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Lars Axelsson

Publications and source records attributed to Lars Axelsson.

16 recordsLinked to original sources
PubMed2006

Improved expression and purification of the correctly folded response regulator PlnC from lactobacilli.

The response regulator PlnC is part of the signal transduction system that plays a key role in the regulation of bacteriocin production in Lactobacillus plantarum C11. In this study, we wanted to express high levels of the response regulator PlnC in a soluble and native form for purification and further studies. The protein was expressed as a fusion protein (fPlnC) containing an N-terminal Flag-tag to facilitate detection and purification. When the fusion gene, fplnC, was expressed in Escherichia coli BL21, nearly all (99%) of the recombinant protein ended up inside inclusion bodies as an incorrectly folded protein. By utilizing two different Gram-positive expression systems (SIP and NICE) in L. plantarum NC8 and Lactobacillus sakei Lb790, the expression of the soluble fPlnC was significantly increased, being 20-40 times more than that in E. coli BL21. Using the N-terminal tag, the expressed protein was purified by immunoprecipitation. By DNA-binding study (EMSA), we demonstrated that the fusion protein purified from the soluble pool was correctly folded as judged by its ability to bind specifically on regulated promoters. Using our approach, we estimate that about 1 mg of fPlnC can be purified from 11 of the bacterial culture.

Bacteriocins↗
PubMed2006

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.

Aminopeptidases↗
PubMed2005

Isolation of constitutive variants of a subfamily 10 histidine protein kinase (SppK) from Lactobacillus using random mutagenesis.

The histidine protein kinase SppK is a peptide pheromone-activated kinase that regulates the production of the bacteriocin sakacin P in Lactobacillus sakei. SppK belongs to subfamily 10 of histidine protein kinases (HPKs), which regulate important processes in Gram-positive bacteria, including virulence, competence and bacteriocin production. To obtain insight into the functional properties of this relatively unknown class of HPKs, we have subjected SppK to random mutagenesis by error-prone PCR, followed by selection for mutants displaying a constitutive phenotype. Most identified mutations were clustered in a predicted coiled coil-like region, which is an important part of the HPK dimer interface and which includes the autophosphorylated histidine. Other mutations were located in the junctions between the dimerization domain and the membrane receptor domain or the catalytic kinase domain. Interestingly, two previously identified constitutive variants of ComD, an SppK homologue involved in competence regulation in Streptococcus pneumoniae, contained single mutations in the same regions.

Amino Acid Sequence↗
PubMed2005

Work ethics and general work attitudes in adolescents are related to quality of life, sense of coherence and subjective health - a Swedish questionnaire study.

BACKGROUND: Working life is an important arena in most people's lives, and the working line concept is important for the development of welfare in a society. For young people, the period before permanent establishment in working life has become longer during the last two decades. Knowledge about attitudes towards work can help us to understand young people's transition to the labour market. Adolescents are the future workforce, so it seems especially important to notice their attitudes towards work, including attitudes towards the welfare system. The aim of this study was to describe and analyse upper secondary school students' work attitudes, and to explore factors related to these attitudes. METHODS: The sample consisted of 606 upper secondary school students. They all received a questionnaire including questions about quality of life (QOL), sense of coherence (SOC), subjective health and attitudes towards work. The response rate was 91%. A factor analysis established two dimensions of work attitudes. Multivariate analyses were carried out by means of logistic regression models. RESULTS: Work ethics (WE) and general work attitudes (GWA) were found to be two separate dimensions of attitudes towards work. Concerning WE the picture was similar regardless of gender or study programme. Males in theoretical programmes appeared to have more unfavourable GWA than others. Multivariate analyses revealed that good QOL, high SOC and good health were significantly related to positive WE, and high SOC was positively related to GWA. Being female was positively connected to WE and GWA, while studying on a practical programme was positively related to GWA only. Among those who received good parental support, GWA seemed more favourable. CONCLUSION: Assuming that attitudes towards work are important to the working line concept, this study points out positive factors of importance for the future welfare of the society. Individual factors such as female gender, good QOL, high SOC and good health as well as support from both parents, positive experience of school and work contacts related positively to attitudes towards work. Further planning and supportive work have to take these factors into account.

Adolescent↗
PubMed2005

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.

Animals↗
PubMed2005

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.

Bacteriocins↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2005

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.

Bacteriocins↗
PubMed2005

Characterization of a new bacteriocin operon in sakacin P-producing Lactobacillus sakei, showing strong translational coupling between the bacteriocin and immunity genes.

Previous studies of genes involved in the production of sakacin P by Lactobacillus sakei Lb674 revealed the presence of an inducible promoter downstream of the known spp gene clusters. We show here that this promoter drives the expression of an operon consisting of a bacteriocin gene (sppQ), a cognate immunity gene (spiQ), another gene with an unknown function (orf4), and a pseudoimmunity gene containing a frameshift mutation (orf5). The leader peptide of the new one-peptide bacteriocin sakacin Q contains consensus elements that are typical for so-called "double-glycine" leader peptides. The mature bacteriocin shows weak similarity to the BrcA peptide of the two-peptide bacteriocin brochocin C. Sakacin Q has an antimicrobial spectrum that differs from that of sakacin P, thus expanding the antimicrobial properties of the producer strain. The genes encoding sakacin Q and its cognate immunity protein showed strong translational coupling, which was investigated in detail by analyzing the properties of a series of beta-glucuronidase fusions. Our results provide experimental evidence that production of the bacteriocin and production of the cognate immunity protein are tightly coregulated at the translational level.

Amino Acid Sequence↗
PubMed2003

Construction of vectors for inducible gene expression in Lactobacillus sakei and L plantarum.

We have constructed vectors for inducible expression of genes in Lactobacillus sakei and Lactobacillus plantarum. The key elements of these vectors are a regulatable promoter involved in the production of the bacteriocins sakacin A and sakacin P and the genes encoding the cognate histidine protein kinase and response regulator that are necessary to activate this promoter upon induction by a peptide pheromone. The vectors are built up of cassettes that permit easy exchange of all parts through restriction enzyme digestion and ligation. Using beta-glucuronidase as a reporter enzyme, variants of these vectors were compared with each other, and with a corresponding system based on genes involved in the production of nisin. Several of the new vectors permitted tightly controlled and efficient expression of beta-glucuronidase in both L. sakei and L. plantarum.

Gene Expression Regulation, Bacterial↗
PubMed2003

Interactions of the bacteriocins sakacin P and nisin with food constituents.

Bacteriocins are amphiphilic peptides susceptible to adsorption to food macromolecules and proteolytic degradation. These properties may limit their use as preservation agents. The aim of the present work has been to elucidate the fate of the bacteriocin sakacin P in food. Nisin was used in a few experiments for comparison. Recovery of bacteriocins was studied in homogenates of cold-smoked salmon, chicken cold cuts and raw chicken, with verification of the results in the corresponding food products. More than 80% of the added sakacin P and nisin were quickly adsorbed to proteins in the food matrix. In foods that had not been heat-treated, proteolytic activity caused a rapid degradation of the bacteriocins, with less than 1% of the total activity left after 1 week in cold-smoked salmon, and even less in raw chicken. In heat-treated foods, the bacteriocin activity was stable for more than 4 weeks. The high fat content in salmon compared to chicken had no adverse effect on bacteriocin recovery or activity. However, mixing of triglyceride oils and bacteriocin solutions caused a considerable loss of activity. No principal differences were observed between sakacin P and nisin, but less nisin was adsorbed to muscle proteins at low pH, and the negative effect of oils was less pronounced for nisin. Growth of Listeria monocytogenes was completely inhibited for at least 3 weeks in both chicken cold cuts and cold-smoked salmon by addition of sakacin P (3.5 microg/g), despite the proteolytic degradation in the salmon.

Adsorption↗
PubMed2002

Anti-idiotypic protein domains selected from protein A-based affibody libraries.

Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.

Antibodies, Anti-Idiotypic↗
PubMed2002

Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication.

Lactic acid bacteria (LAB) fight competing Gram-positive microorganisms by secreting anti-microbial peptides called bacteriocins. Peptide bacteriocins are usually divided into lantibiotics (class I) and non-lantibiotics (class II), the latter being the main topic of this review. During the past decade many of these bacteriocins have been isolated and characterized, and elements of the genetic mechanisms behind bacteriocin production have been unravelled. Bacteriocins often have a narrow inhibitory spectrum, and are normally most active towards closely related bacteria likely to occur in the same ecological niche. Lactic acid bacteria seem to compensate for these narrow inhibitory spectra by producing several bacteriocins belonging to different classes and having different inhibitory spectra. The latter may also help in counteracting the possible development of resistance mechanisms in target organisms. In many strains, bacteriocin production is controlled in a cell-density dependent manner, using a secreted peptide-pheromone for quorum-sensing. The sensing of its own growth, which is likely to be comparable to that of related species, enables the producing organism to switch on bacteriocin production at times when competition for nutrients is likely to become more severe. Although today a lot is known about LAB bacteriocins and the regulation of their production, several fundamental questions remain to be solved. These include questions regarding mechanisms of immunity and resistance, as well as the molecular basis of target-cell specificity.

Amino Acid Sequence↗
PubMed2000

The synthesis of the bacteriocin sakacin A is a temperature-sensitive process regulated by a pheromone peptide through a three-component regulatory system.

Sakacin A is a bacteriocin produced by Lactobacillus sakei Lb706. The gene cluster (sap) encompasses a regulatory unit composed of three consecutive genes, orf4 and sapKR. sapKR encode a histidine protein kinase and a response regulator, while orf4 encodes the putative precursor of a 23-amino-acid cationic peptide (termed Sap-Ph). The authors show that Sap-Ph serves as a pheromone regulating bacteriocin production. Lb706 produced bacteriocin when the growth temperature was kept at 25 or 30 degrees C, but production was reduced or absent at higher temperatures (33.5-35 degrees C). Production was restored by lowering the growth temperature to 30 degrees C, but at temperatures of 33-34 degrees C also by adding exogenous Sap-Ph to the growth medium. A knock-out mutation in orf4 abolished sakacin A production. Exogenously added Sap-Ph complemented this mutation, unambiguously showing the essential role of this peptide for bacteriocin production. Another sakacin A producer, Lactobacillus curvatus LTH1174, had a similar response to temperature and exogenously added Sap-Ph.

Amino Acid Sequence↗
PubMed1996

Analysis of the sakacin P gene cluster from Lactobacillus sake Lb674 and its expression in sakacin-negative Lb. sake strains.

Sakacin P is a small, heat-stable, ribosomally synthesized peptide produced by certain strains of Lactobacillus sake. It inhibits the growth of several Gram-positive bacteria, including Listeria monocytogenes. A 7.6 kb chromosomal DNA fragment from Lb. sake Lb674 encompassing all genes responsible for sakacin P production and immunity was sequenced and introduced into Lb. sake strains Lb790 and Lb706X which are bacteriocin-negative and sensitive to sakacin P. The transformants produced sakacin P in comparable amounts to the parental strain, Lb674. The sakacin P gene cluster comprised six consecutive genes: sppK, sppR, sppA, spiA, sppT and sppE, all transcribed in the same direction. The deduced proteins SppK and SppR resembled the histidine kinase and response regulator proteins of bacterial two-component signal transducing systems of the AgrB/AgrA-type. The genes sppA and spiA encoded the sakacin P preprotein and the putative immunity protein, respectively. The predicted proteins SppT and SppE showed strong similarities to the proposed transport proteins of several other bacteriocins and to proteins implicated in the signal-sequence-independent export of Escherichia coli haemolysin A. Deletion and frameshift mutation analyses showed that sppK, sppT and sppE were essential for sakacin P production in Lb706X. The putative SpiA peptide was shown to be involved in immunity to sakacin P. Analogues of sppR and spiA were found on the chromosomes of Lb. sake Lb706X and Lb790, indicating the presence of an incomplete spp gene cluster in these strains.

Amino Acid Sequence↗