An in vitro initiation of a DNA replication system was developed to study the function of Ø29 terminal protein. Cell free extracts prepared from Bacillus phage Ø29-infected cells catalyzed the formation of a complex between a 30,000 dalton protein and dAMP in the presence of MgCl₂, Ø29 DNA-protein template and α-³²P dATP. Uninfected cell extracts did not support this reaction. The molecular weight of this complex, the nature of linkage between dAMP and 30,000 dalton protein as well as nucleotide specificity for this reaction suggest that the protein moiety of this complex is the terminal protein of Ø29. Similar results were obtained with phages GA-1 and M2Y infected cell extracts. The similar requirements for complex formation and DNA replication in vitro implies that the complex formation is an initiation step in DNA replication. This notion was supported by characterizing the elongation product which formed in the presence of ddCTP. Two distinct antibodies were prepared for analyse the function of the terminal protein in Ø29 DNA replication. These antibodies react with native terminal protein as assayed by immunoprecipitation and ELISA methods. The inhibition of complex formation by these antibodies provides strong evidence that the terminal protein is involved in complex formation. The notion that complex formation is an initial step of DNA replication was demonstrated conclusively by inhibition of anti-TP on DNA replication in vitro. The results presented in this dissertation provide evidence supporting the protein-priming mode of linear Ø29 DNA replication.

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Surfactin, a secondary metabolite produced by Bacillus subtilis, is a surface active compound and antibiotic permeabilizing membrane bilayer. The aim of this study was to reveal the self-resistance strategy at the level of the lipid moiety of cytoplasmic membrane, which B. subtilis employs to combat surfactin in concentrations that are lethal for other bacterial species. Non-producing strain B. subtilis 168 was cultivated in the presence of two different sublethal concentrations of surfactin (350 a 650 µg/ml), which was isolated from the culture broth of B. subtilis ATCC 21332. Presence of surfactin in the medium resulted in a concentration dependent lag phase, which took 40 min (350 µg/ml) and 3 h (650 µg/ml), respectively. Afterwards, the culture grew with the altered doubling time of 44 min (350 µg/ml) and 126 min (650 µg/ml), respectively. Surfactin induced substantial changes in the phospholipid composition of the cytoplasmic membrane. The proportion of the major phospholipid component phosphatidylglycerol decreased and inversely, the level of phosphatidylethanolamine increased. Interestingly, the content of phosphatidic acid rose considerably in the presence of surfactin concentration causing stimulation of B. subtilis growth (350 µg/ml). Liposome leakage assay using phospholipids mimicking...

BACKGROUND: Bacterial RNA polymerase (RNAP) is an extensively studied enzyme required for gene expression. In our Laboratory we found a new protein named HelD. HelD copurifies with B. subtilis RNAP. HelD is a ~90 kDa protein from the UvrD/Rep helicase family, which contains protein with the 3'-5' DNA unwinding activity. The molecular role(s) HelD in cell are still unknown and its potential role in transcription has not been studied so far. OBJECTIVE: The main aim of this Diploma project was to describe HelD. APPROACHES: The characterization was carried out on three levels: (i) bioinformatics analysis in silico was used to identify HelD homologs in other bacteria; (ii) growth tests in vivo were used to determine the phenotype(s) of the HelD-null mutant strain compared to wt; and (iii) biochemical experiments in vitro were utilized to describe the effects of HelD on transcription, and to test whether HelD has DNA binding and DNA unwinding activities. RESULTS: The in silico analysis revealed that HelD is present in Firmicutes, an industrially and medicinally important group of G+ bacteria. The phenotypic experiments showed that HelD is required for rapid adaptations to nutritional changes in the environment. The biochemical experiments showed that HelD stimulates transcription despite the fact that it...

Surfactin is an antibiotic produced by several strains of B. subtilis. Its broad range of biological activities is interesting from perspective of medicine, food industry and bioremediation and is based on its surface-active properties and interaction with biological membranes. The latter means mainly forming ion channels, conductive pores and with increasing concentration eventually disrupting membrane structure in detergent-like manner. Mechanism of resistance of producing strain against its own toxic product is not yet fully understood. This work shows that it could be based on surfactin target modification - which means altering membrane lipid composition. We were able to recognize surfactin-formed ion channels or pores with a broad range of conductivities spanning from 2 pS to 2 nS using BLM method. Liposome leakage assay with carboxyfluorescein revealed few distinct mechanisms of lysis, differing in amplitude, rate of lysis and cooperativity. Increased content of anionic lipids with conical shape, namely cardiolipin and phosphatidic acid led to substantial increased membrane resistance to surfactin-induced permeabilization. Key words: membrane, surfactin, Bacillus subtilis, cardiolipin, black lipid membranes, liposomes

Trensums Food AB är ett företag som producerar ett flertal olika livsmedelsprodukter åt flera globala varumärksägare. Några produkter som produceras av företaget är olika såser bl.a. BBQ-sås och vaniljsås. Genom att tillsätta syra i såserna så att pH blir < 4,2 anses produkterna säkra. Uppdragsgivare till produkten menar att det är viktigt att använda ättiksyra istället för andra syror och att inte pH är helt avgörande för en säker produkt. Syftet med projektet var att undersöka hur effektiva olika organiska svaga syror är på att avdöda olika mikroorganismer i BBQ-sås och mjölkbaserad sås. Det som främst påverkar den antimikrobiella aktiviteten hos en syra är mängden odissocierad syra. Mängden odissocierad syra i produkten beror på syrans pKa-värde, totala halten av syran och det omgivande pH i produkten. pH mättes i produkterna för att se om pH är avgörande för avdödningen eller om det är valet av syra som är viktigast. Syrorna som undersöktes var ättiksyra, citronsyra och mjölksyra på mikroorganismerna Candida utilis, Bacillus subtilis och Enterococcus faecium. BBQ-sås och mjölkbaserad sås tillverkades i olika batcher med viktprocenten 0, 0,12, 0,24 samt 0,36 % av respektive syra. I varje prov ympades respektive mikroorganism till en uppskattad halt på ca 103 CFU/ml. Efter 5 dagars inkubation i rumstemperatur mättes halten CFU/ml. Ättiksyra var den syra som var effektivast både i BBQ-såsen och den mjölkbaserade såsen. I BBQ-såsen reducerade ättiksyra med samtliga viktprocent alla mikroorganismerna till under minsta detektionsgränsen (< 101 CFU/ml). Citronsyra och mjölksyra reducerade enbart E.faecium och B.subtilis till under minsta detektionsgränsen. I den mjölkbaserade såsen som även var kontaminerad av andra mikroorganismer var det bara ättiksyra som synligt reducerade halten med ökad viktprocent syra. pH var lägre i BBQ-såsen med citronsyra och mjölksyra än med ättiksyra. Detta visar på att avdödningen av de undersökta mikroorganismerna inte bara är pH-beroende utan också påverkas av vilken syra som används. Den antimikrobiella aktiviteten beror på syrans pKa-värde och därmed mängden odissocierad syra. Ättiksyra har ett högre pKa-värde än både citronsyra och mjölksyra och befinner sig i högre utsträckning i odissocierad tillstånd än de övriga syrorna i båda produkterna. / Trensums Food AB is a company that produces a wide variety of food products to different global trademark owners. Some of the products produced by Trensums Food AB are different kind of sauces, including BBQ-sauce and vanilla sauce. The sauce products are considered safe when an acid is added resulting in a pH < 4,2. The outsource of the company claims that it is important to use acetic acid instead of other kind of acids and that the pH is not crucial to consider the product safe. The goal of this project was to investigate how effectively different kind of organic acids work against microorganisms in the BBQ-sauce and milk-based sauce produced by Trensums Food AB. The main antimicrobial activity of an acid depends on its concentration of undissociated acid. Which further depends on the pKa-value of the acid and also the surrounding pH in the product. The pH was measured in the products to see if pH is critical for the antimicrobial activity or if the choice of acid is more important. The acids used in this project was acetic acid, citric acid and lactic acid and they were tested on Candida utilis, Bacillus subtilis and Enterococci faecium. BBQ-sauce and milk-based sauce were made in different batches containing 0, 0,12, 0,24 and 0,36 weight percent of respective acid. Each batch contained 103 CFU/ml of respective microorganism. After 5 days of incubation a viable count was performed. The acetic acid was the most effective acid in both type of sauces, reducing all microorganisms below the detection limit (< 101 CFU/ml) in the BBQ-sauce within all batches. The citric acid and lactic acid reduced only E.faecium and B.subtilis to a value below the lowest level of detection. In the milk-based sauce, which was contaminated with additional microorganisms, it was only the acetic acid that showed an increasing effect of antimicrobial activity with increasing concentration of acid. The pH was lower in the BBQ-sauce when using citric acid and lactic acid compared to when using acetic acid. Thus concluding that the antimicrobial activity of the selected microorganisms is not solely dependent on the level of pH but additionally the type of acid being used. The acetic acid has a higher pKa-value compared to the other acids and is therefore to a higher extent in a undissociated state.

Bacterial RNA polymerase (RNAP) is a multisubunit complex essential for transcription of DNA into RNA. As a key enzyme responsible for regulation of gene expression it interprets regulatory signals from the cell and based on these cues RNAP adjusts transcription level of particular genes. This process is affected both by the regular subunits of RNAP as well as other transcription factors (TFs) directly or indirectly interacting with RNAP. The general focus of this Thesis was to extend the knowledge about the complex transcriptional regulatory networks and about the connections between individual pathways. The main specific topic and the main publication of the thesis are focused on the HelD protein, a novel binding partner of RNAP in Bacillus subtilis. We showed that HelD binds between the secondary channel of RNAP and alpha subunits of the core form of the enzyme. We proved that HelD stimulates transcription in an ATP dependent manner by enhancing transcriptional cycling and elongation. We revealed a new connection in the transcription regulatory machinery when we demonstrated that the stimulatory effect of HelD can be amplified by delta, a small subunit of RNAP specific for gram positive (G+) bacteria. Two other publications of the thesis are dealing with the delta subunit. We solved the 3D...

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Determina la actividad antimicrobiana in vitro de los extractos etanólicos del tallo y las hojas del Marrubium vulgare L. (pega pega); así como preparar un gel base al que se le incorporó los extractos que demostraron buenos resultados de inhibición del crecimiento bacteriano. La especie vegetal se recolectó en el departamento de Junín, provincia de Huancayo. El trabajo se desarrolló en 2 etapas: se evaluó la actividad antimicrobiana mediante el método de difusión en agar y se determinó la concentración mínima inhibitoria (CMI) por el método de microdilución colorimétrica frente a Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 y Bacillus subtilis ATCC 6633; asimismo se preparó un gel a base de carbomer al que se le agregó el extracto seco y luego se determinó la actividad antibacteriana del gel mediante el método de difusión en agar. El extracto seco del tallo presentó actividad antibacteriana a una concentración de 100 mg/mL (halo de inhibición ≥18 mm) frente a Staphylococcus epidermidis ATCC 12228 y Bacillus subtilis ATCC 6633 por el método de difusión en agar, mientras que en el caso del extracto de las hojas presento acción frente a todas las bacterias en estudio excepto para Escherichia coli ATCC 25922, por el método de microdilución, el extracto seco de las hojas presentó actividad antibacteriana significativa frente a Staphylococcus aureus ATCC 25923 (CMI = 666,67 ± 243,98 µg/mL), Staphylococcus epidermidis ATCC 12228 (CMI = 633,33 ± 228,87 µg/mL) y Bacillus subtilis ATCC 6633 (CMI = 300 ± 103,51 µg/mL), mientras que en el caso del tallo se observó un CMI de 733,33 ± 238,2 µg/mL para el Bacillus subtilis ATCC 6633. La preparación del gel se realizó en base a la consistencia y estabilidad, al ser incorporado los extractos a las concentraciones de 1 %, 2 % y 4 %, presentó mayor acción antibacteriana frente a Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228 y Bacillus subtilis ATCC 6633 al 2 % y 4 % tanto en tallos como en hojas respectivamente. / Tesis