Metabolic pathways are complex dynamic systems whose response to perturbations and environmental challenges are governed by multiple interdependencies between enzyme properties, reactions rates, and substrate levels. Understanding the dynamics arising from such a network can be greatly enhanced by the construction of a computational model that embodies the properties of the respective system. Such models aim to incorporate mechanistic details of cellular interactions to mimic the temporal behavior of the biochemical reaction system and usually require substantial knowledge of kinetic parameters to allow meaningful conclusions. Several approaches have been suggested to overcome the severe data requirements of kinetic modeling, including the use of approximative kinetics and Monte-Carlo sampling of reaction parameters. In this work, we employ a probabilistic approach to study the response of a complex metabolic system, the central metabolism of the lactic acid bacterium Lactococcus lactis, subject to perturbations and brief periods of starvation. Supplementing existing methodologies, we show that it is possible to acquire a detailed understanding of the control properties of a corresponding metabolic pathway model that is directly based on experimental observations. In particular, we delineate the role of enzymatic regulation to maintain metabolic stability and metabolic recovery after periods of starvation. It is shown that the feedforward activation of the pyruvate kinase by fructose-1,6-bisphosphate qualitatively alters the bifurcation structure of the corresponding pathway model, indicating a crucial role of enzymatic regulation to prevent metabolic collapse for low external concentrations of glucose. We argue that similar probabilistic methodologies will help our understanding of dynamic properties of small-, medium- and large-scale metabolic networks models.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• biologické modely MeSH
• fruktosadifosfáty metabolismus   MeSH
• Lactococcus lactis metabolismus   MeSH
• metabolické sítě a dráhy MeSH
• metabolismus sacharidů * MeSH
• metoda Monte Carlo MeSH
• počítačová simulace MeSH
• statistické modely MeSH
• zpětná vazba fyziologická MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine.

• MeSH
• albuminy metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• ELISA MeSH
• HeLa buňky MeSH
• imobilizované proteiny metabolismus   MeSH
• Lactococcus lactis metabolismus   MeSH
• lidé MeSH
• metody zobrazení buněčného povrchu MeSH
• podjednotky proteinů metabolismus   MeSH
• povrchová plasmonová rezonance MeSH
• proteinové domény MeSH
• průtoková cytometrie MeSH
• rekombinace genetická genetika   MeSH
• rekombinantní proteiny metabolismus   MeSH
• ribozomy metabolismus   MeSH
• sekvenční homologie aminokyselin MeSH
• shiga toxin 1 chemie   metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed. Genes for the infrared fluorescent protein and for the human IgG-binding DARPin were cloned in all possible combinations to assess the protein yield. The dual promoter plasmid pNZDual enabled balanced expression of the two model proteins. It was exploited for the development of a single-plasmid inducible CRISPR-Cas9 system (pNZCRISPR) by using a nisin promoter, first to drive Cas9 expression and, secondly, to drive single guide RNA transcription. sgRNAs against htrA and ermR directed Cas9 against genomic or plasmid DNA and caused changes in bacterial growth and survival. Replacing Cas9 by dCas9 enabled CRISPR interference-mediated silencing of the upp gene. The present study introduces a new series of plasmids for advanced genetic modification of lactic acid bacterium L. lactis.

• MeSH
• antibakteriální látky farmakologie   MeSH
• CRISPR-Cas systémy MeSH
• editace genu metody   MeSH
• fermentace MeSH
• genetické inženýrství metody   MeSH
• genom bakteriální * MeSH
• guide RNA, Kinetoplastida genetika   metabolismus   MeSH
• imunoglobulin G genetika   metabolismus   MeSH
• klonování DNA MeSH
• Lactococcus lactis genetika   metabolismus   MeSH
• lidé MeSH
• methyltransferasy genetika   metabolismus   MeSH
• nisin farmakologie   MeSH
• plazmidy chemie   metabolismus   MeSH
• promotorové oblasti (genetika) účinky léků   MeSH
• proteiny teplotního šoku genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• transgeny * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

IL-23-mediated Th-17 cell activation and stimulation of IL-17-driven pro-inflammatory axis has been associated with autoimmunity disorders such as Inflammatory Bowel Disease (IBD) or Crohn’s Disease (CD). Recently we developed a unique class of IL-23-specific protein blockers, called ILP binding proteins that inhibit binding of IL-23 to its cognate cell-surface receptor (IL-23R) and exhibit immunosuppressive effect on human primary blood leukocytes ex vivo. In this study, we aimed to generate a recombinant Lactococcus lactis strain which could serve as in vivo producer/secretor of IL-23 protein blockers into the gut. To achieve this goal, we introduced ILP030, ILP317 and ILP323 cDNA sequences into expression plasmid vector containing USP45 secretion signal, FLAG sequence consensus and LysM-containing cA surface anchor (AcmA) ensuring cell-surface peptidoglycan anchoring. We demonstrate that all ILP variants are expressed in L. lactis cells, efficiently transported and secreted from the cell and displayed on the bacterial surface. The binding function of AcmA-immobilized ILP proteins is documented by interaction with a recombinant p19 protein, alpha subunit of human IL-23, which was assembled in the form of a fusion with Thioredoxin A. ILP317 variant exhibits the best binding to the human IL-23 cytokine, as demonstrated for particular L.lactis-ILP recombinant variants by Enzyme-Linked ImmunoSorbent Assay (ELISA). We conclude that novel recombinant ILP-secreting L. lactis strains were developed that might be useful for further in vivo studies of IL-23-mediated inflammation on animal model of experimentally-induced colitis.

• MeSH
• buňky Th17 účinky léků   MeSH
• ELISA MeSH
• interleukin-23 metabolismus   MeSH
• Lactococcus lactis metabolismus   MeSH
• lidé MeSH
• proteiny genetika   metabolismus   farmakologie   MeSH
• průtoková cytometrie MeSH
• rekombinantní proteiny genetika   metabolismus   farmakologie   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

AIM: To compare pH and conductivity used in the determination of growth in reconstituted skim milk (RSM), to determine whether the presence of one or two plasmids in Lactococcus lactis had any influence on growth, and whether AbiS improved bacteriophages resistance of L. lactis.

• MeSH
• analýza kolonii tvořících jednotek MeSH
• bakteriální proteiny genetika   MeSH
• bakteriální transformace MeSH
• bakteriofágy fyziologie   MeSH
• elektrická vodivost MeSH
• fermentace MeSH
• financování organizované MeSH
• koncentrace vodíkových iontů MeSH
• Lactococcus lactis genetika   růst a vývoj   MeSH
• mléko mikrobiologie   MeSH
• plazmidy aplikace a dávkování   MeSH
• potravinářská mikrobiologie MeSH
• sýr MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH

The widespread Mn2+-sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch aptamer from Xanthomonas oryzae at 2.96 Å resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >100 µs of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.

• MeSH
• bakteriální RNA chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   MeSH
• hořčík metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• krystalografie rentgenová MeSH
• Lactococcus lactis genetika   metabolismus   MeSH
• ligandy MeSH
• mangan metabolismus   MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• mutace MeSH
• regulace genové exprese u bakterií MeSH
• riboswitch fyziologie   MeSH
• signální transdukce * MeSH
• simulace molekulární dynamiky MeSH
• vazebná místa MeSH
• Xanthomonas metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Out of six samples of wastewater produced in the dairy industry, taken in 2017 at various places of dairy operations, 86 bacterial strains showing decarboxylase activity were isolated. From the wastewater samples, the species of genera Staphylococcus, Lactococcus, Enterococcus, Microbacterium, Kocuria, Acinetobacter, Pseudomonas, Aeromonas, Klebsiella and Enterobacter were identified by the MALDI-TOF MS and biochemical methods. The in vitro produced quantity of eight biogenic amines (BAs) was detected by the HPLC/UV-Vis method. All the isolated bacteria were able to produce four to eight BAs. Tyramine, putrescine and cadaverine belonged to the most frequently produced BAs. Of the isolated bacteria, 41% were able to produce BAs in amounts >100 mg L-1. Therefore, wastewater embodies a potential vector of transmission of decarboxylase positive microorganisms, which should be taken into consideration in hazard analyses within foodstuff safety control. The parameters of this wastewater (contents of nitrites, nitrates, phosphates, and proteins) were also monitored.

• MeSH
• Acinetobacter MeSH
• Aeromonas MeSH
• biogenní aminy chemie   MeSH
• chemické látky znečišťující vodu chemie   izolace a purifikace   MeSH
• Enterobacter MeSH
• Enterococcus MeSH
• karboxylyasy chemie   MeSH
• Klebsiella MeSH
• Lactobacillus MeSH
• Lactococcus MeSH
• Microbacterium MeSH
• mikrobiologie vody MeSH
• mlékárenství * MeSH
• odpadní voda analýza   mikrobiologie   MeSH
• Pediococcus MeSH
• Pseudomonas MeSH
• spektrofotometrie ultrafialová MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• Staphylococcus MeSH
• Streptococcus MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Evropa MeSH

Lactococcus lactis, a probiotic bacterium of food origin, has recently been demonstrated as a suitable strain for the production and in vivo delivery of therapeutically important proteins into the gut. We aimed to engineer recombinant L. lactis cells producing/secreting REX binding proteins that have been described as IL-23 receptor (IL-23R) blockers and IL-23R antagonists suppressing the secretion of cytokine IL-17A, a pivotal step in the T-helper Th17-mediated pro-inflammatory cascade, as well as in the development of autoimmune diseases, including inflammatory bowel disease (IBD). To reach this goal, we introduced cDNA sequences coding for REX009, REX115, and REX125 proteins into plasmid vectors carrying a Usp45 secretion signal, a FLAG tag sequence consensus, and a LysM-containing cA surface anchor (AcmA), thus allowing cell-surface peptidoglycan anchoring. These plasmids, or their non-FLAG/non-AcmA versions, were introduced into L. lactis host cells, thus generating unique recombinant L. lactis-REX strains. We demonstrate that all three REX proteins are expressed in L. lactis cells and are efficiently displayed on the bacterial surface, as tested by flow cytometry using an anti-FLAG antibody conjugate. Upon 10-fold concentration of the conditioned media, a REX125 secretory variant can be detected by Western blotting. To confirm that the FLAG/non-FLAG REX proteins displayed by L. lactis retain their binding specificity, cell-surface interactions of REX proteins with an IL-23R-IgG chimera were demonstrated by flow cytometry. In addition, statistically significant binding of secreted REX009 and REX115 proteins to bacterially produced, soluble human IL-23R was confirmed by ELISA. We conclude that REX-secreting L. lactis strains were engineered that might serve as IL-23/IL-23R blockers in an experimentally induced mouse model of colitis.

• Publikační typ
• časopisecké články MeSH

Recently, high interest has been attracted to the research of inflammatory bowel diseases (IBD). Recombinant probiotic bacteria may represent an interesting way to influence the course of IBD. Their benefits include cheap and simple production and easy manipulation of the genetic material. Several gene therapy and probiotic approaches already showed promising results in the past. The aim of this study was to test the probiotic potential of IL-10-expressing Escherichia coli Nissle 1917 in a mouse model of IBD and to compare it with control bacterial strains. The dextran sulphate sodium (DSS) model of colitis was examined for this purpose. Animals received control probiotic bacteria or modified probiotics (expressing IL-10) via gastric gavage. Body weight, stool consistency, food and water consumption were monitored. At the end of the experiment, the parameters of inflammation, oxidative stress and carbonyl stress were analysed in the samples and statistical analysis was performed. We prepared an anti-inflammatory probiotic Escherichia coli strain that we designated Nissle 1917/pMEC-IL10 and proved its anti-inflammatory properties, which are similar to those of the control probiotic strains Nissle 1917 and Lactococcus lactis/pMEC-IL10 in vivo. The probiotic therapy was successful according to several parameters, including colon length, and oxidative and carbonyl stress. Bacterially produced IL-10 was detected in the plasma. The potential of bacterial anti-inflammatory therapy of IBD using modified probiotics was outlined. The results opened a way for upcoming studies using modified probiotics for therapy of systemic diseases.

• MeSH
• feces mikrobiologie   MeSH
• fruktosamin metabolismus   MeSH
• interleukin-10 krev   farmakologie   terapeutické užití   MeSH
• karbonylace proteinů účinky léků   MeSH
• kolitida krev   farmakoterapie   mikrobiologie   patologie   MeSH
• kolon účinky léků   patologie   MeSH
• malondialdehyd metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• oxidační stres účinky léků   MeSH
• probiotika farmakologie   terapeutické užití   MeSH
• rekombinantní proteiny farmakologie   terapeutické užití   MeSH
• tělesná hmotnost účinky léků   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH