Lactococcus lactis Resistance to Aureocin A53- and Enterocin L50-Like Bacteriocins and Membrane-Targeting Peptide Antibiotics Relies on the YsaCB-KinG-LlrG Four-Component System.

Resistance to nonribosomally synthesized peptide antibiotics affecting the cell envelope is well studied and mostly associated with the action of peptide-sensing and detoxification (PSD) modules, which consist of a two-component system (TCS) and an ATP-binding cassette (ABC) transporter. In contrast, the mechanisms of resistance to ribosomally synthesized bacterial toxic peptides (bacteriocins), which also affect the cell envelope, are studied to a lesser extent, and the possible cross-resistance between them and antibiotics is still poorly understood. In the present study, we investigated the development of resistance of Lactococcus lactis to aureocin A53- and enterocin L50-like bacteriocins and cross-resistance with antibiotics. First, 19 spontaneous mutants resistant to their representatives were selected and also displayed changes in sensitivity to peptide antibiotics acting on the cell envelope (bacitracin, daptomycin, and gramicidin). Sequencing of their genomes revealed mutations in genes encoding the ABC transporter YsaCB and the TCS KinG-LlrG, the emergence of which induced the upregulation of the dltABCD and ysaDCB operons. The ysaB mutations were either nonsense or frameshift mutations and led to the generation of truncated YsaB but with the conserved N-terminal FtsX domain intact. Deletions of ysaCB or llrG had a minor effect on the resistance of the obtained mutants to the tested bacteriocins, daptomycin, and gramicidin, indicating that the development of resistance is dependent on the modification of the protein rather than its absence. In further corroboration of the above-mentioned conclusion, we show that the FtsX domain, which functions effectively when YsaB is lacking its central and C-terminal parts, is critical for resistance to these antimicrobials.

Cyclic di-AMP Oversight of Counter-Ion Osmolyte Pools Impacts Intrinsic Cefuroxime Resistance in Lactococcus lactis.

The broadly conserved cyclic di-AMP (c-di-AMP) is a conditionally essential bacterial second messenger. The pool of c-di-AMP is fine-tuned through diadenylate cyclase and phosphodiesterase activities, and direct binding of c-di-AMP to proteins and riboswitches allows the regulation of a broad spectrum of cellular processes. c-di-AMP has a significant impact on intrinsic ß-lactam antibiotic resistance in Gram-positive bacteria; however, the reason for this is currently unclear. In this work, genetic studies revealed that suppressor mutations that decrease the activity of the potassium (K+) importer KupB or the glutamine importer GlnPQ restore cefuroxime (CEF) resistance in diadenylate cyclase (cdaA) mutants of Lactococcus lactis Metabolite analyses showed that glutamine is imported by GlnPQ and then rapidly converted to glutamate, and GlnPQ mutations or c-di-AMP negatively affects the pools of the most abundant free amino acids (glutamate and aspartate) during growth. In a high-c-di-AMP mutant, GlnPQ activity could be increased by raising the internal K+ level through the overexpression of a c-di-AMP-insensitive KupB variant. These results demonstrate that c-di-AMP reduces GlnPQ activity and, therefore, the level of the major free anions in L. lactis through its inhibition of K+ import. Excessive ion accumulation in cdaA mutants results in greater spontaneous cell lysis under hypotonic conditions, while CEF-resistant suppressors exhibit reduced cell lysis and lower osmoresistance. This work demonstrates that the overaccumulation of major counter-ion osmolyte pools in c-di-AMP-defective mutants of L. lactis causes cefuroxime sensitivity.IMPORTANCE The bacterial second messenger cyclic di-AMP (c-di-AMP) is a global regulator of potassium homeostasis and compatible solute uptake in many Gram-positive bacteria, making it essential for osmoregulation. The role that c-di-AMP plays in ß-lactam resistance, however, is unclear despite being first identified a decade ago. Here, we demonstrate that the overaccumulation of potassium or free amino acids leads to cefuroxime sensitivity in Lactococcus lactis mutants partially defective in c-di-AMP synthesis. It was shown that c-di-AMP negatively affects the levels of the most abundant free amino acids (glutamate and aspartate) in L. lactis Regulation of these major free anions was found to occur via the glutamine transporter GlnPQ, whose activity increased in response to intracellular potassium levels, which are under c-di-AMP control. Evidence is also presented showing that they are major osmolytes that enhance osmoresistance and cell lysis. The regulatory reach of c-di-AMP can be extended to include the main free anions in bacteria.

The Lactococcal dgkB (yecE) and dxsA Genes for Lipid Metabolism Are Involved in the Resistance to Cell Envelope-Acting Antimicrobials.

The emergence of antibiotic-resistant bacteria led to an urgent need for next-generation antimicrobial agents with novel mechanisms of action. The use of positively charged antimicrobial peptides that target cytoplasmic membrane is an especially promising strategy since essential functions and the conserved structure of the membrane hinder the development of bacterial resistance. Aureocin A53- and enterocin L50-like bacteriocins are highly cationic, membrane-targeting antimicrobial peptides that have potential as next-generation antibiotics. However, the mechanisms of resistance to these bacteriocins and cross-resistance against antibiotics must be examined before application to ensure their safe use. Here, in the model bacterium Lactococcus lactis, we studied the development of resistance to selected aureocin A53- and enterocin L50-like bacteriocins and its correlation with antibiotics. First, to generate spontaneous resistant mutants, L. lactis was exposed to bacteriocin BHT-B. Sequencing of their genomes revealed single nucleotide polymorphisms (SNPs) in the dgkB (yecE) and dxsA genes encoding diacylglycerol kinase and 1-deoxy-D-xylulose 5-phosphate synthase, respectively. Then, selected mutants underwent susceptibility tests with a wide array of bacteriocins and antibiotics. The highest alterations in the sensitivity of studied mutants were seen in the presence of cytoplasmic membrane targeting bacteriocins (K411, Ent7, EntL50, WelM, SalC, nisin) and antibiotics (daptomycin and gramicidin) as well as lipid II cycle-blocking bacteriocins (nisin and Lcn972) and antibiotics (bacitracin). Interestingly, decreased via the SNPs accumulation sensitivity to membrane-active bacteriocins and antibiotics resulted in the concurrently increased vulnerability to bacitracin, carbenicillin, or chlortetracycline. It is suspected that SNPs may result in alterations to the efficiency of the nascent enzymes rather than a total loss of their function as neither deletion nor overexpression of dxsA restored the phenotype observed in spontaneous mutants.

Engineering Lactococcus lactis as a multi-stress tolerant biosynthetic chassis by deleting the prophage-related fragment.

BACKGROUND: In bioengineering, growth of microorganisms is limited because of environmental and industrial stresses during fermentation. This study aimed to construct a nisin-producing chassis Lactococcus lactis strain with genome-streamlined, low metabolic burden, and multi-stress tolerance characteristics. RESULTS: The Cre-loxP recombination system was applied to reduce the genome and obtain the target chassis strain. A prophage-related fragment (PRF; 19,739 bp) in the L. lactis N8 genome was deleted, and the mutant strain L. lactis N8-1 was chosen for multi-stress tolerance studies. Nisin immunity of L. lactis N8-1 was increased to 6500 IU/mL, which was 44.44% higher than that of the wild-type L. lactis N8 (4500 IU/mL). The survival rates of L. lactis N8-1 treated with lysozyme for 2 h and lactic acid for 1 h were 1000- and 10,000-fold higher than that of the wild-type strain, respectively. At 39 â„ƒ, the L. lactis N8-1 could still maintain its growth, whereas the growth of the wild-type strain dramatically dropped. Scanning electron microscopy showed that the cell wall integrity of L. lactis N8-1 was well maintained after lysozyme treatment. Tandem mass tags labeled quantitative proteomics revealed that 33 and 9 proteins were significantly upregulated and downregulated, respectively, in L. lactis N8-1. These differential proteins were involved in carbohydrate and energy transport/metabolism, biosynthesis of cell wall and cell surface proteins. CONCLUSIONS: PRF deletion was proven to be an efficient strategy to achieve multi-stress tolerance and nisin immunity in L. lactis, thereby providing a new perspective for industrially obtaining engineered strains with multi-stress tolerance and expanding the application of lactic acid bacteria in biotechnology and synthetic biology. Besides, the importance of PRF, which can confer vital phenotypes to bacteria, was established.

Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP.

Multidrug transporters can confer drug resistance on cells by extruding structurally unrelated compounds from the cellular interior. In transport assays, Hoechst 33342 (referred to as Hoechst) is a commonly used substrate, the fluorescence of which changes in the transport process. With three basic nitrogen atoms that can be protonated, Hoechst can exist as cationic and neutral species that have different fluorescence emissions and different abilities to diffuse across cell envelopes and interact with lipids and intracellular nucleic acids. Due to this complexity, the mechanism of Hoechst transport by multidrug transporters is poorly characterised. We investigated Hoechst transport by the bacterial major facilitator superfamily multidrug-proton antiporter LmrP in Lactococcus lactis and developed a novel assay for the direct quantitation of cell-associated Hoechst. We observe that changes in Hoechst fluorescence in cells do not always correlate with changes in the amount of Hoechst. Our data indicate that chemical proton gradient-dependent efflux by LmrP in cells converts populations of highly fluorescent, membrane-intercalated Hoechst in the alkaline interior into populations of less fluorescent, cell surface-bound Hoechst in the acidic exterior. Our methods and findings are directly relevant for the transport of many amphiphilic antibiotics, antineoplastic agents and cytotoxic compounds that are differentially protonated within the physiological pH range.

Baseline gut microbiome composition predicts metformin therapy short-term efficacy in newly diagnosed type 2 diabetes patients.

BACKGROUND: The study was conducted to investigate the effects of metformin treatment on the human gut microbiome's taxonomic and functional profile in the Latvian population, and to evaluate the correlation of these changes with therapeutic efficacy and tolerance. METHODS: In this longitudinal observational study, stool samples for shotgun metagenomic sequencing-based analysis were collected in two cohorts. The first cohort included 35 healthy nondiabetic individuals (metformin dose 2x850mg/day) at three time-points during metformin administration. The second cohort was composed of 50 newly-diagnosed type 2 diabetes patients (metformin dose-determined by an endocrinologist) at two concordant times. Patients were defined as Responders if their HbA1c levels during three months of metformin therapy had decreased by ≥12.6 mmol/mol (1%), while in Non-responders HbA1c were decreased by <12.6 mmol/mol (1%). RESULTS: Metformin reduced the alpha diversity of microbiota in healthy controls (p = 0.02) but not in T2D patients. At the species level, reduction in the abundance of Clostridium bartlettii and Barnesiella intestinihominis, as well as an increase in the abundance of Parabacteroides distasonis and Oscillibacter unclassified overlapped between both study groups. A large number of group-specific changes in taxonomic and functional profiles was observed. We identified an increased abundance of Prevotella copri (FDR = 0.01) in the Non-Responders subgroup, and enrichment of Enterococcus faecium, Lactococcus lactis, Odoribacter, and Dialister at baseline in the Responders group. Various taxonomic units were associated with the observed incidence of side effects in both cohorts. CONCLUSIONS: Metformin effects are different in T2D patients and healthy individuals. Therapy induced changes in the composition of gut microbiome revealed possible mediators of observed short-term therapeutic effects. The baseline composition of the gut microbiome may influence metformin therapy efficacy and tolerance in T2D patients and could be used as a powerful prediction tool.

BacSJ-Another Bacteriocin with Distinct Spectrum of Activity that Targets Man-PTS.

Lactic acid bacteria produce diverse antimicrobial peptides called bacteriocins. Most bacteriocins target sensitive bacteria by binding to specific receptors. Although a plethora of bacteriocins have been identified, for only a few of them the receptors they recognize are known. Here, we identified permease IIC and surface protein IID, two membrane subunits of the mannose-specific quaternary phosphotransferase system (Man-PTS), as a receptor for BacSJ, a subclass IId bacteriocin produced by Lactobacillus paracasei subsp. paracasei BGSJ2-8. BacSJ shares 45% identity with another Man-PTS binding bacteriocin, garvicin Q (GarQ). Similarly to GarQ, BacSJ has a relatively broad activity spectrum acting against several Gram-positive bacteria, such as Lactococcus lactis and Listeria monocytogenes, harboring fairly similar Man-PTSs, but not against Lactococcus garvieae. To identify specific Man-PTS amino acids responsible for the L.lactis sensitivity to BacSJ, and thus likely involved in the interaction with this bacteriocin, we generated eight independent BacSJ resistant L.lactis mutants harboring five distinct missense mutations in the ptnC or ptnD genes encoding the IIC and IID subunits. Concurrently with the resistance to BacSJ, the mutants efficiently utilized mannose as a carbon source, which indicated functionality of their mutated Man-PTS. The amino acid substitutions in the mutants localized to the intracellular region of the IIC permease or to the extracellular parts of IID. This localization coincides with regions targeted by GarQ and some other Man-PTS-binding garvicins, pointing to similarities between all these bacteriocins in the mechanism of their interaction with Man-PTS. During the attack by these bacteriocins, subunits IID and IIC are assumed to function sequentially as a docking and an entry module allowing the toxic peptide to bind the cell and then open the pore. However, since not all of the BacSJ-resistant mutants exhibited cross-resistance to GarQ, we propose that BacSJ interacts with Man-PTS in a manner slightly different from that of GarQ.

Genomics and transcriptomics analysis reveals the mechanism of isobutanol tolerance of a laboratory evolved Lactococcus lactis strain.

Isobutanol, in spite of its significant superiority over ethanol as a biofuel, remains commercially non-viable due to the non-availability of a suitable chassis which can handle the solvent toxicity associated with its production. To meet this challenge, we chose Lactococcus lactis which is known for its ability to handle environmental stress and carried out Adaptive laboratory evolution (ALE) in a continuous stirred tank reactor (CSTR) to evolve an isobutanol tolerant strain. The strain was grown for more than 60 days (> 250 generations) while gradually increasing the selection pressure, i.e. isobutanol concentration, in the feed. This led to the evolution of a strain that had an exceptionally high tolerance of up to 40 g/l of isobutanol even though a scanning electron microscope (SEM) study as well as analysis of membrane potential revealed only minor changes in cellular morphology. Whole genome sequencing which was done to confirm the strain integrity also showed comparatively few mutations in the evolved strain. However, the criticality of these mutations was reflected in major changes that occurred in the transcriptome, where gene expression levels from a wide range of categories that involved membrane transport, amino acid metabolism, sugar uptake and cell wall synthesis were significantly altered. Analysing the synergistic effect of these changes that lead to the complex phenotype of isobutanol tolerance can help in the construction of better host platforms for isobutanol production.

Systemic understanding of Lactococcus lactis response to acid stress using transcriptomics approaches.

During fermentation, acid stress caused by the accumulation of acidic metabolites seriously affects the metabolic activity and production capacity of microbial cells. To elucidate the acid stress-tolerance mechanisms of microbial cells, we performed genome mutagenesis combined with high-throughput technologies to screen acid stress-tolerant strains. Mutant strain Lactococcus lactis WH101 showed a 16,000-fold higher survival rate than that of the parent strain after 5 h of acid shock at pH 4.0 and maintained higher ATP, NH4+, and intracellular pH (pHi) levels during acid stress. Additionally, comparative transcriptomics analysis revealed enhanced regulation of carbohydrate metabolism and sugar transport to provide additional energy, amino acid metabolism and transport to maintain pHi homeostasis and ATP generation, and fatty acid metabolism to enhance cellular acid tolerance. Moreover, overexpression of identified components resulted in 12.6- and 12.9-fold higher survival rates after acid shock for 3 h at pH 4.0 in L. lactis (ArcB) and L. lactis (MalQ) compared to the control strain, respectively. These findings provide valuable insight into the acid stress-response mechanisms of L. lactis and promote the further development of robust industrial strains.

Ampicillin-treated Lactococcus lactis MG1363 populations contain persisters as well as viable but non-culturable cells.

Lactococcus lactis is used as cell-factory and strain selections are regularly performed to improve production processes. When selection regimes only allow desired phenotypes to survive, for instance by using antibiotics to select for cells that do not grow in a specific condition, the presence of more resistant subpopulations with a wildtype genotype severely slows down the procedure. While the food grade organism L. lactis is not often exposed to antibiotics we characterized its response to ampicillin in more detail, to better understand emerging population heterogeneity and how this might affect strain selection procedures. Using growth-dependent viability assays we identified persister subpopulations in stationary and exponential phase. Growth-independent viability assays revealed a 100 times larger subpopulation that did not grow on plates or in liquid medium, but had an intact membrane and could maintain a pH gradient. Over one third of these cells restored their intracellular pH when we induced a temporary collapse, indicating that this subpopulation was metabolically active and in a viable but non-culturable state. Exposure of L. lactis MG1363 to ampicillin therefore results in a heterogeneous population response with different dormancy states. These dormant cells should be considered in survival-based strain selection procedures.

Bypassing lantibiotic resistance by an effective nisin derivative.

The need for new antibiotic compounds is rising and antimicrobial peptides are excellent candidates to fulfill this object. The bacteriocin subgroup lantibiotics, for example, are active in the nanomolar range and target the membranes of mainly Gram-positive bacteria. They bind to lipid II, inhibit cell growth and in some cases form pores within the bacterial membrane, inducing rapid cell death. Pharmaceutical usage of lantibiotics is however hampered by the presence of gene clusters in human pathogenic strains which, when expressed, confer resistance. The human pathogen Streptococcus agalactiae COH1, expresses several lantibiotic resistance proteins resulting in resistance against for example nisin. This study presents a highly potent, pore forming nisin variant as an alternative lantibiotic which bypasses the SaNSR protein. It is shown that this nisin derivate nisinC28P keeps its nanomolar antibacterial activity against L. lactis NZ9000 cells but is not recognized by the nisin resistance protein SaNSR. NisinC28P is cleaved by SaNSR in vitro with a highly decreased efficiency, as shown by an cleavage assay. Furthermore, we show that nisinC28P is still able to form pores in the membranes of L. lactis and is three times more efficient against SaNSR-expressing L. lactis cells than wildtype nisin.

Adaption to glucose limitation is modulated by the pleotropic regulator CcpA, independent of selection pressure strength.

BACKGROUND: A central theme in (micro)biology is understanding the molecular basis of fitness i.e. which strategies are successful under which conditions; how do organisms implement such strategies at the molecular level; and which constraints shape the trade-offs between alternative strategies. Highly standardized microbial laboratory evolution experiments are ideally suited to approach these questions. For example, prolonged chemostats provide a constant environment in which the growth rate can be set, and the adaptive process of the organism to such environment can be subsequently characterized. RESULTS: We performed parallel laboratory evolution of Lactococcus lactis in chemostats varying the quantitative value of the selective pressure by imposing two different growth rates. A mutation in one specific amino acid residue of the global transcriptional regulator of carbon metabolism, CcpA, was selected in all of the evolution experiments performed. We subsequently showed that this mutation confers predictable fitness improvements at other glucose-limited growth rates as well. In silico protein structural analysis of wild type and evolved CcpA, as well as biochemical and phenotypic assays, provided the underpinning molecular mechanisms that resulted in the specific reprogramming favored in constant environments. CONCLUSION: This study provides a comprehensive understanding of a case of microbial evolution and hints at the wide dynamic range that a single fitness-enhancing mutation may display. It demonstrates how the modulation of a pleiotropic regulator can be used by cells to improve one trait while simultaneously work around other limiting constraints, by fine-tuning the expression of a wide range of cellular processes.

A new furan derivative from an endophytic Aspergillus tubingensis of Decaisnea insignis (Griff.) Hook.f. & Thomson.

A new furan derivative named 3-(5-oxo-2,5-dihydrofuran-3-yl) propanoic acid (1) was isolated for the first time. Its structure was elucidated by UV, IR, NMR, HR-ESI-MS and the single-crystal X-ray diffraction spectroscopic data. Meanwhile, the antifungal and antibacterial activities of compound 1 was tested, it exhibited potent antifungal activity against Fusarium graminearum with MIC value of 16 µg/mL and medium antibacterial activity against Streptococcus lactis with MIC value of 32 µg/mL.

Method of preservation and type of protective agent strongly influence probiotic properties of Lactococcus lactis: A complete process of probiotic preparation manufacture and use.

In order to assess the essential probiotic properties of a strain dedicated for administration in humans and animals, characteristics of finally formulated products, rather than the cells solely, seems to be of crucial importance. In this study, composition of protective blends for manufacture of L. lactis probiotic powders was optimized using a statistical experimental design. The powders, generated by either spray- or freeze-drying techniques, were subsequently subjected to storage testing, and in vitro digestion in simulated stomach and small intestine. Finally, maintenance of adherence capability to human enterocyte-like cell lines, was evaluated. Our data demonstrated that 10% trehalose ensures the highest viability of L. lactis bacteria upon both drying techniques (viability of 60-68%). Moreover, skimmed milk-protected spray-dried cells exhibit the highest resistance to harsh environmental conditions of stomach (53.9 ±â€¯7.6% survival rate) and higher adhesion ability to HT-29 cell line after digestion (528 ±â€¯29 cells per 100 epithelial cells).

Reverse micelles as nano-carriers of nisin against foodborne pathogens. Part II: The case of essential oils.

During the last years, the food industry is working on the replacement of high energy methodologies with more sustainable techniques for the encapsulation of natural preservatives, in order to enhance their effectiveness as food additives. In the present study, nisin, an antimicrobial agent, was encapsulated in essential oil-containing microemulsions. More specifically, rosemary, thyme, oregano, and dittany essential oil-containing microemulsions were formulated to encapsulate nisin enhancing the system's overall antimicrobial activity. The systems were investigated for the interfacial properties and size of the surfactants' monolayer using electron paramagnetic resonance spectroscopy and dynamic light scattering. Subsequently, nisin-loaded microemulsions were tested for their antimicrobial activity against Lactococcus lactis, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus, using the well diffusion assay. Finally, this technique was validated by a killing assay. Overall, this study provides important information on the antibacterial activity of nisin-loaded nano-carriers enhanced by essential oils, in relation to the microemulsions' structure.

Infective endocarditis caused by Lactococcus lactis subsp. lactis and Pediococcus pentosaceus: A case report and literature review.

RATIONALE: Lactococcus lactis and Pediococcus pentosaceus are rare pathogens which rarely caused infections in humans. Several cases with L. lactis endocarditis have been reported in the literature, among them few were caused by L. lactis subsp. Lactis. Opportunistic P. pentosaceus infections were rarely reported. PATIENT CONCERNS: A 66-year-old man presented to our hospital due to persistent fever for 15 days. A physical checking revealed a grade II holosystolic murmur at the heart apex. A chest computed tomography (CT) scan suggested bronchitis. L. lactis subsp. lactis was identified in blood cultures. Transthoracic and transesophageal echocardiography revealed the presence of a large hyperechogenic mass in the left atrium, and a large floating vegetation on the mitral valve with a severe mitral regurgitation. DIAGNOSIS: Infectious endocarditis caused by L. lactis subsp. Lactis was diagnosed. INTERVENTIONS: Levofloxacin (0.5 g/day) was used for 20 days; however, L. lactis subsp. lactis remained to be isolated from blood culture. Therefore, vancomycin (2 g/day) was used to replace levofloxacin. Six days after the treatment with vancomycin, the blood culture revealed no L. lactis subspecies lactis, but yielded a growth of gram-positive and non-spore forming cocci; and P. pentosaceus was identified. Antimicrobial susceptibility test revealed P. pentosaceus was sensitive to penicillin and levofloxacin. Vancomycin was discontinued, and levofloxacin (0.5 g/day) was restarted and treated for another 7 days. The patient recovered with negative blood culture results, and discharged from the hospital. OUTCOMES: The patient recovered with negative blood culture results, and discharged from the hospital. LESSONS: Our patient had a long-period of antibiotic treatment with strategy alterations. Standard interpretation criteria of Clinical and Laboratory Standards Institute (CLSI) for L. lactis should be established, and molecular tests will increase the identification rate of L. lactis infections.

Secretion of tumoricidal human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by recombinant Lactococcus lactis: optimization of in vitro synthesis conditions.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively eliminates tumor cells. However, the short biological half-life of this molecule limits its potential use in the clinic. Our aim was to construct a recombinant strain of nonpathogenic Lactococcus lactis bacteria as a vector for effective and prolonged human TRAIL production. Herein, we examined the expression and secretion conditions leading to the production of biologically active protein in vitro. RESULTS: The human soluble TRAIL-cDNA (hsTRAIL-cDNA) with optimized codons was designed to fit the codon usage pattern (codon bias) of the L. lactis host. This cDNA construct was synthesized and cloned in lactococcal plasmid secretion vector pNZ8124 under the control of the nisin-induced PnisA promoter. The pNZ8124-hsTRAIL plasmid vector was transformed into the L. lactis NZ9000 host strain cells by electroporation. Secretion of the protein occurred at the neutral pH during induction, with optimized concentration of the inducer and presence of serine proteases inhibitor. Using Western blotting and amino acid sequencing method we found that TRAIL was secreted in two forms, as visualized by the presence of two distinct molecular size bands, both deprived of the usp45 protein, the bacterial signal peptide. By the use of MTS assay we were able to prove that hsTRAIL present in supernatant from L. lactis (hsTRAIL+) broth culture was cytotoxic to human HCT116 colon cancer cells but not to normal human fibroblasts. Flow cytometry analysis revealed TRAIL-induced apoptosis of cancer cells. CONCLUSIONS: We designed recombinant L. lactis bacteria, which efficiently produce biologically active, anti-tumorigenic human TRAIL in vitro. Further studies in tumor-bearing NOD-SCID mice will reveal whether the TRAIL-secreting L. lactis bacteria can be used as a safe carrier of this protein, capable of inducing effective elimination of human colon cancer cells in vivo.

Rapid Screening of Lanthipeptide Analogs via In-Colony Removal of Leader Peptides in Escherichia coli.

Most native producers of ribosomally synthesized and post-translationally modified peptides (RiPPs) utilize N-terminal leader peptides to avoid potential cytotoxicity of mature products to the hosts. Unfortunately, the native machinery of leader peptide removal is often difficult to reconstitute in heterologous hosts. Here we devised a general method to produce bioactive lanthipeptides, a major class of RiPP molecules, in Escherichia coli colonies using synthetic biology principles, where leader peptide removal is programmed temporally by protease compartmentalization and inducible cell autolysis. We demonstrated the method for producing two lantibiotics, haloduracin and lacticin 481, and performed analog screening for haloduracin. This method enables facile, high throughput discovery, characterization, and engineering of RiPPs.

Cheese brines from Danish dairies reveal a complex microbiota comprising several halotolerant bacteria and yeasts.

The Danish Danbo cheese is a surface ripened semi-hard cheese, which before ripening is submerged in brine for up to 24 h. The brining is required in order to obtain the structural and organoleptic properties of the cheeses. Likewise, the content of NaCl in the cheese will influence especially the surface microbiota being of significant importance for flavour development and prevention of microbial spoilage. Even though the microbiota on cheese surfaces have been studied extensively, limited knowledge is available on the occurrence of microorganisms in cheese brine. The aim of the present study was to investigate by both culture-dependent and -independent techniques the brine microbiota in four Danish dairies producing Danbo cheese. The pH of the brines varied from 5.1 to 5.6 with a dry matter content from 20 to 27% (w/w). The content of lactate varied from 4.1 to 10.8 g/L and free amino acids from 65 to 224 mg/L. Bacteria were isolated on five different media with NaCl contents of 0.85-23.0% (w/v) NaCl. The highest count of 6.3 log CFU/mL was obtained on TSA added 4% (w/v) NaCl. For yeasts, the highest count was 3.7 log CFU/mL on MYGP added 8% (w/v) NaCl. A total of 31 bacterial and eight eukaryotic species were isolated including several halotolerant and/or halophilic species. Among bacteria, counts of ≥6.0 log CFU/mL were obtained for Tetragenococcus muriaticus and Psychrobacter celer, while counts between ≥4.5 and < 6.0 log CFU/mL were obtained for Lactococcus lactis, Staphylococcus equorum, Staphylococcus hominis, Chromohalobacter beijerinckii, Chromohalobacter japonicus and Microbacterium maritypicum. Among yeasts, counts of ≥3.5 log CFU/mL were only obtained for Debaryomyces hansenii. By amplicon-based high-throughput sequencing of 16S rRNA gene and ITS2 regions for bacteria and eukaryotes respectively, brines from the same dairy clustered together indicating the uniqueness of the dairy brine microbiota. To a great extent the results obtained by amplicon sequencing fitted with the culture-dependent technique though each of the two methodologies identified unique genera/species. Dairy brine handling procedures as e.g. microfiltration were found to influence the brine microbiota. The current study proves the occurrence of a specific dairy brine microbiota including several halotolerant and/or halophilic species most likely of sea salt origin. The importance of these species during especially the initial stages of cheese ripening and their influence on cheese quality and safety need to be investigated. Likewise, optimised brine handling procedures and microbial cultures are required to ensure an optimal brine microbiota.

Effects of fermented green-loofah and green-papaya on nitric oxide secretion from murine macrophage raw 264.7 cells.

To clarify the effect of lactic acid bacteria (LAB) fermentation on the immunomodulation capacity of green-loofah and green-papaya, aqueous suspensions prepared from the fresh and dry-powdered vegetables were fermented by Lactococcus lactis subsp. lactis Uruma-SU1 and Lactobacillus plantarum Uruma-SU4. Fermented and non-fermented suspensions were added to murine macrophage RAW264.7 culture with and without Escherichia coli O111 lipopolysaccharide (LPS). In the absence of LPS, nitric oxide (NO) secretion was elevated significantly in LAB fermented suspensions compared to that in non-fermented suspensions. NO production in fermented suspensions was observed even at low sample concentrations, but it was attenuated in the centrifuged supernatant. With LPS treatment, inhibition of NO secretion was shown with the high concentration of the non-fermented and also fermented samples. These results suggest that fermented green-loofah and green-papaya suspensions can play both immunostimulatory and anti-inflammatory roles at low and high doses, respectively.