The fate of phage-infected bacteria is determined by the holin, a small membrane protein that triggers disruption of the membrane at a programmed time, allowing a lysozyme to attack the cell wall. S2168, the holin of phage 21, has two transmembrane domains (TMDs) with a predicted N-in, C-in topology. Surprisingly, TMD1 of S2168 was found to be dispensable for function, to behave as a SAR ("signal-anchor-release") domain in exiting the membrane to the periplasm, and to engage in homotypic interactions in the soluble phase. The departure of TMD1 from the bilayer coincides with the lethal triggering of the holin and is accelerated by membrane depolarization. Basic residues added at the N-terminus of S2168 prevent the escape of TMD1 to the periplasm and block hole formation by TMD2. Lysis thus depends on dynamic topology, in that removal of the inhibitory TMD1 from the bilayer frees TMD2 for programmed formation of lethal membrane lesions. Like the holin S of λ, the holin of lambdoid phage 21 (S21) controls lysis by forming holes in the membrane. However, unlike Sλ, these holes are small, serving only to depolarize the membrane facilitating the release and activation of the SAR endolysin, R21. We were able to demonstrate that, unlike Sλ, S2168 forms a “pinhole”, thus macromolecules easily pass through Sλ but not S21 holes. This result again supports our interpretation: when S21 triggers, it only needs to collapse the membrane potential, thus causing release and activation of the membrane-tethered inactive SAR endolysin, but does not form holes in the membrane large enough to allow passage of a pre-folded, active cytoplasmic endolysin. The lysis defective S2168 mutant alleles were isolated throughout the S21 gene. Although the majority of lysis defective mutations occurred in the codons for the TMD2 domain, two mutations were found in the codons for the TMD1. This result suggests that only the TMD2 domain of S2168 is likely to participate in actual hole formation. One can assume that two mutant alleles of TMD1 are involved in two different interactions: (a) TMD1-TMD1 intermolecular interaction, (b) TMD1-TMD2 intramolecular interaction. We showed that there is a specific TMD1-TMD2 interaction. In terms of TMD1-TMD2 interaction, the mutated residues of the two TMD1 mutants might prevent a departure of TMD1 from TMD2, resulting in the lysis defective phenotype. Hopefully, these findings deliver some hints about the mechanism of S2168 hole formation and further provoke more extensive work which is required to provide a definite answer to many questions regarding this matter.

Bacteriophages (phages) are viruses that infect bacteria. The phages that are described by this dissertation encompass 2 classes, double-stranded DNA phages and single-stranded RNA phages. While both of these phages infect similar bacteria, they have adopted different mechanisms to lyse, or destroy, the cell in order to release phage progeny. dsDNA phages have large genomes (> 20 kb) and use multiple lysis proteins (holin, endolysin, and spanin complex) to lyse the cell. ssRNA phages, on the other hand, have small genomes (< 6 kb) and only encode one lysis protein. The two X-ray crystallography projects outlined here deal with the phage proteins involved in these lysis mechanisms. The project described in the first study deals with the holin (T) and the antiholin (RI) of the ds-DNA phage T4, the major players of the lysis inhibition (LIN) phenomenon. Crystal structures of the holin and of the holin-antiholin complex are presented. The structures provide new molecular level insights into the phenomenon of LIN in bacteriophage T4 and the T-even phages in general. The second investigation describes ongoing efforts at structural characterization of A2, the maturation protein of the ssRNA bacteriophage Qbeta that inhibits E. coli MurA. In addition, the structure of Bacillus subtilis MurA, which is not recognized by A2, is presented. The crystal structure of B. subtilis MurA, the first structure of MurA from a Gram-positive organism, allows for a direct comparison of Gram-positive and Gram-negative homologs and for identification of any significant structural differences. The more flexible catalytic loop of B. subtilis MurA protrudes farther out compared to the loop of E. coli MurA and creates enough hindrance to prevent A2 from establishing secure contact points.

The timing of host lysis is the only decision made in the bacteriophage lytic cycle. To optimize timing, double-stranded DNA phages use a 2-component lysis system consisting of a muralytic enzyme, the endolysin, and a small membrane protein, the holin, which controls the timing of lysis. The best characterized holin gene to date is the S gene of bacteriophage λ. One unusual feature of the S gene is that it produces two proteins of opposing function: the holin, S105, and the antiholin, S107. Raab et al isolated and characterized a number of S mutants, but all of them expressed both the holin and the antiholin; it is possible, then, that the true extent of the holin-holin interactions were masked by interactions with the antiholin. Thus, a large number of S105 mutants were created, and their phenotypes characterized in the absence of the antiholin. The interaction between those mutants and the wild-type were examined in an attempt to better understand what determines the timing of hole formation by S105. S105 and S107 differ only by two amino acids at the N-terminus; S107 has an additional Met-Lys sequence. Previous studies have shown that S107 may have a different topology to S105, where the N-terminus of S107 is located in the cytoplasm and is cannot flip through the membrane because of the extra cationic side chain. This study investigates the role of the N-terminal transmembrane domain of the S proteins in terms of hole formation and its role in the antiholin character of S107. Previous results suggest that S105 forms hole via a large oligomeric structure termed the “death raft”. The death raft model states that after S105 is inserted into the membrane, it forms “rafts”, which grow in size until a spontaneous channel forms leading to depolarization of the membrane and hole formation. This study investigates the pathway of hole formation at the single-cell level, using a C-terminal fusion of S105 and green fluorescent protein, and attempts to address several of the predictions posed by the death raft model.

The 105aa lambda S protein is the prototype holin, S accumulates in the cytoplasmic membrane during late gene expression until, at a time programmed into its primary structure, it disrupts the membrane and allows the lambda lysozyme, R, to attack the cell wall. In this study, a zwitterionic detergent Empigen BB, was used to extract and purify the lambda holin S. In Empigen BB, CD analysis on S gave 54% alpha helical content, consistent with 3 TM domains, which has been reported by other in vivo studies. Empigen BB-purified S can be exchanged into a chaotropic solution by dialysis and reconstituted into preformed lipid vesicles for activity assays. When diluted to fluorescein-loaded suspensions of liposomes, different chaotrope-solubilized S alleles caused dye release reflective of their in vivo phenotypes. The problem was the low efficiency of delivery of S to the liposomes. Unfortunately, dye loaded liposomes are highly sensitive to any detergent, making it necessary to find other ways to solubilize S. GroEL, a chaperonin from E. coli, is responsible for folding and refolding globular proteins in vitro. It has also been reported that GroEL improves the ability of a membrane protein synthesized in vitro to insert post-translationally into liposomes. This work will investigate the behavior of GroEL towards membrane proteins. The first of two membrane proteins studied in this respect is Bacteriorhodopsin (BR), a membrane proton pump, from H. halibium. The second is the105aa S protein, a prototype holin from bacteriophage lambda. Holin and BR subjected to detergent removal in the presence of GroEL remained in solution, while in the control sample (without GroEL) S and BR precipitated. "GroELsolubilized" holin still retained its lesion forming activity and solubilized BR maintained its proton pumping ability, detected by using a liposome dye activity assay unique to each protein. This approach may be applicable to other systems requiring detergent- or chaotrope-free preparation of membrane proteins. Finally, these results suggest that GroEL may be involved in the insertion of integral membrane proteins into the lipid bilayer, a role heretofore unsuspected.

Double stranded DNA bacteriophages achieve release of phage progeny by disrupting the cell envelope of the host cell. This is accomplished by two phage-encoded proteins, the holin and the endolysin. In bacteriophage lambda, the S holin is a small three TMD membrane protein that creates a lesion in the inner membrane of the host at a specific time, programmed in its primary structure. Lesion formation permits the cytoplasmic endolysin R access to the murein cell wall for degradation and cell lysis. Although it has been shown that S oligomerizes in the membrane, the structural nature of this complex has not been elucidated. In this study the S holin was purified using a mild non-ionic detergent and the structure of a ring complex formed by the holin was determined by electron microscopy and single particle analysis at a resolution of 2.6 nm. Biochemical characterization of the rings suggests that such a complex might represent the assembly formed by S in the membrane. Protein translocation in all organisms allows the export of proteins destined for localization outside the cytoplasm. In eubacteria, newly synthesized proteins are directed to the heterotrimeric membrane complex SecYEG by signals embedded in their sequence. The driving force through this complex is provided by the cytoplasmic ATPase SecA which combines ATP hydrolysis to mechanically insert proteins through the protein conducting channel. Using electron microscopy and single particle analysis we have obtained the structure of SecA from M. tuberculosis. The structure indicates that four SecA monomers assemble to form an elongated molecule with D2 symmetry. Docking of the EM map to the crystal structure of tb SecA confirms this arrangement of the subunits. This finding, that M. tuberculosis SecA forms a tetramer raises intriguing possibilities about SecA function.

Double stranded DNA bacteriophages achieve release of phage progeny by disrupting the cell envelope of the host cell. This is accomplished by two phage-encoded proteins, the holin and the endolysin. In bacteriophage lambda, the S holin is a small three TMD membrane protein that creates a lesion in the inner membrane of the host at a specific time, programmed in its primary structure. Lesion formation permits the cytoplasmic endolysin R access to the murein cell wall for degradation and cell lysis. Although it has been shown that S oligomerizes in the membrane, the structural nature of this complex has not been elucidated. In this study the S holin was purified using a mild non-ionic detergent and the structure of a ring complex formed by the holin was determined by electron microscopy and single particle analysis at a resolution of 2.6 nm. Biochemical characterization of the rings suggests that such a complex might represent the assembly formed by S in the membrane. Protein translocation in all organisms allows the export of proteins destined for localization outside the cytoplasm. In eubacteria, newly synthesized proteins are directed to the heterotrimeric membrane complex SecYEG by signals embedded in their sequence. The driving force through this complex is provided by the cytoplasmic ATPase SecA which combines ATP hydrolysis to mechanically insert proteins through the protein conducting channel. Using electron microscopy and single particle analysis we have obtained the structure of SecA from M. tuberculosis. The structure indicates that four SecA monomers assemble to form an elongated molecule with D2 symmetry. Docking of the EM map to the crystal structure of tb SecA confirms this arrangement of the subunits. This finding, that M. tuberculosis SecA forms a tetramer raises intriguing possibilities about SecA function.

Lysis of the host by bacteriophage 21 requires two proteins: the pinholin S21 (forms pinholes in the cytoplasmic membrane and controls lysis timing) and the endolysin (degrades the cell wall). S21 has a dual-start motif, encoding a holin, S2168, and a weak antiholin, S2171. Both proteins have two transmembrane domains (TMD) and adopt an N-in, C-in topology. The topology of S2168 is dynamic because TMD1 is a signal-anchor-release (SAR) domain which, while initially integrated into the cytoplasmic membrane, is eventually released into the periplasm. TMD1 is dispensable because the truncated protein, S2168?TMD1, retains the holin function. Adding two positive charges to N-terminus of S2168 by an irs tag (RYIRS) prevents the release of TMD1. The irsS2168 protein not only has lost its holin function, but is a potent antiholin and blocks the function of S2168. In this dissertation, the structure of S2168 was suggested by incorporating electron-microscopy, biochemical, and computational approaches. The results suggest that S2168 forms a symmetric heptamer, with the hydrophilic side of TMD2 lining the channel of ~ 15 A in diameter. This model also identifies two interacting surfaces, A and B, of TMD2. A model for the pinhole formation pathway was generated from analyzing phenotypes of an extensive collection of S21 mutants. In this model, the individually folded and inserted S21 molecules first form the inactive dimer, with the membrane-inserted TMD1 inhibiting the lethal function of TMD2 both inter- and intra-molecularly. A second inactive dimer may form, with one TMD1 released. When both TMD1s are released, the activated dimer is formed, with the homotypic interfaces A:A interaction of the TMD2s. However, this interaction might not be stable, which will shift to heterotypic A:B interactions, allowing TMD2 to oligomerize. Finally, the pinhole forms, possibly driven by the hydration of lumenal hydrophilic residues. In addition, the localization of pinholes was visualized by fusing the green fluorescent protein (GFP) to the C-terminus of pinholins. The results showed that pinholins form numerous small aggregates, designated as rafts, spread all over the cell body. The antiholin irsS2168 not only inhibits the triggering of S2168GFP, but inhibits the rafts formation as well.

Bacteriophage holins are a diverse group of proteins that are responsible for the spontaneous and specifically-timed triggering of host cell lysis. The best-studied holin, S105 of phage lambda, is known to form lesions, or “holes”, in the inner membrane of E. coli which are large enough to allow the endolysin through to the periplasm. S105 has been studied extensively by both genetic and biochemical approaches; however, little is known about the mechanism of hole formation or the structure of the lambda holin and its inner membrane lesion. An in vitro system for reconstituting hole formation by S105 was developed in which liposomes containing a self-quenched fluorophore served as artificial cell membranes (1-2). Upon delivery of solubilized S105 to the liposomes, an increase in fluorescence was observed, indicating that the fluorophore within the liposomes had escaped into the surrounding media via an S105-mediated hole in the membrane. This in vitro system, which has been optimized in this work, has been a valuable biochemical tool for analysis and reconstitution of the pathway to S105 hole formation in the cell membrane. Due to the difficulty associated with over-expression and purification of toxic membrane proteins, there are no solved structures of bacteriophage holins. Sample preparation and experimental conditions for NMR spectroscopy were optimized and structural information about a lambda holin mutant protein was obtained. Specifically, micellar contacts of transmembrane domain regions versus water contacts of the C-terminal region, secondary structure, and backbone dynamics were determined. Cryo-electron microscopy was used to visualize the inner membrane lesions formed by phage holins [lambda] S105, P2 Y, and T4 T. Therefore, the large holes initially seen in cells expressing S105 are not specific to the lambda holin, nor to class I holins. The S105 holes average ~340 nm (3), and are the largest membrane lesions ever observed in biology. They are stable at their original size, and are not localized to a specific region of the membrane. In addition, missense mutants of S105 were used to correlate hole size, protein accumulation, and lysis timing in a current model for the S105 hole formation pathway.

The aim of the thesis was to analyze and subsequent analysis of forest regeneration for the period 2003 - 2012 at the Training Forest Enterprise Masaryk Forest Křtiny. The bases for the analysis were the data from forest management records. Based on the data obtained there was made a comparison between natural and artificial regeneration of forest, it was compared to the proportion of first and repeated reforestation. Thesis also analyzes the recovery in relation to stand conditions and trees.

There is an increasing incidence of antimicrobial-resistant organisms isolated from food and food products. Coupled with that rising incidence is increased media scrutiny and coverage of outbreaks of foodborne illnesses. Consequently, consumers increasingly demand safer food, and that the antimicrobial measures used be other than antimicrobial drugs. A possible solution is to use bacteriophages, or the purified holin and endolysin proteins that make them lethal and lytic, as antimicrobial food treatments or additives. The bacteriophage Felix O1 is a promising candidate for development as an anti-Salmonella food treatment. This dissertation describes the work done to determine if these proteins could be of value as bioremedial agents. Endolysin treatments of Gram negative bacteria require two agents: the lytic endolysin, and a second agent to permeabilize the outer membrane of the bacterium. The holin protein was proposed as an outer membrane permeabilization agent. Methods used to locate the holin gene included BLAST analysis, analysis of putative Felix O1 proteins for transmembrane domains, and examination of the lysin sequence for an N-terminal signal sequence. Analyses did not reveal a promising candidate. Cloning of rIIA as a potential holin was attempted without success. Results of various analyses are discussed, as are chemical alternatives to the use of purified holin as a permeabilization agent. The endolysin, LysO1, was successfully cloned and characterized. PHYRE analysis predicted that the enzyme structure is composed of α helices arranged into two lobes, with the active site in a cleft between them. The enzyme lysed all tested strains of Salmonella and a tested strain of the foodborne pathogen Escherichia coli. Campylobacter jejuni susceptibility remains ambiguous, and the enzyme had no effect on Listeria monocytogenes or Micrococcus luteus. LysO1 was most active at alkaline pH and low ionic strength; optimal activity was observed in 25 mM buffer at pH 10. If removed from frozen storage, the enzyme was most thermostable at 30 °C. Lytic activity was adversely affected by the presence of the divalent cations calcium, magnesium, and zinc, and by high ionic strength. Considerable time was devoted to development of the activity assay used to further characterize the enzyme, and details of those experiments are provided. Logical extensions of the research project, such as further characterization and testing needed to obtain government approval for widespread use of the treatment, and possible pursuit of treatment based on an enzyme derivative such as an antimicrobial peptide, are discussed. / Ph. D.