Shortening of the Lactobacillus paracasei subsp. paracasei BGNJ1-64 AggLb Protein Switches Its Activity from Auto-aggregation to Biofilm Formation

AggLb is the largest (318.6 kDa) aggregation-promoting protein of Lactobacillus paracasei subsp. paracasei BGNJ1-64 responsible for forming large cell aggregates, which causes auto-aggregation, collagen binding and pathogen exclusion in vitro. It contains an N-terminus leader peptide, followed by six successive collagen binding domains, 20 successive repeats (CnaB-like domains) and an LPXTG sorting signal at the C-terminus for cell wall anchoring. Experimental information about the roles of the domains of AggLb is currently unknown. To define the domain that confers cell aggregation and the key domains for interactions of specific affinity between AggLb and components of the extracellular matrix (ECM), we constructed a series of variants of the aggLb gene and expressed them in Lactococcus lactis subsp. lactis BGKP1-20 using a lactococcal promoter. All of the variants contained a leader peptide, an inter collagen binding-CnaB domain region (used to raise an anti-AggLb antibody), an anchor domain and a different number of collagen binding and CnaB-like domains. The role of the collagen binding repeats of the N-terminus in auto-aggregation and binding to collagen and fibronectin was confirmed. Deletion of the collagen binding repeats II, III and IV resulted in a loss of the strong auto-aggregation, collagen and fibronectin binding abilities whereas the biofilm formation capability was increased. The strong auto-aggregation, collagen and fibronectin binding abilities of AggLb were negatively correlated to biofilm formation

Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials

Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37 degrees C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects

X-ray structural analysis and antitumor activity of new salicylic acid derivatives

This Thesis project is going to be situated in my home town Östersund, 600 kilometers north from Stockholm.It´s a small town with 60 000 people and it is the only city of the region Jämtland in Norrland, Sweden.In the city centre of Östersund there is one existing bus terminal station where the buses arrive with people from the entire region.The task for this Thesis project is to rebuild the existing bus station in Östersund where the actual terminal building is going to be completely demolished and the entire block within the current bus stops is going to be rearranged.The topography of the terminal area is now dividing the regional buses that arrives on the upper level from the city buses that stops at the lower level. Therefore the main challenge of the new proposal is how one would strengthen the junction between these two flows of people and how a new architecture could enable the encounter between the countryside and the urban city.Detta examensprojekt kommer att vara belägen i min hemstad Östersund, 600 kilometer norr om Stockholm. Det är en liten stad med 60 000 personer och det är den enda staden i regionen Jämtland, Norrland. I centrum av Östersund finns en befintlig bussterminalen där bussarna anländer med folk från hela regionen. Uppgiften för detta projekt är att bygga om den befintliga busstationen i Östersund topografin på terminalområdet i dagsläget separerar de regionala bussarna som anländer på den övre nivån från stadsbussarna som stannar på en lägre nivå. Den största utmaningen i det nya förslaget är hur man istället skulle kunna stärka förbindelsen mellan dessa två flöden av människor och hur en ny arkitektur kan möjliggöra mötet mellan glesbyggd och den urbana staden

Functional characterization of the Lactolisterin BU gene cluster of Lactococcus lactis subsp. lactis BGBU1-4

The gene cluster responsible for the production of the aureocin A53-like bacteriocin, lactolisterin BU, is located on plasmid pBU6 in Lactococcus lactis subsp. lactis BGBU1- 4. Heterologous expression of pBU6 confirmed that production and limited immunity to lactolisterin BU were provided by the plasmid. Comparative analysis of aureocin A53- like operons revealed that the structural genes shared a low level of identity, while other genes were without homology, indicating a different origin. Subcloning and expression of genes located downstream of the structural gene, lliBU, revealed that the lactolisterin BU cluster consists of four genes: the structural gene lliBU, the abcT gene encoding an ABC transporter, the accL gene encoding an accessory protein and the immL gene which provides limited immunity to lactolisterin BU. Reverse transcription analysis revealed that all genes were transcribed as one polycistronic mRNA. Attempts to split the lactolisterin BU operon, even when both parts were under control of the PlliBU promoter, were unsuccessful indicate g that expression of lactolisterin BU is probably precisely regulated at the translational level by translational coupling and is possible only when all genes of the operon are in cis constellation. Two ρ-independent transcription terminators were detected in the lactolisterin BU operon: the first in the intergenic region of the lliBU and abcT genes and the second at the end of operon. Deletion of the second transcription terminator did not influence production of the bacteriocin in lactococci

Elongator mutation in mice induces neurodegeneration and ataxia-like behavior

Cerebellar ataxias are severe neurodegenerative disorders with an early onset and progressive and inexorable course of the disease. Here, we report a single point mutation in the gene encoding Elongator complex subunit 6 causing Purkinje neuron degeneration and an ataxia-like phenotype in the mutant wobbly mouse. This mutation destabilizes the complex and compromises its function in translation regulation, leading to protein misfolding, proteotoxic stress, and eventual neuronal death. In addition, we show that substantial microgliosis is triggered by the NLRP3 inflammasome pathway in the cerebellum and that blocking NLRP3 function in vivo significantly delays neuronal degeneration and the onset of ataxia in mutant animals. Our data provide a mechanistic insight into the pathophysiology of a cerebellar ataxia caused by an Elongator mutation, substantiating the increasing body of evidence that alterations of this complex are broadly implicated in the onset of a number of diverse neurological disorders.The authors acknowledge the facilities, and the scientific and technical assistance of the Australian Phenomics Facility (APF), the Australian National University. The APF is supported by the Australian Phenomics Network (APN). The APN is supported by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program. We are very grateful to Jelena Bezbradica Mirkovic and Kate Schroder for providing NLRP3 KO and Caspase-1 KO animals and for their valuable discussion. We also thank Avril Robertson and Matthew Cooper for the gift of MCC950 and Trent Woodruff for advice regarding the administration of MCC950. We acknowledge Ting-Yu Lin and Andrzej Chramiec-Głąbik for providing labeled tRNAs. This work was supported by the POLONEZ1 Grant UMO-2015/19/P/NZ1/02514 from the National Science Centre, Poland and received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 665778 (M.G. and A.S.-K.) and the First Team grant First TEAM/ 2016-1/2 from the Foundation for Polish Science (S.G.)

Lactolisterin BU, a Novel Class II Broad-Spectrum Bacteriocin from Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4

peer-reviewedLactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by 2-propanol and purified to homogeneity by C18 solid-phase extraction and reversed-phase high-performance liquid chromatography. The molecular mass of the purified lactolisterin BU was 5,160.94 Da, and an internal fragment, AVSWAWQH, as determined by N-terminal sequencing, showed low-level similarity to existing antimicrobial peptides. Curing and transformation experiments revealed the presence of a corresponding bacteriocin operon on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4. Analysis of the bacteriocin operon revealed a leaderless bacteriocin of 43 amino acids that exhibited similarity to bacteriocin BHT-B (63%) from Streptococcus ratti, a bacteriocin with analogy to aureocin A.Ministarstvo Prosvete, Nauke i Tehnološkog Razvoj

Functional Characterization of the Lactolisterin BU Gene Cluster of Lactococcus lactis subsp. lactis BGBU1-4

The gene cluster responsible for the production of the aureocin A53-like bacteriocin, lactolisterin BU, is located on plasmid pBU6 in Lactococcus lactis subsp. lactis BGBU1-4. Heterologous expression of pBU6 confirmed that production and limited immunity to lactolisterin BU were provided by the plasmid. Comparative analysis of aureocin A53-like operons revealed that the structural genes shared a low level of identity, while other genes were without homology, indicating a different origin. Subcloning and expression of genes located downstream of the structural gene, lliBU, revealed that the lactolisterin BU cluster consists of four genes: the structural gene lliBU, the abcT gene encoding an ABC transporter, the accL gene encoding an accessory protein and the immL gene which provides limited immunity to lactolisterin BU. Reverse transcription analysis revealed that all genes were transcribed as one polycistronic mRNA. Attempts to split the lactolisterin BU operon, even when both parts were under control of the PlliBU promoter, were unsuccessful indicating that expression of lactolisterin BU is probably precisely regulated at the translational level by translational coupling and is possible only when all genes of the operon are in cis constellation. Two ρ-independent transcription terminators were detected in the lactolisterin BU operon: the first in the intergenic region of the lliBU and abcT genes and the second at the end of operon. Deletion of the second transcription terminator did not influence production of the bacteriocin in lactococci

Evaluation of dormance sunflower seed lots

U Laboratoriji za ispitivaje semena Instituta za kukuruz Zemun Polje, u toku redovnog ispitivanja klijavosti semena suncokreta, uoĉeno je kod dve partije semena veliki broj neklijalog semena. Seme je stavljeno na naklijavanje dva puta sa razliĉitim temperaturnim režimom. U toku prvog ispitivanja seme je stavljeno na klijanje metodom između filter papira, hlađenje semena je trajalo 6 dana, energija klijanja je oĉitana ĉetvrtog dana a ukupna klijavost desetog dana. Seme je naklijavano u klijalištu na 200C sa režmom svetlosti 8 sati dan, 16 sati noć. U toku drugog ispitivanja, seme suncokreta je postavljeno na klijanje istom metodom između filter papira, hlađeno je 6 dana, energija klijanja je oĉitana ĉetvrti dan a završna klijavost deseti dan. Naklijavanje je obavljeno u sobi klijalištu na naizmeniĉnoj temperaturi 20300C, sa svetlosnim režimom 8 sati dan, 16 sati noć. Urađen je i TTZ - tetrazolium test. Analizom dobijenih rezultata uoĉeno je da na kraju prvog ispitivanja izdvojen veliki broj neklijalog semena koje je bilo sveže bez simptoma bolesti. Na kraju drugog ispitivanja taj procenat je bio manji u odnosu na prvo ispitivanje. Naklijavanje semena na većoj, naizmeniĉnoj temperaturi 20300C, u toku drugog ispitivanja, smanjilo je broj neklijalog semena. TTZ testom je potvrđena vitalnost neklijalog semena.A great number of nongerminated sunflower seed of two lots were observed during regular testing in the Seed Testing Laboratory of the Maize Research Institute, Zemun Polje. Seeds were germinated twice under different temperature regimes. In the course of the first test, seeds were germinated between filter paper, cooling lasted 6 days, the first count was done on the fourth day, while total germination was determined on the 10th day. Seeds were germinated in the germination cabinet at 200oC with 8 h (light) to16 h (night) regime. In the course of the second test, the method applied was the same: sunflower seeds were germinated between filter paper, cooling lasted 6 days, the first count was done on the fourth day, while total germination was determined on the 10th day. Germination was performed in the germination cabinet at the alternating temperature of 20300oC, and the light regime of 8 h (light) to16 h (night). The TTZ -tetrazolium test was also performed. The analysis of obtained results showed that a great number of nongermination, but fresh and disease-free seeds were detected at the end of the first test. The corresponding percentage at the end of the second test was smaller. Seed germination at higher, alternating temperatures of 20300oC, in the course of the second test, reduced the number of nongerminated seeds. Seed viability was confirmed by the TTZ test