Fuel molecules are organic solvents that have disruptive effects on the bacterial membrane. This is a significant barrier in biofuel production, as it limits the fuel concentration that can be achieved through fermentation. One potential way of overcoming this barrier is to identify lipid compositions that can better withstand solvent stress, for which it is important to understand how organic solvents disrupt the membrane. Use of biophysical characterization techniques to quantify physical properties like fluidity and thickness will enable us to understand the mechanism by which solvents disrupt membranes. Native membranes are very complex, and we sought to develop in-vitro models for the membrane of the bacterium Bacillus subtilis that use pure phospholipids. Toward this goal, a number of the unusual B. subtilis fatty acids were synthesized, partial synthesis of the membrane phospholipids was achieved, and preliminary assessment of solvent effects on standard lipids was performed using a fluorescence technique.

The master's thesis is focused on study of specific protective proteins from animal products. Two different types of antimicrobial egg white proteins were studied in detail - antimicrobial protein ovotransferrin (conalbumin) and enzyme lysozyme. Ovotransferrin belongs to transferrin group of proteins and exhibits activities similar to milk protective protein lactoferrin. The main effects of ovotransferrin are antiviral, anticancer and immunomodulatory. Antimicrobial activity of ovotransferrin based on the possibility to bind iron is still a subject of interest. For comparison the second egg protein lysozyme (N-acetyl muramidglycan hydrolase) was used. Lysozyme is a hydrolytic enzyme which primary attack cell wall of bacteria. In the theoretical part of the thesis an overview of the specific antimicrobial proteins in selected animal products was introduced mainly focused on ovotransferrin and lysozyme. The experimental part of this work was focused on optimization of methods for the determination of antimicrobial activity, protein concentration and purity. For quantitative analysis of total proteins, optimized Hartree – Lowry spectrophotometric method was used. For the determination of molecular weight and purity SDS-PAGE was used and stained by Coomassie Brilliant Blue G250 and silver. In experimental part the real sample of egg white was compared with samples of lyophilized antimicrobial proteins and therapeutical pills supplied by industrial partner. Protein composition and purity of these preparative has been determined. Antimicrobial activity of ovotransferrin was studied on cultures of G+ bacterium Bacillus subtilis and for comparison on G– E. coli. Ovotransferrin showed antimicrobial effect only at very high concentrations of about 75 mg/ml (Bacillus subtilis) and 50 mg/ml (E coli) even with addition of high amount (100 mM) of hydrogen carbonate ions. The inhibitory effect was most evident in liquid media. On the other hand, lysozyme exhibited significant inhibitory activity from 0.3 mg/ml on gram positive bacteria. Inhibitory effect on E. coli was not observed. Another part of study was focused on isolation of ovotransferrin from egg white using gel permeation chromatography on Sephadex G100. As mobile phases 0.1 M phosphate buffer and 0.05 M Tris-HCl buffer were tested. By SDS-PAGE the purity of ovotransferin comparing to standard was evaluated. Finally, the encapsulation of ovotransferrin and lysozyme was tested. Ovotransferrin and lysozyme was encapsulated into liposome and chitosan particles. Particles stability, distribution and average size distribution were studied by dynamic light scattering and zeta potential measurement. The stability of particles in the model physiological conditions was studied too.

Bacterial DNA-dependent RNA polymerase (RNAP) is a key enzyme of bacterial transcription. Its activity must be tightly regulated. This could be done on the level of promoter DNA topology recognition, by changing the intracellular levels of metabolites, or by binding proteins, known as transcription factors. Even though the RNAP regulatory network has been intensively studied for decades, new regulators are still being described. The main focus of this Thesis is to characterize some of them: i) HelD, a novel RNAP interacting factor, with so far unknown protein 3D structure; ii) RNase J1, an enzyme with a unique mechanism of functioning; iii) Spx, a major regulator of gene expression in Bacillus subtilis, with still new roles to be defined and iv) the effect of the topological state of promoters on transcription. We identified HelD as an interacting protein of RNAP in Bacillus subtilis and described its biochemical properties. It stimulates transcription in an ATP-dependent manner, by enhancing recycling of RNAP molecules (Publication I). We published the first insight into the HelD structure by SAXS (small angle X-ray scattering) and deepened the understanding of HelD domain composition (Publication III). And finally, we were able to solve the cryo-EM structure of HelD:RNAP complexes from...

No description available.

Bacterial DNA-dependent RNA polymerase (RNAP) is a key enzyme of bacterial transcription. Its activity must be tightly regulated. This could be done on the level of promoter DNA topology recognition, by changing the intracellular levels of metabolites, or by binding proteins, known as transcription factors. Even though the RNAP regulatory network has been intensively studied for decades, new regulators are still being described. The main focus of this Thesis is to characterize some of them: i) HelD, a novel RNAP interacting factor, with so far unknown protein 3D structure; ii) RNase J1, an enzyme with a unique mechanism of functioning; iii) Spx, a major regulator of gene expression in Bacillus subtilis, with still new roles to be defined and iv) the effect of the topological state of promoters on transcription. We identified HelD as an interacting protein of RNAP in Bacillus subtilis and described its biochemical properties. It stimulates transcription in an ATP-dependent manner, by enhancing recycling of RNAP molecules (Publication I). We published the first insight into the HelD structure by SAXS (small angle X-ray scattering) and deepened the understanding of HelD domain composition (Publication III). And finally, we were able to solve the cryo-EM structure of HelD:RNAP complexes from...

No description available.

No description available.

Water-responsive biological materials make up a large fraction of the earth’s biomass. Organisms can exchange water with their environment to actuate organ movement, and this process has inspired engineers to mimic this for technological use. Hygroscopic biological materials are chemically and phylogenetically diverse, implying that there may be fundamental physical principles which can explain their mechanics. In this thesis I will detail the development of a theory, the hygroelastic model, that explains a number of surprising mechanical behaviors exhibited by the hygroscopic bacterial spore of Bacillus subtilis. The hygroelastic model relies on the idea that the nanoconfinement of water molecules near interfaces influences the mechanics of nanoporous biological materials. The effects associated with this restructuring are collectively referred to as Hydration Forces. I will explain how these forces give rise to the equilibrium, nonequilibrium, and hygroscopic mechanical behaviors of the bacillus subtilis spore. Further, I will explain how hydration forces predict a previously unrecognized mechanical transition in the spore that emerges under rapid compression. The predicted mechanical behaviors of the model were validated experimentally through the use of the Atomic Force Microscope (AFM). By modifying the traditional Hertz formula to account for a strain-dependent elastic modulus, we show that the hygroelastic model well explains the anomalous force-indentation curves collected on bacterial spores. We also confirm the existence of the mechanical transition which appears under rapid indentation. Using multiple AFM operational modes, we collected force-indentation curves across a wide range of contact times ranging from near a second to 10’s of microseconds. These experiments showed a rapid increase in elastic modulus occurring near the predicted timescale of the hygroelastic transition. Though these unique mechanical properties are uncommon in materials, the underlying assumptions of the hygroelastic theory are general. Because nanoporous hygroscopic matter is commonly found in nature, it is possible that hygroelastic model could be applied to a number of other biological structures as well. Notably, the hygroelastic model predicts that bacterial spores owe their elastic response to hydration forces, which emerge from a disruption of water structure near the porous interface. These ‘hydration solids,’ may represent a paradigm in materials. Their mechanical properties may find use in engineered materials, with tailored elasticity, dissipation, nonlinear response, and frequency response.

Bacillus subtilis strain 3610 is an ancestral undomesticated strain. It diers from the laboratory strain 168 in many aspects. One dierence in strain 3610 is the presence of plasmid pBS32 encoding the sigma factor N (σN). This σ factor is activated when DNA damage occurs and induces the bacteria's cell death. The aim of the Thesis was a systematic characterisation of σN-dependent transcription. First, I showed that plasmid-borne but not chromosome-borne predicted σN-dependent promoters were ac- tive in transcription in vitro. Next, the anities of RNAP with σN for DNA, initiating NTP (iNTP) were determined for both relaxed and supercoiled DNA templates. Sur- prisingly, the activity of RNAP on relaxed σN-dependent promoters was higher than on their supercoiled versions, an opposite trend than displayed by RNAP associated with other σ factors. This property of σN-dependent promoters was not encoded by the core promoter sequence. In summary, this Thesis contributed to our understanding of the bacterial transcription apparatus. 1

<p> Deletion in the chromosome of Bacillus subtilis strain Sp-H12-3 was demonstrated by Hg-Cs(2)SO(4) density gradient centrifugation. The base composition of the deleted DNA segments and transcription of m-RNA from these DNA segments were investigated. Physiological and biochemical studies of the mutant Sp-H12-3 yielded information on uridine derivatives which may be intimately associated with the process of sporulation. </p> / Thesis / Doctor of Philosophy (PhD)