting mutant chromosomes ᮊ 1999 Blackwell Science Ltd, Molecular Microbiology, 31, 533-543 Em r , pAM␤1 replication origin Kiewiet et al (1993) are shown schematically in Fig. 1.…”

Section: Resultsmentioning

confidence: 99%

Ecs, an ABC transporter of Bacillus subtilis: dual signal transduction functions affecting expression of secreted proteins as well as their secretion

Leskelä

Wahlström

Hyyryläinen

et al. 1999

Molecular Microbiology

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Summaryecs is a three-cistron operon of Bacillus subtilis, encoding proteins with similarity to the ATPase (EcsA) and hydrophobic components (EcsB) of ABC transporters. The ecsA26 point mutation was shown to cause a strong processing defect of a secreted ␣-amylase precursor (preAmyQ) and of three other exoproteins. Northern analysis of the level of amyQ mRNA showed that ecsA26 also decreases amyQ transcription. This effect too was pleiotropic, as judged by a drastic decrease in the expression from an exoprotease promoter of a reporter protein. A knockout mutation of the ecsB cistron caused a processing defect similar to ecsA26 but, unlike ecsA26, did not affect amyQ transcription. There was also no defect in transcription in the ecsA ecsB double mutant. Thus, an intact ecsB product was required for the downregulation of amyQ by the mutant ecsA. These results suggest a dual regulatory function for Ecs, in which Ecs, possibly as part of a signal transduction mechanism, regulates some component(s) of the protein secretion apparatus as well as secretory protein transcription in a co-ordinated fashion.

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Section: Resultsmentioning

confidence: 99%

Ecs, an ABC transporter of Bacillus subtilis: dual signal transduction functions affecting expression of secreted proteins as well as their secretion

Leskelä

Wahlström

Hyyryläinen

et al. 1999

Molecular Microbiology

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“…1991. Assays for segregational plasmid stability were performed as described by Kiewiet et al (1993).…”

Section: Copy Number Determination Detection Of Single-stranded Dna mentioning

confidence: 99%

Theta replication of the lactococcal plasmid pWVO2

Kiewiet¹,

Bron²,

Jonge³

et al. 1993

Molecular Microbiology

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pWVO2 is a 3.8 kb narrow-host-range plasmid from Lactococcus lactis ssp. cremoris Wg2, which does not replicate in Bacillus subtilis or Escherichia coli. Single-stranded pWVO2 DNA was not observed in lactococcal cells, indicating that this plasmid does not replicate via a rolling-circle mechanism. The sequence of pWVO2 neither showed the structural organization typical for rolling-circle plasmids, nor were sequence similarities with known rolling-circle plasmids present. By 2-D agarose gel electrophoresis of replication intermediates, it was shown that pWVO2 replicates via a theta mechanism. This is the first proof for the existence of theta-replicating plasmids in lactococci. The pWVO2 minimal replicon is strongly related to that of several other lactococcal plasmid replicons. It contains one open reading frame encoding the replication protein, which is preceded by a 22 bp sequence tandemly repeated three and a half times. Further upstream is another 10 bp direct repeat present in an A/T-rich sequence. This structural organization resembles that of several iteron-containing theta-type plasmids from E. coli. Derivatives of pWVO2 were stably maintained in L. lactis and are good candidates for the development of stable food-grade cloning vectors for this organism.

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“…This result reveals that the mode of plasmid replication determines consistent stability both in P-and F-cells regardless of the presence of cryptic plasmid. Kiewiet et al (1993) reported that the theta-type plasmids are segregationally stable in host cells and we have found that the pCB vector replicate by theta-type replication mode .…”

Section: Plasmid Segregational Stabilitymentioning

confidence: 62%

Plasmid curing resulted in improved heterologous gene expression inLeuconostoc citreumEFEL2700

Jang

Kim

Seo

et al. 2019

Lett Appl Microbiol

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Leuconostoc citreum EFEL2700 isolated from kimchi was used as a host strain for genetic and metabolic engineering in our previous studies, but the cells of EFEL2700 contained a cryptic plasmid (P‐cells). Thus, we created plasmid‐free cells (F‐cells) using the CRISPR/Cas9 system. In this study, we compared the microbial characteristics of P‐ and F‐cells in terms of growth rate, biochemical properties, transformation efficiency, plasmid copy number and protein expression level. When the growth rate was measured in MRS medium at 30°C, no significant difference (P > 0·01) was observed. Biochemical properties, tested using an API 50CHL kit, showed no differences. Transformation efficiency of F‐cells, measured using pCB4270, was higher (1·3 × 104 CFU per μg DNA) than that of P‐cells (5·0 × 103 CFU per μg DNA). Copy number after transformation of pCBBgl was 4‐fold higher for F‐cells than for P‐cells. When β‐glucosidase activity was assayed in the above experiment, F‐cells showed 3·4‐fold higher values than P‐cells. In conclusion, this study demonstrates that plasmid curing in L. citreum EFEL2700 improves its characteristics as a gene expression host. Significance and Impact of the Study Leuconostoc citreum EFEL2700 (P‐cell) isolated from kimchi is a useful food‐grade host for expressing heterologous genes. The presence of a cryptic plasmid is thought to limit efficient gene expression. In this study, we compared the microbial and genetic changes after plasmid curing in this strain. The plasmid‐free strain showed improved levels of transformation efficiency, copy number and heterologous gene expression without alterations in phenotypes such as the growth rates and biochemical properties. The resulting strain of L. citreum EFEL2701 (F‐cell) can be used as an efficient host for genetic engineering.

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The Mode of Replication Is a Major Factor in Segregational Plasmid Instability in Lactococcus lactis

Cited by 100 publications

References 36 publications

Ecs, an ABC transporter of Bacillus subtilis: dual signal transduction functions affecting expression of secreted proteins as well as their secretion

Ecs, an ABC transporter of Bacillus subtilis: dual signal transduction functions affecting expression of secreted proteins as well as their secretion

Theta replication of the lactococcal plasmid pWVO2

Plasmid curing resulted in improved heterologous gene expression inLeuconostoc citreumEFEL2700

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