Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1

Leenhouts, Kees; Tolner, Berend; Bron, Sierd; Kok, Jan; Venema, Gerard; Seegers, Jos F. M. L.

doi:10.1016/0147-619x(91)90036-v

Cited by 158 publications

(112 citation statements)

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“…This could preclude the construction of a mutation in any gene if it is followed by an essential gene under the same transcriptional control. In the pORI plasmids the repC promoter (PrepC) from pWV01 (Leenhouts et al 1991c) is present between Ori> and the mcs (Fig. 1).…”

Section: Gene Replacement In¸ Lactismentioning

confidence: 99%

“…A 601 bp ¹aqI fragment of pWV01, which carries the plus origin of replication (Ori>) but lacks the gene encoding the replication initiation protein (repA), was also treated with Klenow enzyme and both fragments were ligated, resulting in pORI24. The XhoI fragment of pORI24 carrying the (Leenhouts et al 1991c)] Tc gene was replaced by the Em gene from plasmid pUC19E, located on a 1 kb SalI fragment, which resulted in plasmid pORI28. Vectors like pORI24 and pORI28 have a low copy number in the E. coli RepA> strain EC1000, in contrast to RepA> pWV01 derivatives, which have a high copy number in E. coli (Kok et al 1984).…”

Section: Construction Of Pori240 and Pori280mentioning

confidence: 99%

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A general system for generating unlabelled gene replacements in bacterial chromosomes

et al. 1996

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A general system for generating unlabelled gene replacements in bacterial chromosomes Leenhouts, K.; Buist, Girbe; Bolhuis, A.; Berge, A. ten; Kiel, Jan; Mierau, I.; Dabrowska, M.; Venema, G.; Kok, Jan IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document VersionPublisher's PDF, also known as Version of record Publication date: 1996Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Leenhouts, K., Buist, G., Bolhuis, A., Berge, A. T., Kiel, J., Mierau, I., ... Kok, J. (1996). A general system for generating unlabelled gene replacements in bacterial chromosomes. Molecular and General Genetics MGG, 253(1-2), 217-224. DOI: 10.1007/s004380050315 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Abstract A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using¸actococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

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Section: Gene Replacement In¸ Lactismentioning

confidence: 99%

Section: Construction Of Pori240 and Pori280mentioning

confidence: 99%

A general system for generating unlabelled gene replacements in bacterial chromosomes

et al. 1996

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“…The RC plasmids are classified on the basis of homologies within the region of the double-stranded origin and the gene encoding the replication initiation protein. Two classes of RC plasmid are evident in Lactococcus, pE194-like and pC194-like, of which pWV01 (26,28) and pWC1 (33), respectively, are the prototypes. In general, replicons are classified according to their structural organization and the requirement for host-encoded proteins in the replication process.…”

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confidence: 99%

Identification and Characterization of an Active Plasmid Partition Mechanism for the Novel Lactococcus lactis Plasmid pCI2000

2000

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The replication region of the lactococcal plasmid pCI2000 was subcloned and analyzed. The nucleotide sequence of one 5.6-kb EcoRI fragment which was capable of supporting replication when cloned on a replication probe vector revealed the presence of seven putative open reading frames (ORFs). One ORF exhibited significant homology to several replication proteins from plasmids considered to replicate via a theta mode. Deletion analysis showed that this ORF, designated repA, is indeed required for replication. The results also suggest that the origin of replication is located outside repA. Upstream and divergently transcribed from repA, an ORF that showed significant (48 to 64%) homology to a number of proteins that are required for faithful segregation of chromosomal or plasmid DNA of gram-negative bacteria was identified. Gene interruption and transcomplementation experiments showed that this ORF, designated parA, is required for stable inheritance of pCI2000 and is active in trans. This is the first example of such a partitioning mechanism for plasmids in gram-positive bacteria.Lactococcus lactis strains are of considerable industrial and economic importance, as they are widely used in the production of a variety of fermented dairy products. A characteristic of Lactococcus strains is that they typically possess an abundance of plasmid DNA on which a number of significant technological traits are encoded. These include bacteriophage resistance, bacteriocin production, lactose assimilation, citrate utilization, and proteinase activity (11). Therefore, an extensive knowledge of lactococcal plasmid replication, partition, and stability functions is essential in order to ensure the stable maintenance of these traits. This information can also be applied for the generation of novel stable food-grade cloning and expression vectors for the manipulation of these hosts.There are two modes of bacterial plasmid replication, rolling circle (RC) and theta ( ), both of which have been identified in Lactococcus. The RC plasmids are classified on the basis of homologies within the region of the double-stranded origin and the gene encoding the replication initiation protein. Two classes of RC plasmid are evident in Lactococcus, pE194-like and pC194-like, of which pWV01 (26, 28) and pWC1 (33), respectively, are the prototypes. In general, replicons are classified according to their structural organization and the requirement for host-encoded proteins in the replication process. Originally, the best-characterized replicating plasmids were of gram-negative origin, and three classes designated A, B, and C were identified. Class A plasmids possess an origin of replication (ori), which consists of an AT-rich region, adjacent to a number of iterons which are essential in cis for replication initiation. This mode of replication requires a plasmid-encoded replication initiation protein (Rep) and is DNA polymerase I independent. Class B and C replicons do not harbor a typical ori sequence and require host-encoded DNA poly-

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“…ssDNA intermediates indicative of rolling-circle replication were detected for pVE6007 (Fig. 1B and C), a plasmid containing the pWV01 replication origin which replicates through a rolling-cycle mechanism (36). However, no ssDNA intermediates were detected during the replication of pLS203 (Fig.…”

Section: Resultsmentioning

confidence: 96%

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118

Fang

Flynn²,

et al. 2008

Appl Environ Microbiol

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The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli.Lactic acid bacteria and in particular members of the genus Lactobacillus are the most common microbes used as probiotics. They may beneficially affect the host upon ingestion by a variety of potential or proven mechanisms (13,41). Similarly to bifidobacteria, lactobacilli are normal inhabitants of human and animal intestines. Among the more than 100 species of the genus Lactobacillus that have been identified, those that are used as . rhamnosus, and L. salivarius (32). Genome sequence availability considerably facilitates the identification of probiotic characteristics of these bacteria and the prediction of their behaviors in the human gastrointestinal (GI) tract. To date, 11 Lactobacillus genome sequences have been published, and at least 11 additional sequencing projects are in progress (6). This information has dramatically improved our understanding of the metabolic processes and genetics of these microorganisms, as well as their potential roles in health promotion of their hosts. However, for the targeted analysis of genes that contribute to probiotic characteristics, the development of molecular tools for these lactobacilli is of paramount importance.Plasmids, autonomously replicating extrachromosomal genetic elements, are widely present in the genus Lactobacillus. About 38% of the species in this genus contain plasmids (60). Endogenous plasmids from Lactobacillus are of interest because of the traits they confer upon the host. For example, these plasmids may harbor genes encoding resistance to antibiotics (39, 54) and metal ions (55), genes encoding bacteriocins (30, 44), gene clusters for conjugation (55), genes involved in adherence and biotin metabolism (10), and genes encoding toxin-antitoxin (TA) pr...

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Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1

Cited by 158 publications

References 40 publications

A general system for generating unlabelled gene replacements in bacterial chromosomes

A general system for generating unlabelled gene replacements in bacterial chromosomes

Identification and Characterization of an Active Plasmid Partition Mechanism for the Novel Lactococcus lactis Plasmid pCI2000

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118

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