Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06 Archiv

All living cells are highly organized and exhibit complex cellular machineries facilitating biochemical reactions. Compartmentalization is a prerequisite to allocate an appropriate environment for these processes. In this work, compartments that are involved in Bacillus subtilis membrane organization and cell division were studied. B. subtilis division site selection is dependent on the nucleoid occlusion and the Min system. The B. subtilis Min system consists of four components. MinC is the actual inhibitor of the tubulin homologue FtsZ that is a crucial component of the divisome, forming the so called Z-ring. MinC is bound to the ATPase MinD that is tethered via the adapter protein MinJ to DivIVA. DivIVA senses membrane curvature and was supposed to be stably tethered to the cell poles. Thereby a stable, static DivIVA / MinJDC gradient with minimum concentration at midcell is formed. Using advanced microscopy techniques like single cell time lapse microscopy, fluorescence recovery after photo bleaching and by utilization of photo-activatable / convertible fluorophores we could demonstrate that DivIVA is in vegetative cells recruited from the cell pole to mature septa. These data provide first evidence that the role of the B. subtilis Min system is not to define midcell, but prevents reinitiation of Z-ring constriction after fulfilled division. Utilizing single cell time lapse microscopy we could further demonstrate that proteins crucial to condense the chromosome are vital for correct chromosome segregation during cell division by influencing the replication fork velocity or resolution. As a second compartment B. subtilis flotillin dependent membrane microdomains were studied. These domains are likely scaffolded by the membrane protein flotillin. This protein is pinned to the membrane via a hairpin loop as shown by SNAP–tag labelling experiments. Utilizing the anisotropic dye Laurdan we could further show spectroscopically and microscopically that flotillins prevent condensation of microdomains. Flotillin deletion strains also exhibit a generally more liquid ordered membrane compared to wild type cells. Using co–immunoprecipitation experiments several proteins interacting with flotillin were identified. These interactions were confirmed with microscopical co–localization analysis. B. subtilis flotillin was additionally heterologously purified via affinity chromatography. The purified protein creates large homo–oligomers likely in mega Dalton size. Using truncation mutants it could be shown that flotillin oligomerizes via a flotillin specific domain, namely the PHB domain. Though, contrary to eukaryotic cells, B. subtilis PHB domain does not contribute to lipid binding. However, several cellular machineries that interact with flotillins, as exemplary shown for the secretion machinery, are impaired in their functionality in absence of flotillins. These data provide first evidence that prokaryotic flotillins are elements that scaffold the plasma membrane and thereby provide a lipid environment that is vital for correct functionality of diverse cellular machineries.

Bacteria constantly need to monitor their environment and adapt the bacterial group-coordinated behaviour to changing habitats like nutrition alterations or host variations. Commonly cell-cell communication via acyl homoserine lactones (AHLs)is used to synchronise the behaviour of a bacterial population dependent on cell size. This process is referred to as quorum sensing (QS) and predominantly occurs in Gram-negative bacteria. The typical QS system consists of a LuxI-synthase that synthesises AHLs, and a LuxR-type receptor, which then responds to these AHLs. Upon AHL-binding, the LuxR-type receptor regulates the expression of different target genes and thus influences several processes, like biofilm formation, virulence, antibiotic production or cell-cell interaction. Interestingly, many proteobacteria possess additional LuxR homologs, but lack a cognate LuxI-type synthase. Those LuxR-type receptors are referred to as LuxR orphans or LuxR solos and can expand the regulatory QS network. Photorhabdus species are insect pathogenic bacteria, living in symbiosis with entomopathogenic nematodes. They all possess an exceptionally high number of LuxR solos, but lack LuxI homologs and therefore do not produce AHLs. The function of these LuxR solos, their role in cell-cell communication and the identification of their cognate signalling molecules in Photorhabdus species is the main focus of this work. In this thesis a novel signalling molecule used for QS could be identified for the first time in P. luminescens. This novel QS molecule is an α-pyrone named photopyrone (PPY) and produced endogenously by the photopyrone synthase (PpyS). The PPYs are specifically recognized by the LuxR solo regulator PluR, which then activates expression of the pcf (Photorhabdus clumping factor) operon leading to cell clumping of P. luminescens cells. Moreover, the PpyS/PluR quorum sensing system and its induced cell clumping contribute to the overall toxicity of P. luminescens. Furthermore, a second novel signalling molecule sensed by a LuxR solo of Photorhabdus species could be identified besides PPYs. The insect and human pathogenic bacteria P. asymbiotica lacks a PpyS homolog as well as a LuxI homolog, but harbours a pcf operon and a homologue to PluR, which is named PauR. The signalling molecule sensed by the LuxR-type receptor PauR could be identified, which is neither an AHL nor a PPY. PauR recognises a 2,5-dialkylresorcinol (DAR) produced by the DarABC pathway. Upon binding of the cognate signalling molecule, Summary XII PauR activates expression of the pcf operon. This also leads to cell clumping in P. asymbiotica. Furthermore, the DarABC/PauR QS system also contributes to the overall pathogenicity of P. asymbiotica against Galleria mellonella insect larvae. A bioinformatics approach revealed a high number of LuxR solos present in P. temperata and P. asymbiotica like in P. luminescens. Thereby, several conserved motives of amino acids could be identified, which are potentially important for signalbinding and -specificity. Variations in these amino acid motifs are assumed to reflect the overall variety of signals that can be sensed by LuxR solos. Furthermore, the specificity of the two LuxR solos PluR and PauR towards their cognate signalling molecules, PPYs and DARs, respectively, was analysed. Thereby, it could be shown that the previously identified conserved amino acid motives in the signal-binding domain (SBD), the TYDQCS-motif of PluR and the TYDQYI-motif of PauR, are essential but not sufficient for ligand-binding. Similar as for AHLs, it was unclear how the signalling molecules PPYs and DARs can cross the bacterial cell membrane. In the last part of this thesis the import mechanism for the Photorhabdus-specific signalling compounds PPYs and DARs were identified. Initial evidence could be provided that the membrane-integrated transporter FadL is mainly involved in the import of these hydrophobic compounds, and that they are not transported via simple diffusion across the cell membrane, which is assumed for AHLs. In conclusion, the data that is compiled presents two LuxR solos of Photorhabdus species adapted to sense and respond to novel non-AHL signalling molecules used for QS. Therefore, this thesis reveals that cell-cell communication via LuxR-type receptors goes far beyond AHL-signalling in nature.

Corynebacterium glutamicum is a Gram positive soil bacterium with high industrial importance in ton scale production of amino acids. Apart from that, it becomes more and more important for medical studies, where it serves as model organism due to its close relation to bacteria causing several pathogens such as tuberculosis, diphtheria and leprosy. C. glutamicum, like Mycobacterium tuberculosis, has a distinct cell wall which is composed of a peptidoglycan layer (murein) with covalently bound polysaccharide layers that are capped with mycolic acids. In addition, both organisms have a polar cell wall synthesis machinery which is spatially regulated by DivIVA (Wag31 in M. tuberculosis). The present study shows that DivIVA regulates cell wall synthesis upon direct interaction with the lipid II flippase RodA. RodA determines morphology and growth in C. glutamicum and is localized to the poles and septa. The absence of rodA results in growth defects and cell shape alterations as well as altered lipid II proliferation of the poles (polar cell growth is sustained). DivIVA is furthermore involved in chromosome segregation upon direct interaction with the partitioning ParB protein, which binds to parS sites on the chromosome, thus tethering the replicated nucleoids to the cell poles. Interactions of DivIVA with ParB and RodA were identified in a synthetic in vivo protein-protein interaction assay where fluorescently labeled proteins of interest are expressed in E. coli cells and interaction is analyzed microscopically. A decisive improvement of this assay is the application of FRET, which is more sensitive and allows quantification of interaction. In order to test whether ParB and RodA compete for the same interaction site in DivIVA, we mapped interaction sites of both proteins. It turned out that ParB binds to a middle region of DivIVA, whereas RodA binds to the N-terminal domain of DivIVA where one lysine residue is essential for interaction. To fight bacterial infections, that cause thousands of casualties each year, it is mandatory to understand mechanisms in cellular processes, such as cell division and growth, to find new targets for antibiotic intervention. Unfortunately, bacteria are able to develop resistances against many antibiotics. The mycolic acid or arabinan layer and synthesis machinery are good candidates for new antibiotics. Amongst others, two of them have emerged as useful drugs against M. tuberculosis, ethambutol (EMB) and BTZ043. In this study, we investigated the modes of action and antibiotic susceptibility of C. glutamicum after EMB and BTZ043 treatment. We found that both antibiotics, which target the arabinan synthesis pathway, affect exclusively polar elongation growth, as demonstrated in different staining assays. Interestingly, only 10% of the cells were killed and cells in stationary phase were not affected by EMB or BTZ043. Moreover, we used a chromosomal DivIVA-mCherry fusion and found that DivIVA protein level is drastically increased. The cells show asymmetric recovery after treatment, in which one daughter cell acquires the excess DivIVA whereas the other daughter cell exhibits normal cell growth.

Behavioural ecologists aim at providing insights into the evolutionary and ecological processes that shape animal behaviour. Mate choice is a decision faced by most animals that can strongly affect an individual’s reproductive success, an important fitness component. This behaviour has therefore the potential to show many adaptations which have been the subject of a vivid research interest over the last decades. Studies on mate choice have typically focused on female preferences for traits that increase a male's overall attractiveness, which supposedly reflects the male’s absolute quality. Preferences for such traits are expected to provide females with benefits such as good paternal care or ‘good genes’ for their offspring. Nevertheless, in some species, individuals actually show little consensus on what represents a high-quality partner. Such individually-specific preferences are poorly understood, but it has been hypothesized that they have evolved to optimise genetic compatibility (to increase offspring viability) or, although rarely mentioned, to optimise behavioural compatibility (to facilitate joint parental care). The zebra finch is a life-time monogamous passerine whose mating preferences for putative quality traits have been widely studied but where no such quality indicator has been repeatedly shown to influence mating decisions. In this species, preferences seem instead largely idiosyncratic. In this thesis, I investigated whether female zebra finches choose genetically and/or behaviourally compatible social mates (Chapters 2 and 3). More precisely, I measured, in a large-scale breeding design, the fitness consequences of free mate choice for compatibility while experimentally controlling for effects of overall quality (Chapter 3). In this system, genetic incompatibility results in embryo mortality and therefore hatching failure, while behavioural incompatibility results in offspring mortality. I therefore expected social mate choice for genetic and/or behavioural compatibility to optimise embryo and offspring survival (Chapter 3). Moreover, I tested whether siblings who grew up together avoid choosing each other as social partner; in other words, if they avoid inbreeding depression by choosing a genetically more compatible partner (Chapter 2). In addition, I performed a meta-analysis of published experiments on zebra finches that allow for different mechanisms of kin discrimination to take place (Chapter 2). Finally, I investigated whether females enhance their extra-pair behaviour when paired to an apparently genetically incompatible male to obtain compatible genes benefits (Chapter 1). Indeed, extra-pair behaviour is largely hypothesized to be an adaptive response that would allow females to compensate for a potentially sub-optimal social mate choice. Specifically, I tested whether female extra-pair mating evolved as a counter strategy when females experience low hatching success with their social partner, that is to say whether female extra-pair mate choice targets fertility benefits and/or compatible genes benefits (Chapter 1). Contrary to all expectations derived from optimality, zebra finches were unable to identify partners with whom they would minimise embryo mortality (Chapter 3), or to recognize unfamiliar kin on the basis of genetic similarity per se to minimize inbreeding depression (meta-analysis of Chapter 2), and did not adjust their extra-pair mating behaviour in response to repeated hatching failure (Chapter 1). This suggests that, in zebra finches, individuals have not evolved any ability (other than avoiding familiar kin, experiment of Chapter 2) to judge genetic compatibility of any kind, despite the adaptive value of such behaviour. Finally, allowing free social mate choice did enhance pair fitness due to direct compatibility benefits (Chapter 3). This thesis provides therefore the first evidence of mate choice for behavioural compatibility. Nevertheless, the adaptive significance of the underlying choosiness remains speculative (Chapter 3). A null model consisting of random non-adaptive genetic variation in sensory systems and variation in phenotypes that are better at stimulating some sensory systems than others might well account for the observed fitness differences between compatible and incompatible pairs. This study highlights that there are limits to adaptation.