Organization of the bacterial nucleoid by DNA-bridging proteins and globular crowders

The genomic DNA of bacteria occupies only a fraction of the cell called the nucleoid, although it is not bounded by any membrane and would occupy a volume hundreds of times larger than the cell in the absence of constraints. The two most important contributions to the compaction of the DNA coil are the cross-linking of the DNA by nucleoid proteins (like H-NS and StpA) and the demixing of DNA and other abundant globular macromolecules which do not bind to the DNA (like ribosomes). The present work deals with the interplay of DNA-bridging proteins and globular macromolecular crowders, with the goal of determining the extent to which they collaborate in organizing the nucleoid. In order to answer this question, a coarse-grained model was developed and its properties were investigated through Brownian dynamics simulations. These simulations reveal that the radius of gyration of the DNA coil decreases linearly with the effective volume ratio of globular crowders and the number of DNA bridges formed by nucleoid proteins in the whole range of physiological values. Moreover, simulations highlight the fact that the number of DNA bridges formed by nucleoid proteins depends crucially on their ability to self-associate (oligomerize). An explanation for this result is proposed in terms of the mean distance between DNA segments and the capacity of proteins to maintain DNA--bridging in spite of the thermal fluctuations of the DNA network. Finally, simulations indicate that non-associating proteins preserve a high mobility inside the nucleoid while contributing to its compaction, leading to a DNA/protein complex which looks like a liquid droplet. In contrast, self-associating proteins form a little deformable network which cross-links the DNA chain, with the consequence that the DNA/protein complex looks more like a gel

Post-translational modification of nucleoid-associated proteins: an extra layer of functional modulation in bacteria?

Post-translational modification (PTM) of histones has been investigated in eukaryotes for years, revealing its widespread occurrence and functional importance. Many PTMs affect chromatin folding and gene activity. Only recently the occurrence of such modifications has been recognized in bacteria. However, it is unclear whether PTM of the bacterial counterparts of eukaryotic histones, nucleoid-associated proteins (NAPs), bears a comparable significance. Here, we scrutinize proteome mass spectrometry data for PTMs of the four most abundantly present NAPs in Escherichia coli (H-NS, HU, IHF and FIS). This approach allowed us to identify a total of 101 unique PTMs in the 11 independent proteomic studies covered in this review. Combined with structural and genetic information on these proteins, we describe potential effects of these modifications (perturbed DNA-binding, structural integrity or interaction with other proteins) on their function.Macromolecular Biochemistr

DNA binding properties of histone-like protein HU from Deinoccus radiodurans suggest involvement in DNA recombination

The Histone-like protein HU is ubiquitous in eubacteria. Usually with a length of ~90 amino acids, they are predominantly homodimeric, with sequence and structural homology. Escherichia coli HU is involved in DNA repair and recombination. The crystal structure of Anabaena HU shows that it binds DNA with prolines intercalating into the DNA backbone, introducing two kinks at a spacing of 9 bp and bending the DNA through a variable angle of 105-140°. Deinococcus radiodurans is a gram positive mesophile, capable of reconstituting its genome from 1000-2000 double strand breaks incurred due to exposure to environmental extremes. In the first study, D. radiodurans HU (DrHU) is characterized in terms of its DNA binding properties. The binding site size of DrHU is the largest so far reported, ~50 bp. DrHU binds preferentially to four-way junction DNA with half-maximal saturation of 18 ± 2 nM. In distinct contrast to E. coli HU, DrHU has no marked preference for DNA with nicks or gaps compared to perfect duplex DNA, nor is it able of mediating circularization of linear duplex DNA. In the second study, the N-terminus of DrHU was truncated, generating ΔDrHU, and the functional role of the N-terminus investigated. ΔDrHU exhibits a binding site size of 17 ± 1 bp similar to HU homologs from other mesophiles. ΔDrHU also binds preferentially to four-way junction DNA, but protects the crossover rather than the junction arms protected by DrHU. The melting temperature of ΔDrHU of 46.4 ± 0.1°C is similar to that of HU from mesophiles. DrHU interacts with other D. radiodurans proteins(s) in the presence of four-way junction DNA, suggesting its role in DNA recombination. In a similar study with the HU homolog from Helicobacter pylori (HpyHU), the protein binds stably to four-way junction DNA with half-maximal saturation of 5.0 ± 0.5 nM. Thermal denaturation of HpyHU measured by circular dichroism spectroscopy yields a Tm = 56.4 ± 0.1°C suggesting greater than average thermal stability. Mutagenesis of HpyHU suggests that a differential target site selection of HU proteins is achieved through their individual capacity for inducing the required DNA bend

Interplay between the bacterial nucleoid protein H-NS and macromolecular crowding in compacting DNA

In this dissertation we discuss H-NS and its connection to nucleoid compaction and organization. Nucleoid formation involves a dramatic reduction in coil volume of the genomic DNA. Four factors are thought to influence coil volume: supercoiling, DNA charge neutralization, macromolecular crowding and DNA deformation by NAPs. This study focuses mainly on the latter two factors, and on their interplay. We investigate both direct and indirect changes in DNA coil volume as a result of H-NS binding to DNA. H-NS / DNA binding is thought to be influenced by the self-association of H-NS, hence DNA self-association (both in bulk and on DNA) has also been investigated. Chapter 2 focuses on the known cooperative character of H-NS-DNA binding. The molecular origin of the cooperativity is poorly understood. High concentrations of H-NS are known to oligomerize extensively in the absence of DNA. Some models propose that cooperativity is caused by the same protein-protein interactions that cause oligomerization in solution, whereas others propose cooperativity may be induced by the DNA substrate. We have mutated some parts of H-NS we believed to be important in oligomerization to investigate the role of H-NS protein-protein interactions in cooperative DNA binding, and studied the oligomerization and DNA-binding properties of these mutants. The D68VD71V mutant has two aspartic acids in the linker region replaced by valines, making the linker significantly more hydrophobic. The double linker mutation D68VD71V dramatically enhances H-NS oligomerization in solution, and its temperature-dependence is changed as well in vitro. Yet there is only a moderate effect on DNA binding properties, which does point in the direction of enhanced cooperativity, as expected. This suggests that protein-protein interactions have a much larger effect on H-NS self-association in solution than on the DNA binding properties. Chapter 3 discusses the influence of the bacterial nucleoid protein H-NS on DNA coil sizes in solution, using Light Scattering, for both supercoiled and linear pUC18 DNA. We clearly find H-NS binding: the intensity of light scattered by the DNA coils increased upon the addition of H-NS. But, H-NS did not have a significant effect on the effective hydrodynamic radius of the coils. Our results suggest that under the conditions of our experiment (in particular the buffer conditions: 10mM Sodium Phosphate buffer, pH 7, 100mM NaCl), the H-NS proteins most likely did not cause extensive bridging of dsDNA, since this most certainly would have led to a significant effect on the DNA coil sizes. This absence of bridging in the absence of multivalent cations is consistent with single molecule DNA force measurements performed for similar buffer conditions by other authors. We also find that, although H-NS alone does not have a dramatic effect on DNA coil sizes in solution (for our solution conditions), it does have an interesting synergetic effect on polymer-induced condensation of DNA. Condensation of H-NS/DNA complexes was measured by their sedimentability in solutions of polyethylene glycol (PEG). In the absence of H-NS the critical concentration of PEG needed to condense DNA is approximately 15%, whereas the critical concentration is remarkably lower, about 3.5%, at near saturation concentrations of H-NS. Chapter 4 is concerned with the effect of binding of H-NS and macromolecular crowding on nucleoid compaction. An osmotic shock method using ampicillin was used to isolate the Escherichia coli nucleoids intact, disrupting the peptidoglycan layer. These nucleoids were stained with DAPI and photographed using confocal microscopy. This showed a decrease in the volume of the isolated nucleoids when polyethylene glycol (PEG) concentrations became higher. The addition of small amounts of H-NS appeared to enhance the compaction due to macromolecular crowding induced by PEG. Remarkably, in the absence of PEG, H-NS did not affect the compaction of the nucleoids even at higher concentrations. The results are consistent with previous experiments done on DNA-binding proteins HU and Sso7d by other research groups. Therefore, our results confirm a general enhancement of macromolecular crowding effect of cytoplasm by nucleoid-associated proteins binding. In Chapter 5 we focus on an archaeal NAP. Like bacteria, archaea have NAPs that bend DNA and form extended helical protein-DNA fibers. These do not condense the genomic DNA directly, but some NAPS strongly promote DNA condensation by macromolecular crowding, such as the bacterial HU. Using theoretical arguments, we show that this synergy can be explained by the larger diameter and lower net charge density of protein-covered DNA filaments compared to naked DNA. Therefore the effect should be nearly universal in prokaryotes. We illustrate this general effect by demonstrating that Sso7d, a 7 kDa basic DNA-bending protein from the archaeon Sulfolobus Solfataricus, does not significantly condense DNA by itself, using light-scattering to determine coil volumes. However, the Sso7d-coated DNA fibres are much more susceptible to macromolecular crowding-induced condensation. Clearly, if DNA-bending nucleoid proteins fail to condense DNA in dilute solution, this does not mean that they do not contribute to DNA condensation in the context of the crowded living cell. </p

Histone-like protein from Mycobacterium smegmatis has two DNA binding domains and is localized to the nucleoid in vivo

Eubacteria encode numerous small basic histone-like proteins (such as HU, H-NS and Fis) that are required for nucleoid organization and for regulation of DNA-dependent processes. One of these histone-like proteins, HU from Escherichia coli has been shown to associate with the nucleoid and to regulate processes such as DNA repair and recombination. In contrast, the divergent HU homologs encoded by mycobacteria have been variously identified as involved in the physiology of dormancy, in the response to cold shock or as laminin binding proteins associated with the cell envelope. Using indirect fluorescent antibody microscopy, contrary to previous reports, it is shown that the HU-related histone-like protein Hlp from Mycobacterium smegmatis is nucleoid-associated. No evidence of surface exposed Hlp was found in cells treated for cell wall permeabilization. Quantitative Western blots indicate that exponentially growing cells contain ~120 molecules per cell, with up-regulation of Hlp after cold shock estimated to be ~10 fold. Hlp binds both DNA and RNA in vitro and protects DNA from hydroxyl radical- or DNase I-mediated damage. Hlp, which in addition to the HU fold, has a basic C-terminal tail composed of PAAK and PAKK repeats, has extremely high affinity for DNA. The binding affinity of Hlp for 76 bp linear DNA is greater, Kd = 0.037 ± 0.001 nM, compared to Hlp lacking the C-terminal repeats, Kd = 2.5 ± 0.05 nM and the C-terminal repeat domain, Kd = 0.82 ± 0.17 nM. Hlp lacking the entire C-terminal domain does not bind DNA up to 0.5 µM protein concentration. Hlp does not constrain DNA supercoil in the presence of Topoisomerase I but enhances DNA end-joining in the presence of T4 DNA ligase, and this property is mediated by the C-terminal repeats. At \u3c100 nM concentration, Hlp represses transcription by T7 RNA polymerase in vitro whereas the individual N- and C-terminal domains do not, even when added together. These data indicate Hlp domains contribute to high-affinity DNA binding. Combined, the data suggest that its primary functional role may be the DNA dependent responses to environmental stress rather than nucleoid organization

Master of Science

thesisThe histone-like nucleoid-structuring protein (H-NS) is well known as a global regulator of transcription. A number of studies have suggested that H-NS also positively influences the function of the flagellar motor, but the details of its motility-regulating action remain unclear. In an effort to characterize the actions of H-NS in the flagellar motor, we sought to test the effects of specific mutations in H-NS that are predicted to alter its state of multimerization. As a foundation for this work, we examined the effects of H-NS expression in strains that expressed the flagellar regulatory proteins FlhDC at various levels, from various plasmids. The results gave indications that certain plasmids previously used to provide FlhDC constitutively did not, in fact, express the proteins at levels sufficient to stimulate flagellar assembly. This complicates the interpretation of previous work, because the cells retained the chromosomal copies of the flhDC genes whose expression is known to be influenced by H-NS. Thus, effects in the previous experiments may have been the result of up-regulation of chromosomal flhDC rather than direct actions at the flagellar motor. To overcome this problem, I constructed new strains in which the chromosomal copies of flhDC were deleted, and revisited the question of HNS action in the motor. For these experiments, the flhDC genes were expressed from a regulatable plasmid that had been verified by complementation of the flhDC deletion strain, and H-NS was expressed from a second regulatable plasmid. The results indicate that H-NS contributes to flagellar motility in ways other than its stimulatory effect flhDC iv expression, as was suggested on the basis of the previous work. Details of its action are different from those reported previously. An analysis of mutants altered at interfaces needed for H-NS multimerization gives evidence that H-NS must act as a dimer or larger multimer, in both its gene-regulatory and motility regulating

Transcriptional Silencing and Anti-Silencing of Virulence Genes in the Bacterial Pathogen Shigella Flexneri: VIRB, DNA Supercoiling, and the Histone-Like Nucleoid Structuring Protein

Transcriptional silencing and anti-silencing affect many aspects of bacterial physiology, including virulence in bacterial pathogens. In Shigella species, a group of gram-negative pathogens that cause bacillary dysentery in humans, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences virulence genes found on the large virulence plasmid while VirB anti-silences these genes. However, the mechanistic details of their interplay are not fully understood. To elucidate their regulatory mechanisms, I use the icsP virulence locus, which shares a long intergenic region with the divergently transcribed ospZ gene (1535 bp from TSS to TSS). Prior to this work, two discrete H-NS binding regions had been identified, suggesting H-NS-mediated bridging of these two regions as the mechanism of silencing. However, I show that changes to the spacing and helical phasing designed to disrupt the potential bridging were tolerated, suggesting an alternate mechanism of silencing is at play. In addition to H-NS, two other H-NS homologs found in S. flexneri, StpA and Sfh, can also silence the icsP promoter. Interestingly, VirB counters transcriptional silencing mediated by these other H-NS homologs. The site required for VirB-dependent anti-silencing of the icsP promoter is located over 1 kb upstream of the TSS, and nearly 500 bp upstream of the ospZ promoter, but exactly how VirB accomplishes this long-range regulation is not known. I show that VirB docks to this recognition site in vitro and has a high specificity for this site in vivo. Using a combination of 1D and 2D chloroquine-based agarose gel electrophoresis, I demonstrate that, upon docking to its recognition site, VirB triggers a loss of negative supercoiling of our VirB-dependent PicsP-lacZ reporter; importantly, this phenomenon occurs with native VirB levels in S. flexneri. Because H-NS is sensitive to DNA topology at some promoters, it is tantalizing to envision that VirBmediated changes in supercoiling alleviate H-NS-mediated silencing of virulence genes in Shigella. Although anti-silencing proteins in other bacteria, including related pathogens, bear little sequence homology to VirB, the possibility that changes to DNA supercoiling mechanistically unite this group of proteins requires further consideration when studying transcriptional silencing and anti-silencing processes in bacteria

Characterization of function and regulation of the subtilase cytotoxin and Shiga toxin of pathogenic Escherichia coli

Food-borne diseases caused by enterohemorrhagic Escherichia coli (EHEC) constitute a great threat to human health worldwide. Pathogenicity of EHEC strongly depends on the ability to produce virulence factors such as amongst others bacterial toxins. One of these toxins are the so-called Shiga toxins (Stx), which is why EHEC are assigned to the group of Shiga toxin-producing Escherichia coli (STEC). Stx belong to the family of AB5 protein toxins consisting of two subunits. One of them, the StxA-subunit causes depurination of the 28S rRNA in eukaryotic ribosomes by exhibiting N-glycosidase activity subsequently leading to inhibition of the protein biosynthesis followed by apoptosis of the host cell. The second one is the homopentameric B-subunit, which mediates binding to the host cell surface via the receptor glycolipid globotriaosylceramide (Gb3). Besides Stx, the subtilase cytotoxin (SubAB) has been described in STEC in recent years. SubAB, also assigned to the family of AB5 toxins, generates its cytotoxic activity via cleavage of the endoplasmic chaperone binding immunoglobulin protein (BiP) by its A-subunit. This cleavage leads to an unfolded protein response, resulting in apoptosis of the host cell. The B-subunit forms a ring-like homopentameric structure which is responsible for the binding to the receptor N-glycolylneuraminic acid (Neu5Gc) and other O-glycans. Although the mode of cytotoxicity of AB5 toxins have been studied extensively, some mechanisms remain unsolved. The scope of this thesis was to analyze further the mode of action of AB5 toxins and the gene regulation of stx and subAB. Both publications included in this thesis combine the characterization of the cytotoxic activity of AB5 toxins, the regulation of their genes, their subunits, and the combination of subunits of Stx and SubAB. In the first publication the regulation of gene expression of AB5 toxins was investigated in more detail. In this study, the gene expression of subAB1 was analyzed with a luciferase reporter gene assay and by quantitative real-time polymerase chain reaction. To unravel the regulatory mechanisms, both the laboratory E. coli strain DH5&#945; and the STEC O113:H21 strain TS18/08 were used. Expression of subAB1 and promoter activity was studied using standard cultivation methods. Moreover, this work shed light on the impact of the global regulatory proteins host factor of bacteriophage Q&#946; (Hfq) and histone-like nucleoid structuring protein (H-NS) on subAB1 gene expression. Therefore, isogenic deletion mutants of hfq and hns gene were generated in the respective strains. Afterwards, plasmid-based complementation was conducted to verify that the observed effects were due to the deletion. Analysis of subAB1 promoter activity revealed impact of both Hfq and H-NS during different growth phases in both strains. In addition, the influence of both regulatory proteins on the expression toxin genes in STEC strain TS18/08 was investigated. This study did not only focus on the expression of stx2a and subAB1, but also the gene expression of the gene of the cytolethal distending toxin V (cdtV) was analyzed. Interestingly, all three toxin genes studied were upregulated in the deletion mutants of &#916;hfq and &#916;hns. Those results demonstrate the impact of global regulatory proteins on AB5 toxin gene expression and show that all three toxin genes investigated are integrated into the same regulatory network. In the second publication, the mode of action of AB5 toxins on the example of Stx2a was analyzed in more detail. The paradigm of AB5 toxin was known as the receptor binding B-subunit which mediates uptake of the enzymatic A-subunit and the subsequent cytotoxic activity. Previous studies have questioned this paradigm by showing cytotoxic effects of the SubA-subunit in absence of its corresponding B-subunit. This work analyzed whether this cytotoxic effect of the A-subunit is not only true for SubAB, but also for Stx. Thus, seperate recombinant expression of StxA2a subunits and subsequent His tag-based purification was performed. Both StxA2a-His and StxB2a-His were analyzed on cytotoxicity separately or in combination with the other subunit. Strikingly, cytotoxic effects of the StxA2a-His was observed in the absence of its corresponding B-subunit cell-type independently on HeLa, Vero B4, and HCT-116 cells. Studies on the B-subunit revealed no cytotoxicity on all cell lines. Additionally, combinations of different A- and B-subunits of Stx2a and SubAB1 proteins were analyzed. The hybrid combination showed that the cytotoxic effect of StxA2a-His on HeLa and HCT-116 cells could be reduced in the presence of the SubB1-His. Contrary, the cytotoxic effects of SubA1- His were unaltered in combination with StxB2a-His. Those results give the assumption that the Stx2aA-subunit binds to a target cell receptor blocked by SubB1-His. Additional experiments on the binding capacity of the Stx2a-subunits to Gb3 revealed that while StxB2a-His was able to bind to the receptor, no binding of the recombinant A-subunit was observed. The results indicate a cytotoxic effect of StxA2a on different cell types in absence of its corresponding B-subunit, which is designated as single-A effect in this work. The role of this effect in STEC pathogenicity, the uptake mechanism and subsequent transport inside the host cells of StxA-subunit need to be further analyzed in the future.Durch enterohämorrhagische Escherichia coli (EHEC) verursachten Lebensmittel-assoziierte Krankheiten stellen weltweit ein ernstes Problem dar. Dabei wird die Pathogenität der EHEC vor allem durch die Fähigkeit der Produktion von Virulenzfaktoren, wie z.B. Toxinen, definiert. Eines dieser Toxine ist das sogenannte Shiga Toxin (Stx), weshalb die EHEC zur Gruppe der Shiga Toxin-produzierenden E. coli (STEC) zugeordnet sind. Stx gehören zur Familie der AB5 Toxine und bestehen aus zwei Untereinheiten. Davon hemmt die StxA-Untereinheit die Proteinbiosynthese durch die Depurinierung der 28S rRNA der eukaryotischen Ribosomen, indem sie eine N-Glycosidase Aktivität aufweist und anschließend zur Apoptose der Zielzelle führt. Die zweite Untereinheit ist die homopentamere B-Untereinheit, welche an den Glykolipid-Rezeptor Globotriaosylceramid Gb3 auf der Membran der Zielzelle. In den letzten Jahren wurde in STEC neben dem Stx das Subtilase Zytotoxin (SubAB) beschrieben. Auch SubAB gehört zu der Familie der AB5 Toxine, wobei die A-Untereinheit die zytotoxische Aktivität durch die Spaltung des endoplasmatischen Chaperons Immunoglobulin-Bindeprotein (BiP) aufweist. Diese Spaltung führt zur unfolded protein response (dt. ungefaltete Protein-Antwort) und infolgedessen zum Zelltod. Die B-Untereinheit besteht aus fünf identischen Molekülen, welche ringartig angeordnet sind, und bindet an den Rezeptor N-glykolyneuraminsäure (Neu5Gc) und andere O-Glykane. Obwohl die Mechanismen der Zytotoxizität der AB5 Toxine bereits untersucht wurden, bleiben einige Mechanismen ungeklärt. Dabei stellt das Ziel dieser Dissertation die Charakterisierung der zytotoxischen Aktivität und Regulation der Genexpression von stx und subAB dar. Beide Publikationen im Rahmen dieser Dissertation kombinieren dabei die Charakterisierung der Zytotoxizität dieser AB5 Toxine, der Regulation ihrer Gene, ihrer Untereinheiten und der Kombination der Untereinheiten von SubAB und Stx. In der ersten Publikation wurde die Fragestellung der Regulation der Genexpression von AB5 Toxinen untersucht. Die Genexpression des subAB1 Gens wurde in dieser Studie mit Hilfe der Methoden des Luciferase-Reportergenassays und quantitativer Echtzeit-Polymerase-kettenreaktion (quantitative real time polymerase chain reaction) analysiert. Um die Fragestellung der regulatorischen Mechanismen zu untersuchen, wurden sowohl der Laborstamm E. coli DH5&#945; als auch der STEC O113:H21 Stamm TS18/08 verwendet. Die Expression und Promoteraktivität des subAB1 wurden unter Standard-Kulturbedingungen gemessen. Des Weiteren wurden vor allem der Einfluss der globalen Regulatorproteinen host factor of bacteriophage Q&#946; (Hfq) und des Histone-ähnlichen Nukleoid bindenden Proteins (H-NS) auf die subAB1 Genexpression untersucht. Dafür wurden isogene Deletionsmutanten der Gene hfq und hns in den beiden genannten Stämmen erstellt. Um zu beweisen, dass die gemessenen Effekte auf diesen Deletionen basieren, wurde anschließend eine Plasmid-basierte Komplementation durchgeführt. Die Analyse der Promoteraktivität zeigte, dass beide Regulatorproteine Hfq und H-NS Einfluss auf diese während unterschiedlichen Wachstumsphasen in beiden untersuchten Stämmen haben. Zusätzlich wurde der Einfluss der beiden Regulatorproteine auf die Genexpression der Toxingene im STEC TS18/08 untersucht. Dabei wurden in dieser Arbeit nicht nur die Gene stx2a, subAB1 untersucht, sondern auch das Gen des Cytolethalen Distending Toxins V (cdtV) betrachtet. Interessanterweise wurden alle drei untersuchten Gene in den Deletionsmutanten &#916;hfq und &#916;hns hochreguliert. Die Ergebnisse dieser Studie zeigen den Einfluss von globalen Regulatorproteinen auf die Genexpression von AB5 Toxinen und weisen auf eine Regulation der untersuchten Gene in einem gemeinsamen Netzwerk hin. In der zweiten Publikation wurde das Wirkprinzip der AB5 Toxine am Beispiel von Stx2a näher untersucht. Das bisher geltende Paradigma der AB5 Toxine umfasst den Wirkmechanismus der Rezeptorbindenden B-Untereinheit, welche die Aufnahme der enzymatischen A-Untereinheit und die damit verbundene Zytotoxizität vermittelt. Dieses Paradigma wurde erstmals durch vorangegangene Studien in Frage gestellt, welche die zytotoxische Aktivität der SubA-Untereinheit in Abwesenheit ihrer zugehörigen B-Untereinheit aufgezeigt hatten. In dieser Arbeit wurde geprüft, ob diese zytotoxische Aktivität der A-Untereinheit nicht nur für SubAB, sondern auch für Stx gilt. Daher wurden die Stx-Untereinheiten rekombinant exprimiert und anschließend separate His-Tag basierte Reinigungen durchgeführt. Sowohl StxA2a-His als auch StxB2a-His wurden auf ihre Zytotoxizität einzeln oder in Kombination mit der jeweils anderen Untereinheit analysiert. Auffallend war dabei die Zelltyp-unabhängige zytotoxische Aktivität der StxA2a-His Untereinheit in Abwesenheit der B-Untereinheit auf HeLa, Vero B4 und HCT-116 Zellen. Versuche mit StxB2a-His resultierten in keinerlei Zytotoxizität auf allen Zelllinien. Zusätzlich wurde die hybride Kombination aus verschiedenen Untereinheiten von SubAB1 und Stx2a untersucht. Dabei wurde gezeigt, dass die durch StxA2a-His ausgelösten Effekte auf HeLa und HCT-116 Zellen durch SubB1-His reduziert wurden. Im Gegensatz dazu blieb die durch SubA1-His induzierte Zytotoxizität bei Kombination mit StxB2a-His unverändert. Diese Versuche lassen auf eine StxA2a-His Zielstruktur auf der Zelloberfläche schließen, welche durch SubB1-His blockiert wird. Zusätzliche Versuche zur Bindungskapazität der Stx2a-Untereinheiten an Gb3 zeigten, dass obwohl eine Bindung von StxB2a-His zum Rezeptor nachgewiesen wurden, keine Bindung der rekombinanten A-Untereinheit an diesen festgestellt werden konnte. Die Ergebnisse dieser Studie weisen auf einen zytotoxischen Effekt der StxA2a-Untereinheit in Abwesenheit ihrer B-Untereinheit auf, welcher hier als Single-A Effekt bezeichnet wird. Zukünftig soll die Rolle dieses Effektes auf die Pathogenität der STEC, der Aufnahmemechanismus und der nachfolgende Transport der StxA-Untereinheit analysiert werden

Chromosome Organization and Segregation in Pseudomonas Aeruginosa

The faithful propagation of genetic information from a mother to its progeny is one of the most fundamental aspects of life. Encoding the entirety of an organisms' genetic information onto chromosomes poses a unique set of problems that cells are required to overcome for proper genetic flow. In bacteria, one or more DNA molecules are condensed almost 1000-fold in order to fit within the small vicinity of a single cell. While undergoing significant compaction, the chromosome must also retain its accessibility in order to perform various DNA dependent processes. Although several key elements of chromosome organization have been identified, our knowledge regarding this process remains limited. In order to maintain genetic integrity, newly replicated chromosomes must faithfully segregate into daughter cells before the completion of cell division. Unlike in eukaryotes, chromosome replication and segregation in bacteria occur concurrently. How a bacterial chromosome maintains coordination between replication, segregation, and cell division is still unclear. The role of chromosome organization in segregation is also not fully understood. Therefore, it is of the utmost importance to acquire a better understanding of these complex biological processes which will in-turn illuminate our comprehension of the most basic and fundamental aspects of life. Elucidation of such processes will enable the potential to better manipulate chromosomes which can have various applications including but not limited to anti-microbial drug discovery, anti-cancer therapy, and creating programmable artificial cells.This study investigated chromosome segregation in an opportunistic human pathogen, Pseudomonas aeruginosa. Pseudomonas related infections are one of the major causes of death in newly born babies, burn victims, cystic fibrosis patients, and patients with suppressed immune system. The ability of this organism to differentiate into different morphological states allows it to survive in various ecological niches. Its intrinsic multi-drug resistance and ability to form biofilms make it difficult to control. With the emergence of multi-drug resistant strains of Pseudomonas aeruginosa, the discovery of new drugs is imperative in order to prevent further transmission of this organism. A better understanding of chromosome dynamics can help identify and exploit novel drug targets.To determine the segregation pattern in the P. aeruginosa strain PAO1, a fluorescent repressor-operator system was used. The data indicate that the PAO1 chromosome is longitudinally organized between the origin of replication site, oriC to the sister chromosome resolution site, dif. In PAO1, both replication and segregation initiate at oriC and progress bidirectionally. Interestingly, chromosome segregation but not replication ends at the dif site. Proteins of the condensin family play a major role in global chromosome organization in both prokaryotes and eukaryotes. In Pseudomonas aeruginosa, two different families of condensins are present: MksBEF and SMC-ScpAB. These two proteins localize on different regions of the chromosome and differentially affect chromosome segregation. Finally, the study uncovered a novel co-ordination between condensin mediated global chromosome organization and ParABS mediated chromosome segregation, where the presence of at least one of them is necessary for cell viability