Plasmid p256 from Lactobacillus plantarum represents a new type of replicon in lactic acid bacteria, and contains a toxin–antitoxin-like plasmid maintenance system

Sørvig, Elisabeth; Skaugen, Morten; Naterstad, Kristine; Eijsink, Vincent G. H.; Axelsson, Lars

doi:10.1099/mic.0.27389-0

Cited by 45 publications

(38 citation statements)

References 66 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As described by Sørvig et al (2005), this 688 bp fragment contains features which indicate that p256 replicates via a theta mechanism. As an alternative in this study, we used the pSH71 replicon (de Vos, 1987).…”

Section: Methodsmentioning

confidence: 99%

“…To achieve these goals, we have tested the effect of exchanging the inducible promoters present in the prototype pSIP vectors with other regulated promoters present in the spp and sap regulons. In addition, we have studied the effects of varying the reporter gene (pepN and gusA), the host strain (L. sakei and L. plantarum), and the replicon [the narrow-host-range Lactobacillus replicon from plasmid p256 (Sørvig et al, 2005) and the broadhost-range, high-copy-number pSH71 replicon (de Vos, 1987)]. The resulting optimized pSIP vectors permitted controlled, high-level expression of b-glucuronidase and aminopeptidase N from Lc.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors

Sørvig

Mathiesen

Naterstad³

et al. 2005

Microbiology

Self Cite

182

185

View full text Add to dashboard Cite

Vectors have been developed for inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum in which expression of the gene of interest is driven by strong, regulated promoters from bacteriocin operons found in L. sakei strains. The activity of these promoters is controlled via a two-component signal transduction system, which responds to an externally added peptide pheromone. The vectors have a modular design, permitting easy exchange of all essential elements: the inducible promoter, the cognate regulatory system, the gene of interest, the antibiotic resistance marker and the replicon. Various variants of these so-called 'pSIP' vectors were constructed and tested, differing in terms of the bacteriocin regulon from which the regulatory elements were derived (sakacin A or sakacin P), the regulated promoter selected from these regulons, and the replicon (derived from p256 or pSH71). Using b-glucuronidase (GusA) and aminopeptidase N (PepN) as reporters, it was shown that the best vectors permitted inducible, pheromone-dose-dependent gene expression at very high levels, while displaying moderate basal activities when not induced. The most effective set-up was obtained using a vector containing the pSH71 replicon, the orfX promoter from the sakacin P regulon, and the cognate regulatory genes, in a L. sakei host. GusA levels obtained with this set-up were approximately ten times higher than the levels obtained with prototype pSIP versions, whereas PepN levels amounted to almost 50 % of total cellular protein. INTRODUCTIONMany Lactobacillus species are important in the food industry, where they are used in a variety of products. Lactobacillus sakei is used in meat and vegetable fermentations and has a broad range of possible habitats. The versatility of L. sakei can partly be explained by its ability to survive and grow under adverse conditions, such as low temperature and pH, high salt concentration, smoke, ethanol, low water activity and radiation. Lactobacillus plantarum is also found in many habitats, including dairy and meat products, plant and vegetable fermentations, and in the gastrointestinal tract and oral cavity of humans (Axelsson & Ahrné, 2000). Some L. plantarum strains have probiotic effects on human health (Alander et al., 1999;Schultz et al., 2002;Mercenier et al., 2003). Lactobacilli have great potential as food-grade cell factories and as delivery vehicles for interesting proteins, such as antigens, antibodies and growth factors (Pouwels et al., 1996(Pouwels et al., , 2001Pavan et al., 2000; Krüger et al., 2002;Scheppler et al., 2002). Thus, there is considerable interest in the development of genetic tools for efficient and controllable gene expression in lactobacilli (de Vos, 1999;Mercenier et al., 2000).Many lactic acid bacteria (LAB) produce antimicrobial peptides called bacteriocins, and their production is often regulated via quorum-sensing mechanisms based on a secreted peptide pheromone (Eijsink et al., 2002;Quadri, 2002). The pheromone activates a two-component regulatory s...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors

Sørvig

Mathiesen

Naterstad³

et al. 2005

Microbiology

Self Cite

182

185

View full text Add to dashboard Cite

show abstract

“…Two putative TA system (24) gene pairs were present in pSF118-44 (LSL_1994 and LSL_1995 and LSL_1996 and LSL_1997) while we annotated one such system (LSL_1984 and LSL_1985) in pSF118-20. The TA systems in pSF118-44 are similar to those of the relB and relE family (21), while the single TA system in pSF118-20 (LSL_1984 and LSL_1985) encodes proteins showing 99% and 96% identity to those encoded by pemI and pemK from p256 (53). pemI and pemK are the type II TA system in which the antitoxin is a protein and the toxin, PemK, is an endoribonuclease, which cleaves cellular mRNAs and blocks protein synthesis (62).…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, a new strategy for curing pSF118-44 will be required. TA systems present on pSF118-20 and pSF118-44 are the second-reported plasmid-encoded TA loci in lactobacilli, while the first was from L. plantarum plasmid p256 (53). Bacterial TA systems were initially identified in plasmids (17) and are presumed to maintain the stability of the plasmid in corresponding hosts (24).…”

Section: Discussionmentioning

confidence: 99%

“…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) proteins for plasmid maintenance (53). In addition to encoding such interesting traits, endogenous plasmids are the most commonly used systems to construct genetic tools especially for gene cloning and gene expression purposes (7,52) due to their ability to replicate in the original hosts.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118

Fang

Flynn²,

et al. 2008

Appl Environ Microbiol

View full text Add to dashboard Cite

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...

show abstract