Microarray-based transcriptome of Listeria monocytogenes adapted to sublethal concentrations of acetic acid, lactic acid, and hydrochloric acid.

Listeria monocytogenes , an important foodborne pathogen, commonly encounters organic acids in food-related environments. The transcriptome of L. monocytogenes L502 was analyzed after adaptation to pH 5 in the presence of acetic acid, lactic acid, or hydrochloric acid (HCl) at 25 °C, representing a condition encountered in mildly acidic ready-to-eat food kept at room temperature. The acid-treated cells were compared with a reference culture with a pH of 6.7 at the time of RNA harvesting. The number of genes and magnitude of transcriptional responses were higher for the organic acids than for HCl. Protein coding genes described for low pH stress, energy transport and metabolism, virulence determinates, and acid tolerance response were commonly regulated in the 3 acid-stressed cultures. Interestingly, the transcriptional levels of histidine and cell wall biosynthetic operons were upregulated, indicating possible universal response against low pH stress in L. monocytogenes. The opuCABCD operon, coding proteins for compatible solutes transport, and the transcriptional regulator sigL were significantly induced in the organic acids, strongly suggesting key roles during organic acid stress. The present study revealed the complex transcriptional responses of L. monocytogenes towards food-related acidulants and opens the roadmap for more specific and in-depth future studies.

Genome sequence of the naturally plasmid-free Lactobacillus plantarum strain NC8 (CCUG 61730).

Lactobacillus plantarum is a highly versatile lactic acid bacterium found in various ecological niches, such as fermented vegetable, meat, and dairy products and the gastrointestinal tract. We sequenced the genome of L. plantarum NC8, a naturally plasmid-free strain, which has been used as a model strain in many laboratories worldwide.

In vitro testing of commercial and potential probiotic lactic acid bacteria.

Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host. The objective of this study was to investigate the diversity of selected commercial and potential probiotic lactic acid bacteria using common in vitro screening assays such as transit tolerance in the upper human gastrointestinal tract, adhesion capacity to human intestinal cell lines and effect on epithelial barrier function. The selected bacteria include strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus farciminis, Lactobacillus sakei, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri and Pediococcus pentosaceus. Viable counts after simulated gastric transit tolerance showed that L. reuteri strains and P. pentosaceus tolerate gastric juice well, with no reduction of viability, whereas L. pentosus, L. farciminis and L. sakei strains lost viability over 180min. All strains tested tolerate the simulated small intestinal juice well. The bacterial adhesion capacity to human intestinal cells revealed major species and strain differences. Overall, L. plantarum MF1298 and three L. reuteri strains had a significant higher adhesion capacity compared to the other strains tested. All strains, both living and UV-inactivated, had little effect on the epithelial barrier function. However, living L. reuteri strains revealed a tendency to increase the transepithelial electrical resistance (TER) from 6 to 24h. This work demonstrates the diversity of 18 potential probiotic bacteria, with major species and strain specific effects in the in vitro screening assays applied. Overall, L. reuteri strains reveal some interesting characteristics compared to the other strains investigated.

Global transcriptome response in Lactobacillus sakei during growth on ribose.

BACKGROUND: Lactobacillus sakei is valuable in the fermentation of meat products and exhibits properties that allow for better preservation of meat and fish. On these substrates, glucose and ribose are the main carbon sources available for growth. We used a whole-genome microarray based on the genome sequence of L. sakei strain 23K to investigate the global transcriptome response of three L. sakei strains when grown on ribose compared with glucose. RESULTS: The function of the common regulated genes was mostly related to carbohydrate metabolism and transport. Decreased transcription of genes encoding enzymes involved in glucose metabolism and the L-lactate dehydrogenase was observed, but most of the genes showing differential expression were up-regulated. Especially transcription of genes directly involved in ribose catabolism, the phosphoketolase pathway, and in alternative fates of pyruvate increased. Interestingly, the methylglyoxal synthase gene, which encodes an enzyme unique for L. sakei among lactobacilli, was up-regulated. Ribose catabolism seems closely linked with catabolism of nucleosides. The deoxyribonucleoside synthesis operon transcriptional regulator gene was strongly up-regulated, as well as two gene clusters involved in nucleoside catabolism. One of the clusters included a ribokinase gene. Moreover, hprK encoding the HPr kinase/phosphatase, which plays a major role in the regulation of carbon metabolism and sugar transport, was up-regulated, as were genes encoding the general PTS enzyme I and the mannose-specific enzyme II complex (EIIman). Putative catabolite-responsive element (cre) sites were found in proximity to the promoter of several genes and operons affected by the change of carbon source. This could indicate regulation by a catabolite control protein A (CcpA)-mediated carbon catabolite repression (CCR) mechanism, possibly with the EIIman being indirectly involved. CONCLUSIONS: Our data shows that the ribose uptake and catabolic machinery in L. sakei is highly regulated at the transcription level. A global regulation mechanism seems to permit a fine tuning of the expression of enzymes that control efficient exploitation of available carbon sources.

Functional analysis of the role of CggR (central glycolytic gene regulator) in Lactobacillus plantarum by transcriptome analysis.

The level of the central glycolytic gene regulator (CggR) was engineered in Lactobacillus plantarum NC8 and WCFS1 by overexpression and in-frame mutation of the cggR gene in order to evaluate its regulatory role on the glycolytic gap operon and the glycolytic flux. The repressor role of CggR on the gap operon was indicated through identification of a putative CggR operator and transcriptome analysis, which coincided with decreased growth rate and glycolytic flux when cggR was overexpressed in NC8 and WCFS1. The mutation of cggR did not affect regulation of the gap operon, indicating a more prominent regulatory role of CggR on the gap operon under other conditions than tested (e.g. fermentation of other sugars than glucose or ribose) and when the level of the putative effector molecule FBP is reduced. Interestingly, the mutation of cggR had several effects in NC8, i.e. increased growth rate and glycolytic flux and regulation of genes with functions associated with glycerol and pyruvate metabolism; however, no effects were observed in WCFS1. The affected genes in NC8 are presumably regulated by CcpA, since putative CRE sites were identified in their upstream regions. The interconnection with CggR and CcpA-mediated control on growth and metabolism needs to be further elucidated.

Global transcriptional analysis of spontaneous sakacin P-resistant mutant strains of Listeria monocytogenes during growth on different sugars.

Subclass IIa bacteriocins have strong antilisterial activity and can control the growth of Listeria monocytogenes in food. However, L. monocytogenes may develop resistance towards such bacteriocins. In this follow-up study, the transcriptomes of a high level (L502-1) and a low level (L502-6) spontaneous sakacin P-resistant mutant strain of L. monocytogenes were compared to the wild-type (L502). The growth of the resistant strains was reduced on mannose but not affected on cellobiose and the transcriptomics was performed during growth on these sugars. The mannose phosphotransferase system (PTS) encoded by the mptACD operon (mpt) is known for transporting mannose and also act as a receptor to class IIa bacteriocins. The mpt was repressed in L502-1 and this is in accordance with abolition of the bacteriocin receptor with resistance to class IIa bacteriocins. In contrast, the mpt was induced in L502-6. Despite the induction of the mpt, L502-6 showed 1,000 times more resistance phenotype and reduced growth on mannose suggesting the mannose-PTS may not be functional in L502-6. The microarray data suggests the presence of other transcriptional responses that may be linked to the sakacin P resistance phenotype particularly in L502-6. Most of commonly regulated genes encode proteins involved in transport and energy metabolism. The resistant strains displayed shift in general carbon catabolite control possibly mediated by the mpt. Our data suggest that the resistant strains may have a reduced virulence potential. Growth sugar- and mutant-specific responses were also revealed. The two resistant strains also displayed difference in stability of the sakacin P resistance phenotype, growth in the presence of both the lytic bacteriophage P100 and activated charcoal. Taken together, the present study showed that a single time exposure to the class IIa bacteriocin sakacin P may elicit contrasting phenotypic and transcriptome responses in L. monocytogenes possibly through regulation of the mpt.

Primary metabolism in Lactobacillus sakei food isolates by proteomic analysis.

BACKGROUND: Lactobacillus sakei is an important food-associated lactic acid bacterium commonly used as starter culture for industrial meat fermentation, and with great potential as a biopreservative in meat and fish products. Understanding the metabolic mechanisms underlying the growth performance of a strain to be used for food fermentations is important for obtaining high-quality and safe products. Proteomic analysis was used to study the primary metabolism in ten food isolates after growth on glucose and ribose, the main sugars available for L. sakei in meat and fish. RESULTS: Proteins, the expression of which varied depending on the carbon source were identified, such as a ribokinase and a D-ribose pyranase directly involved in ribose catabolism, and enzymes involved in the phosphoketolase and glycolytic pathways. Expression of enzymes involved in pyruvate and glycerol/glycerolipid metabolism were also affected by the change of carbon source. Interestingly, a commercial starter culture and a protective culture strain down-regulated the glycolytic pathway more efficiently than the rest of the strains when grown on ribose. The overall two-dimensional gel electrophoresis (2-DE) protein expression pattern was similar for the different strains, though distinct differences were seen between the two subspecies (sakei and carnosus), and a variation of about 20% in the number of spots in the 2-DE gels was observed between strains. A strain isolated from fermented fish showed a higher expression of stress related proteins growing on both carbon sources. CONCLUSIONS: It is obvious from the data obtained in this study that the proteomic approach efficiently identifies differentially expressed proteins caused by the change of carbon source. Despite the basic similarity in the strains metabolic routes when they ferment glucose and ribose, there were also interesting differences. From the application point of view, an understanding of regulatory mechanisms, actions of catabolic enzymes and proteins, and preference of carbon source is of great importance.

A candidate probiotic with unfavourable effects in subjects with irritable bowel syndrome: a randomised controlled trial.

BACKGROUND: Some probiotics have shown efficacy for patients with irritable bowel syndrome (IBS). Lactobacillus (L.) plantarum MF1298 was found to have the best in vitro probiotic properties of 22 strains of lactobacilli. The aim of this study was to investigate the symptomatic effect of L. plantarum MF1298 in subjects with IBS. Primary outcome was treatment preference and secondary outcomes were number of weeks with satisfactory relief of symptoms and IBS sum score. METHODS: The design was a randomised double blind placebo-controlled crossover trial. 16 subjects with IBS underwent two three-week periods of daily intake of one capsule of 10(10) CFU L. plantarum MF 1298 or placebo separated by a four-week washout period. RESULTS: Thirteen participants (81%; 95% CI 57% to 93%; P = 0.012) preferred placebo to L. plantarum MF1298 treatment. The mean (SD) number of weeks with satisfactory relief of symptoms in the periods with L. plantarum MF1298 and placebo were 0.50 (0.89) and 1.44 (1.26), respectively (P = 0.006). IBS sum score was 6.44 (1.81) in the period with L. plantarum MF1298 treatment compared with 5.35 (1.77) in the period with placebo (P = 0.010). With a clinically significant difference in the IBS sum score of 2 in disfavour of active treatment, the number needed to harm was 3.7, 95% CI 2.3 to 10.9. CONCLUSIONS: This trial shows for the first time an unfavourable effect on symptoms in subjects with IBS after intake of a potential probiotic.

Activation and inhibition of nuclear factor kappa B activity by cereal extracts: role of dietary phenolic acids.

The transcription factor nuclear factor kappa B (NF-kappaB) plays a critical role in stress, immune, and inflammatory responses, and the modulation of its activity can be a potentially effective preventive strategy for controlling certain diseases. Cereal grains contain phenolic compounds in concentrations comparable to those in fruits and vegetables, well-known for their beneficial effect on human health. In this study we aimed to examine the effect of different phenolic extracts from barley, oat, wheat, and buckwheat on the modulation of basal and lipopolysaccharide (LPS)-induced NF-kappaB activity and elucidate the role of phenolic acids in this modulation. Three extracts were prepared: extracts of free phenolic compounds (M1), extracts of free phenolic acids (M2), and extracts of bound phenolic acids (HY). Generally, extracts M2 showed the highest effect on modulation of NF-kappaB activity with strong inhibition of LPS-induced NF-kappaB activity at all concentrations and of the basal NF-kappaB activity at concentrations equal to or lower than 3 mg/mL. Most of extracts M1 and HY slightly increased both the basal and the LPS-induced NF-kappaB activation. However, at the highest concentrations (3 or 15 mg/mL) extracts HY inhibited LPS-induced NF-kappaB activation. Similar experiments with standard solutions of phenolic acids indicated their ability to modulate the NF-kappaB activity.

Complex phenotypic and genotypic responses of Listeria monocytogenes strains exposed to the class IIa bacteriocin sakacin P.

Sakacin P is a class IIa bacteriocin that is active against the food-borne pathogen Listeria monocytogenes, and use of this compound as a biopreservative in foods has been suggested. In the present study, we characterized 30 spontaneous sakacin P-resistant mutants of L. monocytogenes obtained after single exposure to sakacin P. The frequency of development of sakacin P resistance for all strains was in the range from 10(-8) to 10(-9). Using the 50% inhibitory concentration (IC(50)) of sakacin P, the strains could be grouped into strains with high levels of resistance (IC(50), > or =10(4) ng ml(-1)) and strains with low levels of resistance (IC(50), <10(4) ng ml(-1)). Resistant strains belonging to the same IC(50) group also had similar physiological and genetic characteristics. Generally, the resistant strains showed substantial variations in many parameters, such as differences in the stability of the acquired resistance to sakacin P, growth fitness, food-related stress tolerance, and biofilm-forming ability. Fourier transform infrared spectroscopy revealed differences between wild-type and resistant strains in polysaccharide, fatty acid, and, protein regions. A mannose-specific phosphotransferase (PTS) operon has been described for class IIa bacteriocin resistance, and the sakacin P-resistant strains displayed both up- and downregulation of the expression of the mptA gene encoding the PTS system. This is the first comprehensive study of the diversity of a large number of spontaneous resistant mutants obtained after one exposure to a class IIa bacteriocin, particularly to sakacin P. The great diversity among the resistant strains exposed to the same stress conditions suggests that there are different resistance mechanisms.

Diversity of Lactobacillus sakei strains investigated by phenotypic and genotypic methods.

The diversity of 10 strains of Lactobacillus sakei, a commercially important species of lactobacilli, was characterized by studying food isolates. Growth characteristics varied among the strains when examined after growth in a complex medium and a defined medium with either glucose or ribose. A commercial starter culture strain showed the fastest growth rates and high biomass formation on all media, while two of the strains hardly grew on ribose. Based on acidification properties in a meat model, some of the strains had the ability to compete with the indigenous microbiota of the meat batter in addition to being fast acid producers. Carbohydrate-fermentation abilities revealed a relatively wide variation, clustering the strains into two phenotypic groups. The isolates were analyzed using different genetic fingerprinting techniques, demonstrating a distinction between two genetic groups, a grouping consistent with previous studies dealing with L. sakei strains. Comparative genome hybridization (CGH) was introduced for clustering the strains and the same division into two genetic groups was observed. Chromosomal sizes of the strains were estimated by pulsed field gel electrophoresis (PFGE) and were found to vary from 1884 to 2175 kb. The genetic groups did not correlate with the clustering obtained with carbohydrate-fermenting abilities or with chromosomal sizes.

Co-factor engineering in lactobacilli: effects of uncoupled ATPase activity on metabolic fluxes in Lactobacillus (L.) plantarum and L. sakei.

The hydrolytic F(1)-part of the F(1)F(0)-ATPase was over-expressed in Lactobacillus (L.) plantarum NC8 and L. sakei Lb790x during fermentation of glucose or ribose, in order to study how changes in the intracellular levels of ATP and ADP affect the metabolic fluxes. The uncoupled ATPase activity resulted in a decrease in intracellular energy level (ATP/ADP ratio), biomass yield and growth rate. Interestingly, the glycolytic and ribolytic flux increased in L. plantarum with uncoupled ATPase activity compared to the reference strain by up to 20% and 50%, respectively. The ATP demand was estimated to have approximately 80% control on both the glycolytic and ribolytic flux in L. plantarum under these conditions. In contrast, the glycolytic and ribolytic flux decreased in L. sakei with uncoupled ATPase activity.

Characterisation of the gap operon from Lactobacillus plantarum and Lactobacillus sakei.

Glycolysis constitutes the primary energy-generating pathway of most species of lactic acid bacteria. The metabolism ultimately results in massive lactic acid production, which is responsible for the major preservative effect of these organisms. This study reports the identification, sequencing, and characterisation of the central glycolytic operon, the gap operon, from Lactobacillus plantarum NC8 and L. sakei Lb790. The structure of the operons of the two Lactobacillus strains were similar and organised in the order cggR-gap-pgk-tpi-eno, encoding a putative central glycolytic gene regulator and the four glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, triosephosphate isomerase, and enolase, respectively. This operon structure has not been reported in any other bacterial species so far. Transcriptional analysis revealed three major transcripts, the mono-cistronic gap and eno and the tetra-cistronic gap-pgk-tpi-eno.

Triclosan and antimicrobial resistance in bacteria: an overview.

Triclosan is a widely used biocide that is considered as an effective antimicrobial agent against different microorganisms. It is included in many contemporary consumer and personal health-care products, like oral and dermal products, but also in household items, including plastics and textiles. At bactericidal concentrations, triclosan appears to act upon multiple nonspecific targets, causing disruption of bacterial cell wall functions, while at sublethal concentrations, triclosan affects specific targets. During the 1990s, bacterial isolates with reduced susceptibility to triclosan were produced in laboratory experiments by repeated exposure to sublethal concentrations of the agent. Since 2000, a number of studies have verified the occurrence of triclosan resistance amongst dermal, intestinal, and environmental microorganisms, including some of clinical relevance. Of major concern is the possibility that triclosan resistance may contribute to reduced susceptibility to clinically important antimicrobials, due to either cross-resistance or co-resistance mechanisms. Although the number of studies elucidating the association between triclosan resistance and resistance to other antimicrobials in clinical isolates has been limited, recent laboratory studies have confirmed the potential for such a link in Escherichia coli and Salmonella enterica. Thus, widespread use of triclosan may represent a potential public health risk in regard to development of concomitant resistance to clinically important antimicrobials.

A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum.

A synthetic promoter library (SPL) for Lactobacillus plantarum has been developed, which generalizes the approach for obtaining synthetic promoters. The consensus sequence, derived from rRNA promoters extracted from the L. plantarum WCFS1 genome, was kept constant, and the non-consensus sequences were randomized. Construction of the SPL was performed in a vector (pSIP409) previously developed for high-level, inducible gene expression in L. plantarum and Lactobacillus sakei. A wide range of promoter strengths was obtained with the approach, covering 3-4 logs of expression levels in small increments of activity. The SPL was evaluated for the ability to drive beta-glucuronidase (GusA) and aminopeptidase N (PepN) expression. Protein production from the synthetic promoters was constitutive, and the most potent promoters gave high protein production with levels comparable to those of native rRNA promoters, and production of PepN protein corresponding to approximately 10-15 % of the total cellular protein. High correlation was obtained between the activities of promoters when tested in L. sakei and L. plantarum, which indicates the potential of the SPL for other Lactobacillus species. The SPL enables fine-tuning of stable gene expression for various applications in L. plantarum.

Fourier transform infrared and raman spectroscopy for characterization of Listeria monocytogenes strains.

The purpose of this study was to characterize the variation in biochemical composition of 89 strains of Listeria monocytogenes with different susceptibilities towards sakacin P, using Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy. The strains were also analyzed using amplified fragment length polymorphism (AFLP) analysis. Based on their susceptibilities to sakacin P, the 89 strains have previously been divided into two groups. Using the FTIR spectra and AFLP data, the strains were basically differentiated into the same two groups. Analyses of the FTIR and Raman spectra revealed that the strains in the two groups contained differences in the compositions of carbohydrates and fatty acids. The relevance of the variation in the composition of carbohydrates with respect to the variation in the susceptibility towards sakacin P for the L. monocytogenes strains is discussed.

Identification of potential probiotic starter cultures for Scandinavian-type fermented sausages.

Potential probiotic cultures suitable as starter cultures for the Scandinavian-type fermented sausages were identified among strains well-adapted to fermented meats as well as strains originating from a culture collection. From 15 different fermented meat products, 22 strains were isolated as dominant non-starter lactic acid bacteria (NSLAB). The isolates were identified by RAPD, API and sequence analysis of 16S rRNA and showed to be five strains of Lactobacillus sakei, five strains of Lactobacillus farciminis, five strains belonging to the group of Lactobacillus plantarum/pentosus, four strains of Lactobacillus alimentarius, two strains of Lactobacillus brevis and one strain of Lactobacillus versmoldensis. Heterofermentative strains as well as strains not growing at 37 degrees C and not lowering pH below 5.1 in a meat model were excluded leaving 9 strains for further studies. These strains together with 19 strains from a culture collection were evaluated by in vitro methods including survival upon exposure to pH 2.5 or 0.3% oxgall and adhesion to the human colon adenocarcinoma cell line Caco-2 as well as antimicrobial activity against potential pathogens. Strains that fulfilled all the probiotic criteria and showed to be fast acid producers in a meat model included three strains belonging to the group of Lb. plantarum/pentosus (MF1291, MF1298, MF1300) which originated from the dominant NSLAB of fermented meat products. MF1291 and MF 1298 were further identified as Lb. plantarum and MF1300 as Lb. pentosus. The three strains were all successfully applied as starter cultures for the production of fermented sausage. The viable count at the end of the processing period reached high cell numbers (4.7x10(7)-2.9x10(8) cfu/g) and pH of the sausages decreased to pH 4.8-4.9 without any flavour deviation compared to sausage fermented by a commercial meat starter culture.

Sakacin P non-producing Lactobacillus sakei strains contain homologues of the sakacin P gene cluster.

Some strains of Lactobacillus sakei are known to produce the bacteriocin sakacin P, encoded by the spp gene cluster. In strains unable to produce sakacin P, spp homologues were observed. The analysis of 15 strains not producing sakacin P revealed that all contained a region corresponding to a part of sppKR encoding the regulatory elements for sakacin P production. In some strains homologues of sppE and sppT, responsible for sakacin P transport, and the sakacin P structural gene sppA and its immunity gene spiA, were also present. The sequence of the chromosomal spp-related gene cluster was determined in two non-producing strains: L. sakei Lb790 and L. sakei 23K. The L. sakei Lb790 spp gene cluster encompasses genes homologous to sppK, sppR, sppT and sppE. In L. sakei 23K, only sppK and sppR homologues were present. The sppK homologues appeared non-functional as they contained mutations and/or an insertion element. In addition to the spp homologues, several small putative genes were found in the gene clusters of the two strains. Some were similar in both strains, and their organization suggests a mosaic structure resulting from successive rearrangements. Transcriptional analysis showed that the genes of the L. sakei Lb790 spp cluster were expressed when genes encoding an operative sakacin P regulatory system were introduced in this strain, thus complementing the inactive sppK gene. Expression experiments also suggested that some of the spp homologues maintained their function in non-producing strains.

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

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 beta-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.

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

Lactobacillus plantarum NC7 harbours a single 7.2 kb plasmid called p256. This report describes the complete nucleotide sequence and annotation of p256, as well as the identification of the minimal replicon of the plasmid. Based on sequence features in the unusually small (0.7 kb) minimal replicon, and the absence of a gene for a replication-relevant protein, p256 seems to represent a hitherto unknown type of theta replicon in lactic acid bacteria (LAB), with a relatively low copy-number. In addition, a putative toxin-antitoxin (TA) locus was identified. Experiments with variants of p256 indicated that the TA system was involved in plasmid maintenance. Furthermore, controlled expression of the TA genes stabilized vectors derived from the p256 replicon. To the authors' knowledge, this is the first time a TA locus with a demonstrated plasmid maintenance function has been identified in LAB. Transformation of several LAB with plasmids derived from p256 indicated that it has a narrow host range. Several effective expression vectors based on the p256 replicon have been constructed.