Protein profiling of testicular tissue from boars with different levels of hyperactive sperm motility.

Hyperactive sperm motility is important for successful fertilization. In the present study, a proteome profiling approach was performed to identify the differences between Landrace boars with different levels of hyperactive sperm motility in liquid extended semen. Two contrasts were studied: (i) high versus low levels of sperm hyperactivity at semen collection day and (ii) high versus low change in levels of sperm hyperactivity after 96 h semen storage. Testicular samples were analyzed on a Q Exactive mass spectrometer and more than 6000 proteins were identified in the 13 samples. The most significant differentially expressed proteins were mediator complex subunit 28 (MED28), cell division cycle 37 like 1 (CDC37L1), ubiquitin specific peptidase 10 (USP10), zinc finger FYVE-type containing 26 (ZFYVE26), protein kinase C delta (PRKCD), actinin alpha 4 (ACTN4), N(alpha)-acetyltransferase 30 (NAA30), C1q domain-containing (LOC110258309) and uncharacterized LOC100512926. Of the differentially expressed proteins, 11 have previously been identified as differentially expressed at the corresponding mRNA transcript level using the same samples and contrasts. These include sphingosine kinase 1 isoform 2 (SPHK1), serine and arginine rich splicing factor 1 (SRSF1), and tubulin gamma-1 (TUBG1) which are involved in the acrosome reaction and sperm motility. A mass spectrometry approach was applied to investigate the protein profiles of boars with different levels of hyperactive sperm motility. This study identified several proteins previously shown to be involved in sperm motility and quality, but also proteins with no known function for sperm motility. Candidates that are differentially expressed on both mRNA and protein levels are especially relevant as biological markers of semen quality.

Genome-assisted Identification, Purification, and Characterization of Bacteriocins.

Bacteriocins are antimicrobial peptides with activity against antibiotic resistant bacterial pathogens. Here, we describe a set of methods aimed at purifying, identifying, and characterizing new bacteriocins. The purification consists of ammonium sulphate precipitation, cation-exchange chromatography, and reversed-phase chromatography. The yield of the bacteriocin is quantified by bacteriocin antimicrobial activity in a microtiter plate assay after each purification step. The mass of the purified bacteriocin is assessed by MALDI TOF MS analysis of the active fractions after reversed-phase chromatography. The mass is compared with the theoretical mass based on genetic information from the whole genome sequencing of the bacteriocin producer strain. Physicochemical characterization is performed by assessing antimicrobial activity following heat and protease treatments. Fluorescent techniques are used to examine the capacity of the bacteriocin to disrupt membrane integrity. Herein a set of protocols for purification and characterization of the bacteriocin nisin Z is used as a typical example in this paper.

Changes in cardiac proteome and metabolome following exposure to the PAHs retene and fluoranthene and their mixture in developing rainbow trout alevins.

Exposure to polycyclic aromatic hydrocarbons (PAHs) is known to affect developing organisms. Utilization of different omics-based technologies and approaches could therefore provide a base for the discovery of novel mechanisms of PAH induced development of toxicity. To this aim, we investigated how exposure towards two PAHs with different toxicity mechanisms: retene (an aryl hydrocarbon receptor 2 (Ahr2) agonist), and fluoranthene (a weak Ahr2 agonist and cytochrome P450 inhibitor (Cyp1a)), either alone or as a mixture, affected the cardiac proteome and metabolome in newly hatched rainbow trout alevins (Oncorhynchus mykiss). In total, we identified 65 and 82 differently expressed proteins (DEPs) across all treatments compared to control (DMSO) after 7 and 14 days of exposure. Exposure to fluoranthene altered the expression of 11 and 19 proteins, retene 29 and 23, while the mixture affected 44 and 82 DEPs by Days 7 and 14, respectively. In contrast, only 5 significantly affected metabolites were identified. Pathway over-representation analysis identified exposure-specific activation of phase II metabolic processes, which were accompanied with exposure-specific body burden profiles. The proteomic data highlights that exposure to the mixture increased oxidative stress, altered iron metabolism and impaired coagulation capacity. Additionally, depletion of several mini-chromosome maintenance components, in combination with depletion of several intermediate filaments and microtubules, among alevins exposed to the mixture, suggests compromised cellular integrity and reduced rate of mitosis, whereby affecting heart growth and development. Furthermore, the combination of proteomic and metabolomic data indicates altered energy metabolism, as per amino acid catabolism among mixture exposed alevins; plausibly compensatory mechanisms as to counteract reduced absorption and consumption of yolk. When considered as a whole, proteomic and metabolomic data, in relation to apical effects on the whole organism, provides additional insight into PAH toxicity and the effects of exposure on heart structure and molecular processes.

Lipid Body Dynamics in Shoot Meristems: Production, Enlargement, and Putative Organellar Interactions and Plasmodesmal Targeting.

Post-embryonic cells contain minute lipid bodies (LBs) that are transient, mobile, engage in organellar interactions, and target plasmodesmata (PD). While LBs can deliver γ-clade 1,3-ß-glucanases to PD, the nature of other cargo is elusive. To gain insight into the poorly understood role of LBs in meristems, we investigated their dynamics by microscopy, gene expression analyzes, and proteomics. In developing buds, meristems accumulated LBs, upregulated several LB-specific OLEOSIN genes and produced OLEOSINs. During bud maturation, the major gene OLE6 was strongly downregulated, OLEOSINs disappeared from bud extracts, whereas lipid biosynthesis genes were upregulated, and LBs were enlarged. Proteomic analyses of the LB fraction of dormant buds confirmed that OLEOSINs were no longer present. Instead, we identified the LB-associated proteins CALEOSIN (CLO1), Oil Body Lipase 1 (OBL1), Lipid Droplet Interacting Protein (LDIP), Lipid Droplet Associated Protein1a/b (LDAP1a/b) and LDAP3a/b, and crucial components of the OLEOSIN-deubiquitinating and degradation machinery, such as PUX10 and CDC48A. All mRFP-tagged LDAPs localized to LBs when transiently expressed in Nicotiana benthamiana. Together with gene expression analyzes, this suggests that during bud maturation, OLEOSINs were replaced by LDIP/LDAPs at enlarging LBs. The LB fraction contained the meristem-related actin7 (ACT7), "myosin XI tail-binding" RAB GTPase C2A, an LB/PD-associated γ-clade 1,3-ß-glucanase, and various organelle- and/or PD-localized proteins. The results are congruent with a model in which LBs, motorized by myosin XI-k/1/2, traffic on F-actin, transiently interact with other organelles, and deliver a diverse cargo to PD.

Retene, pyrene and phenanthrene cause distinct molecular-level changes in the cardiac tissue of rainbow trout (Oncorhynchus mykiss) larvae, part 2 - Proteomics and metabolomics.

Polycyclic aromatic hydrocarbons (PAHs) are global contaminants of concern. Despite several decades of research, their mechanisms of toxicity are not very well understood. Early life stages of fish are particularly sensitive with the developing cardiac tissue being a main target of PAHs toxicity. The mechanisms of cardiotoxicity of the three widespread model polycyclic aromatic hydrocarbons (PAHs) retene, pyrene and phenanthrene were explored in rainbow trout (Oncorhynchus mykiss) early life stages. Newly hatched larvae were exposed to sublethal doses of each individual PAH causing no detectable morphometric alterations. Changes in the cardiac proteome and metabolome were assessed after 7 or 14 days of exposure to each PAH. Phase I and II enzymes regulated by the aryl hydrocarbon receptor were significantly induced by all PAHs, with retene being the most potent compound. Retene significantly altered the level of several proteins involved in key cardiac functions such as muscle contraction, cellular tight junctions or calcium homeostasis. Those findings were quite consistent with previous reports regarding the effects of retene on the cardiac transcriptome. Significant changes in proteins linked to iron and heme metabolism were observed following exposure to pyrene. While phenanthrene also altered the levels of several proteins in the cardiac tissue, no clear mechanisms or pathways could be highlighted. Due to high variability between samples, very few significant changes were detected in the cardiac metabolome overall. Slight but significant changes were still observed for pyrene and phenanthrene, suggesting possible effects on several energetic or signaling pathways. This study shows that early exposure to different PAHs can alter the expression of key proteins involved in the cardiac function, which could potentially affect negatively the fitness of the larvae and later of the juvenile fish.

Proteome analysis of xylose metabolism in Rhodotorula toruloides during lipid production.

BACKGROUND: Rhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids. RESULTS: Rhodotorula toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose. CONCLUSIONS: The results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through pentose phosphate pathway although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates.

Metaproteomics: Sample Preparation and Methodological Considerations.

Meta-omic techniques have progressed rapidly in the past decade and are frequently used in microbial ecology to study microorganisms in their natural ecosystems independent from culture restrictions. Metaproteomics, in combination with metagenomics, enables quantitative assessment of expressed proteins and pathways from individual members of the consortium. Together, metaproteomics and metagenomics can provide a detailed understanding of which organisms occupy specific metabolic niches, how they interact, and how they utilize nutrients, and these insights can be obtained directly from environmental samples. Here, we outline key aspects of sample preparation, database generation, and other methodological considerations that are required for successful quantitative metaproteomic analyses and we describe case studies on the integration with metagenomics for enhanced functional output.

Methylation of the N-terminal histidine protects a lytic polysaccharide monooxygenase from auto-oxidative inactivation.

The catalytically crucial N-terminal histidine (His1) of fungal lytic polysaccharide monooxygenases (LPMOs) is post-translationally modified to carry a methylation. The functional role of this methylation remains unknown. We have carried out an in-depth functional comparison of two variants of a family AA9 LPMO from Thermoascus aurantiacus (TaLPMO9A), one with, and one without the methylation on His1. Various activity assays showed that the two enzyme variants are identical in terms of substrate preferences, cleavage specificities and the ability to activate molecular oxygen. During the course of this work, new functional features of TaLPMO9A were discovered, in particular the ability to cleave xyloglucan, and these features were identical for both variants. Using a variety of techniques, we further found that methylation has minimal effects on the pKa of His1, the affinity for copper and the redox potential of bound copper. The two LPMOs did, however, show clear differences in their resistance against oxidative damage. Studies with added hydrogen peroxide confirmed recent claims that low concentrations of H2 O2 boost LPMO activity, whereas excess H2 O2 leads to LPMO inactivation. The methylated variant of TaLPMO9A, produced in Aspergillus oryzae, was more resistant to excess H2 O2 and showed better process performance when using conditions that promote generation of reactive-oxygen species. LPMOs need to protect themselves from reactive oxygen species generated in their active sites and this study shows that methylation of the fully conserved N-terminal histidine provides such protection.

Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2.

Enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) play an important role in the conversion of recalcitrant polysaccharides, but their mode of action has remained largely enigmatic. It is generally believed that catalysis by LPMOs requires molecular oxygen and a reductant that delivers two electrons per catalytic cycle. Using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show here that H2O2, rather than O2, is the preferred co-substrate of LPMOs. By controlling H2O2 supply, stable reaction kinetics are achieved, the LPMOs work in the absence of O2, and the reductant is consumed in priming rather than in stoichiometric amounts. The use of H2O2 by a monocopper enzyme that is otherwise cofactor-free offers new perspectives regarding the mode of action of copper enzymes. Furthermore, these findings have implications for the enzymatic conversion of biomass in Nature and in industrial biorefining.

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.

Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression.

A novel proteomics sample preparation method for secretome analysis of Hypocrea jecorina growing on insoluble substrates.

Analysis of the secretomes of filamentous fungi growing on insoluble lignocellulosic substrates is of major current interest because of the industrial potential of secreted fungal enzymes. Importantly, such studies can help identifying key enzymes from a large arsenal of bioinformatically detected candidates in fungal genomes. We describe a simple, plate-based method to analyze the secretome of Hypocrea jecorina growing on insoluble substrates that allows harsh sample preparation methods promoting desorption, and subsequent identification, of substrate-bound proteins, while minimizing contamination with non-secreted proteins from leaking or lysed cells. The validity of the method was demonstrated by comparative secretome analysis of wild-type H.jecorina strain QM6a growing on bagasse, birch wood, spruce wood or pure cellulose, using label-fee quantification. The proteomic data thus obtained were consistent with existing data from transcriptomics and proteomics studies and revealed clear differences in the responses to complex lignocellulosic substrates and the response to pure cellulose. This easy method is likely to be generally applicable to filamentous fungi and to other microorganisms growing on insoluble substrates.

Proteomic Investigation of the Response of Enterococcus faecalis V583 when Cultivated in Urine.

Enterococcus faecalis is a robust bacterium, which is able to survive in and adapt to hostile environments such as the urinary tract and bladder. In this label-free quantitative proteomic study based on MaxQuant LFQ algorithms, we identified 127 proteins present in the secretome of the clinical vancomycin-resistant isolate E. faecalis V583 and we compared proteins secreted in the initial phase of cultivation in urine with the secretome during cultivation in standard laboratory medium, 2xYT. Of the 54 identified proteins predicted to be secreted, six were exclusively found after cultivation in urine including the virulence factor EfaA ("endocarditis specific antigen") and its homologue EF0577 ("adhesion lipoprotein"). These two proteins are both involved in manganese transport, known to be an important determinant of colonization and infection, and may additionally function as adhesins. Other detected urine-specific proteins are involved in peptide transport (EF0063 and EF3106) and protease inhibition (EF3054). In addition, we found an uncharacterized protein (EF0764), which had not previously been linked to the adaptation of V583 to a urine environment, and which is unique to E. faecalis. Proteins found in both environments included a histone-like protein, EF1550, that was up-regulated during cultivation in urine and that has a homologue in streptococci (HlpA) known to be involved in bacterial adhesion to host cells. Up-regulated secreted proteins included autolysins. These results from secretome analyses are largely compatible with previously published data from transcriptomics studies. All in all, the present data indicate that transport, in particular metal transport, adhesion, cell wall remodelling and the unknown function carried out by the unique EF0764 are important for enterococcal adaptation to the urine environment. These results provide a basis for a more targeted exploration of novel proteins involved in the adaptability and pathogenicity of E. faecalis.

New type of antimicrobial protein produced by the plant pathogen Clavibacter michiganensis subsp. michiganensis.

It has previously been shown that the tomato pathogen Clavibacter michiganensis subsp. michiganensis secretes a 14-kDa protein, C. michiganensis subsp. michiganensis AMP-I (CmmAMP-I), that inhibits growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato. Using sequences obtained from tryptic fragments, we have identified the gene encoding CmmAMP-I and we have recombinantly produced the protein with an N-terminal intein tag. The gene sequence showed that CmmAMP-I contains a typical N-terminal signal peptide for Sec-dependent secretion. The recombinant protein was highly active, with 50% growth inhibition (IC50) of approximately 10 pmol, but was not toxic to potato leaves or tubers. CmmAMP-I does not resemble any known protein and thus represents a completely new type of bacteriocin. Due to its high antimicrobial activity and its very narrow inhibitory spectrum, CmmAMP-1 may be of interest in combating potato ring rot disease.

Identification of surface proteins in Enterococcus faecalis V583.

BACKGROUND: Surface proteins are a key to a deeper understanding of the behaviour of Gram-positive bacteria interacting with the human gastro-intestinal tract. Such proteins contribute to cell wall synthesis and maintenance and are important for interactions between the bacterial cell and the human host. Since they are exposed and may play roles in pathogenicity, surface proteins are interesting targets for drug design. RESULTS: Using methods based on proteolytic "shaving" of bacterial cells and subsequent mass spectrometry-based protein identification, we have identified surface-located proteins in Enterococcus faecalis V583. In total 69 unique proteins were identified, few of which have been identified and characterized previously. 33 of these proteins are predicted to be cytoplasmic, whereas the other 36 are predicted to have surface locations (31) or to be secreted (5). Lipid-anchored proteins were the most dominant among the identified surface proteins. The seemingly most abundant surface proteins included a membrane protein with a potentially shedded extracellular sulfatase domain that could act on the sulfate groups in mucin and a lipid-anchored fumarate reductase that could contribute to generation of reactive oxygen species. CONCLUSIONS: The present proteome analysis gives an experimental impression of the protein landscape on the cell surface of the pathogenic bacterium E. faecalis. The 36 identified secreted (5) and surface (31) proteins included several proteins involved in cell wall synthesis, pheromone-regulated processes, and transport of solutes, as well as proteins with unknown function. These proteins stand out as interesting targets for further investigation of the interaction between E. faecalis and its environment.

Characterization of garvicin ML, a novel circular bacteriocin produced by Lactococcus garvieae DCC43, isolated from mallard ducks (Anas platyrhynchos).

Lactococcus garvieae DCC43 produces a bacteriocin, garvicin ML (GarML), with a molecular mass of 6,004.2 Da. Data from de novo amino acid sequencing by tandem mass spectrometry and nucleotide sequencing by reverse genetics suggested that the bacteriocin is synthesized as a 63-amino-acid precursor with a 3-amino-acid leader peptide that is removed by cleavage. Subsequently, a covalent linkage between the N and C termini forms the mature version of this novel 60-amino-acid circular bacteriocin.

Isolation and characterization of a new bacteriocin, termed enterocin M, produced by environmental isolate Enterococcus faecium AL41.

The new bacteriocin, termed enterocin M, produced by Enterococcus faecium AL 41 showed a wide spectrum of inhibitory activity against the indicator organisms from different sources. It was purified by (NH4)2SO4 precipitation, cation-exchange chromatography and reverse phase chromatography (FPLC). The purified peptide was sequenced by N-terminal amino acid Edman degradation and a mass spectrometry analysis was performed. By combining the data obtained from amino acid sequence (39 N-terminal amino acid residues was determined) and the molecular weight (determined to be 4628 Da) it was concluded that the purified enterocin M is a new bacteriocin, which is very similar to enterocin P. However, its molecular weight is different from enterocin P (4701.25). Of the first 39 N-terminal residues of enterocin M, valine was found in position 20 and a lysine in position 35, while enterocin P has tryptophane residues in these positions.

Common mechanisms of target cell recognition and immunity for class II bacteriocins.

The mechanisms of target cell recognition and producer cell self-protection (immunity) are both important yet poorly understood issues in the biology of peptide bacteriocins. In this report, we provide genetic and biochemical evidence that lactococcin A, a permeabilizing peptide-bacteriocin from Lactococcus lactis, uses components of the mannose phosphotransferase system (man-PTS) of susceptible cells as target/receptor. We present experimental evidence that the immunity protein LciA forms a strong complex with the receptor proteins and the bacteriocin, thereby preventing cells from being killed. Importantly, the complex between LciA and the man-PTS components (IIAB, IIC, and IID) appears to involve an on-off type mechanism that allows complex formation only in the presence of bacteriocin; otherwise no complexes were observed between LciA and the receptor proteins. Deletion of the man-PTS operon combined with biochemical studies revealed that the presence of the membrane-located components IIC and IID was sufficient for sensitivity to lactococcin A as well as complex formation with LciA. The cytoplasmic component of the man-PTS, IIAB, was not required for the biological sensitivity or for complex formation. Furthermore, heterologous expression of the lactococcal man-PTS operon rendered the insensitive Lactobacillus sakei susceptible to lactococcin A. We also provide evidence that, not only lactococcin A, but other class II peptide-bacteriocins including lactococcin B and some Listeria-active pediocin-like bacteriocins also target the man-PTS components IIC and IID on susceptible cells and that their immunity proteins involve a mechanism in producer cell self-protection similar to that observed for LciA.

Identification of the DNA-binding site of the Rgg-like regulator LasX within the lactocin S promoter region.

LasX regulates the transcription of the divergent operons lasXY and lasA-W, which specify the production of lactocin S in Lactobacillus sakei L45. Using histidine-tagged LasX, and a DNA fragment containing the complete intergenic lasA-lasX region, electrophoresis mobility-shift (EMSA) analyses were employed to demonstrate that LasX binds to the lasA-lasX intergenic DNA. Two direct heptanucleotide motifs directly upstream of P(lasA-W), and a third imperfect copy of this motif, overlapping the -10 element of P(lasA-W), were identified as possible LasX-binding sites. To assess the role of the direct repeats in the binding of LasX to the intergenic lasA-lasX region, binding experiments were performed using DNA probes with different combinations of the repeats, and with arbitrarily chosen repeat substitutions. The result of these experiments demonstrated that only the middle repeat was required for the binding of LasX to the las-promoter region. This observation correlated with the results of subsequent reporter-gene analyses, thereby weakening the hypothesis of the involvement of the direct repeats in LasX-mediated transcription regulation. By analysing the ability of LasX to bind successively shortened derivatives of the original intergenic fragment, a tentative 19 bp minimum LasX-binding site was identified.

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.

LasX, a transcriptional regulator of the lactocin S biosynthetic genes in Lactobacillus sakei L45, acts both as an activator and a repressor.

The 11 kb las locus, present on the 50 kb plasmid pCIM1, specifies the production of the lantibiotic lactocin S in Lactobacillus sakei L45. The gene cluster is organized into two oppositely orientated operons, lasAMNTUVPJW (lasA-W) and lasXY, the former of which contains the biosynthetic, immunity and transport genes. We have previously shown that inactivation of lasX abolishes lactocin S production and causes a drastic reduction in lasA-specific transcripts (encoding pre-lactocin S). The aim of this study was to determine whether or not the product of lasX, which is significantly similar to Rgg-like regulators, was directly involved in transcriptional regulation of the lactocin S biosynthetic genes. The divergently orientated and overlapping promoters, P(lasA)(-W) and P(lasXY), were transcriptionally fused to the Escherichia coli gusA gene, and the activity of the fusions was assayed in the presence and absence of lasX, which was expressed on a separate plasmid. A significant stimulation of expression (5-6-fold) of the P(lasA-W)-gusA fusion was observed in the presence of lasX, whereas expression of the P(lasXY)-gusA construct was reduced 1.5-2-fold. Our results strongly suggest that LasX is a bifunctional regulatory protein, acting both as an activator of lasA-W transcription and as a repressor of lasXY transcription. While a transcription stimulation activity has been described for several of the Rgg-like proteins, the present study is the first to report an autorepressor function for a member of this protein group.