Esterases and lipases are widely used as industrial enzymes and for the synthesis of chiral drugs. Because of their rich secondary metabolism, Streptomyces species offer a relatively untapped source of interesting esterases and lipases. S. coelicolor and S. avermitilis contain 51 genes annotated as esterases and/or lipases. In this study I have cloned 14 different genes encoding for lipolytic enzymes from S. coelicolor (11 genes) and S. avermitilis (four genes). Some of these genes were over-expressed in E. coli. Three of the produced enzymes, which were produced by the genes SCO 7131, SCO6966 and SCO3644, were characterized biochemically and one of them was subjected for directed evolution. The gene estA (locus SCO 7131) was annotated as a putative lipase/esterase in the genome sequence of S. coelicolor A3(2), but does not have a homologue in the genome sequence of S. avermitilis or in other known Streptomyces sequences. estA was cloned and expressed in E. coli as a His-tagged protein. The protein was purified and could be recovered in its non-tagged form after digestion with factor Xa. The relative molecular weight was estimated to be 35.5kDa. The enzyme was only active towards acetate esters and not on larger substrates. It had a stereospecificity towards α-naphathylacetate. It was thermostable, with a half-life at 50C of 4.5 hours. Est A showed stability over pH range 5.5-10, and had optimum pH of 7.5. Its activity was drastically decreased when it was pre-incubated in 10mM PMSF, Cu+2 and Hg+2. It was not very stable in most organic solvents and had only slight enantioselectivity. Est A belongs to the HSL family whose founder member is the human hormone-sensitive lipase. I have developed a protein profile for the HSL family modifying the conserved motifs found by Arpigny and Jaeger (1999). Due to the presence of several HSL members with known 3D structure and good homology to Est A, I was able to make a homology model of Est A. Five different mutants of Est A were produced through site directed mutagenesis: W87F, V158A, W87F/V158A, M162L and S163A. The mutants M162L and S163A did not produce a significant change either in substrate specificity or enzyme kinetics. The mutants V158A and W87F/V158A could act on the larger substrates p-nitrophenylbutyrate and caproate and tributyrin. The mutant V158A had improved thermostability and its t1/2 at 50ºC increased to 24h. The affinity of V158A towards p-nitrophenyacetate increased 6-fold when compared with the wild type, whereas the affinity of W87F decreased 4-fold. Directed evolution of Est A was done through random mutagenesis and ER-PCR. A library of 6336 mutants was constructed and screened for mutants with a broader spectrum of substrate specificity. The mutant XXVF7 did show alteration in the substrate specificity of Est A. The mutant XXVF7 had 5 amino acids changes L76R, L146P, S196G, W213R and L267R. The gene locus SCO 6966 (estB gene) was cloned and expressed in E. coli as a His-tagged protein. It was not possible to remove the His-tag using factor Xa. The tagged protein had a molecular weight 31.9kDa. Est B was active against short chain fatty acid esters (C2-C6). Its optimum temperature was 30ºC and was stable for 1h at temperatures up to 37ºC. The enzyme had maximum activity at pH 8-8.5 and was stable over pH range 7.5-11 for 24h. It was highly sensitive for PMSF, Cu+2 and Hg+2. The enzymatic activity deceased in presence of organic solvents, however it was fairly stable for 1h in 20% organic solvents solutions. A third esterase was produced from the gene locus SCO 3644. This esterase was a thermosensitive one with optimum temperature of 35ºC. The three characterized enzymes included a thermophilic, mesophilic and psychrophilic ones. This indicates the high variation in the characters of Streptomyces lipolytic enzymes and highlighting Streptomyces as a source for esterases and lipases of interesting catalytic activity. This study was an initial trial to provide a strategy for a comprehensive use of genome data.
In this doctoral thesis, several aspects of neuronal activity in the rat superior olivary complex (SOC), an auditory brainstem structure, were analyzed using optical imaging with voltage-sensitive dyes (VSD). The thesis is divided into 5 Chapters. Chapter 1 is a general introduction, which gives an overview of the auditory brainstem and VSD imaging. In Chapter 2, an optical imaging method for the SOC was standardized, using the VSD RH795. To do so, the following factors were optimized: (1) An extracellular potassium concentration of 5 mM is necessary during the incubation and recording to observe synaptically evoked responses in the SOC. (2) Employing different power supplies reduced the noise. (3) Averaging of 10 subsequent trials yielded a better signal-to-noise ratio. (4) RH795 of 100 µM with 50 min prewash was optimal to image SOC slices for more than one hour. (5) Stimulus-evoked optical signals were TTX sensitive, revealing action potential-driven input. (6) Synaptically evoked optical signals were characterized to be composed of pre- and postsynaptic components. (7) Optical signals were well correlated with anatomical structures. Overall, this method allows the comparative measurement of electrical activity of cell ensembles with high spatio-temporal resolution. In Chapter 3, the nature of functional inputs to the lateral superior olive (LSO), the medial superior olive (MSO), and the superior paraolivary nucleus (SPN) were analyzed using the glycine receptor blocker strychnine and the AMPA/kainate receptor blocker CNQX. In the LSO, the known glutamatergic inputs from the ipsilateral, and the glycinergic inputs from the ipsilateral and contralateral sides, were confirmed. Furthermore, a CNQX-sensitive input from the contralateral was identified. In the MSO, the glutamatergic and glycinergic inputs from the ipsilateral and contralateral sides were corroborated. In the SPN, besides the known glycinergic input from the contralateral, I found a glycinergic input from the ipsilateral and I also identified CNQX-sensitive inputs from the contralateral and ipsilateral sides. Together, my results thus corroborate findings obtained with different preparations and methods, and provide additional information on the pharmacological nature of the inputs. In Chapter 4, the development of glycinergic inhibition for the LSO, the MSO, the SPN, and the medial nucleus of the trapezoid body (MNTB) was studied by characterizing the polarity of strychnine-sensitive responses. In the LSO, the high frequency region displayed a shift in the polarity at P4, whereas the low frequency region displayed at P6. In the MSO, both the regions displayed the shift at P5. The SPN displayed a shift in the polarity at E18-20 without any regional differences. The MNTB lacked a shift between P3-10. Together, these results demonstrate a differential timing in the development of glycinergic inhibition in these nuclei. In Chapter 5, the role of the MSO in processing bilateral time differences (t) was investigated. This was done by stimulating ipsilateral and contralateral inputs to the MSO with different t values. In preliminary experiments, the postsynaptic responses showed a differential pattern in the spread of activity upon different t values. This data demonstrates a possible presence of delay lines as proposed by Jeffress in the interaural time difference model of sound localization. In conclusion, this study demonstrates the usage of VSD imaging to analyze the neuronal activity in auditory brainstem slices. Moreover, this study expands the knowledge of the inputs to the SOC, and has identified one glycinergic and three AMPA/kainate glutamatergic novel inputs to the SOC nuclei.
Prostate cancer preferentially metastasizes to the skeleton and abundant evidence exists that osteoblasts specifically support the metastatic process, including cancer stem cell niche formation. At early stages of bone metastasis, crosstalk of prostate cancer cells and osteoblasts through soluble molecules results in a decrease of cancer cell proliferation, accompanied by altered adhesive properties and increased expression of bone-specific genes, or osteomimicry. Osteoblasts synthesize a plethora of biologically active factors, which comprise the unique bone microenvironment. By means of quantitative real-time RT-PCR it was determined that exposure to the osteoblast secretome induced gene expression changes in prostate cancer cells, including the upregulation of osteomimetic genes such as BMP2, AP, COL1A1, OPG and RANKL. IL6 and TGFbeta1 signaling pathway components also became upregulated at early time points. Moreover, osteoblast-released IL6 and TGFbeta1 contributed to the upregulation of OPG mRNA in LNCaP. Thus, the earliest response of prostate cancer cells to osteoblast-released factors, which ultimately cause metastatic cells to assume an osteomimetic phenotype, involved activation of paracrine and autocrine IL6 and TGFbeta signaling. On the other hand, a microarray analysis showed that osteoblasts exposed to the secretome of prostate cancer cells exhibited gene expression alterations suggestive of repressed proliferation, decreased matrix synthesis and inhibited immune response, which together indicate enhanced preosteocytic differentiation. TGFbeta signaling, known to inhibit osteoblast maturation, was strongly suppressed, as shown by elevated expression of negative regulators, downregulation of pathway components and of numerous target genes. Transcriptional downregulation of osteoblast inhibitory molecules such as DKK1 and FST also occurred, with concomitant upregulation of the osteoinductive molecules ADM, STC1 and BMP2, and of the transcription factors CBFA1 and HES1, which promote osteoblast differentiation. Finally, the mRNA encoding NPPB, the precursor of a molecule implicated in the inhibition of TGFbetaeffects, in bone formation and in stem cell maintenance, became upregulated after coculture both in osteoblasts and in prostate cancer cells. These results provide an insight into potential mechanisms of dysregulated bone formation in metastatic prostate cancer, as well as mechanisms by which osteoblasts might enhance the invasive, osteomimetic and stem cell-like properties of the tumor cells. In particular, the differential modulation of TGFbetasignaling in prostate cancer cells and osteoblasts appears to merit further research.
Herbivory is discussed as a key agent in maintaining dynamics and stability of tropical forested ecosystems. Accordingly increasing attention has been paid to the factors that structure tropical herbivore communities. The aim of this study was (1) to describe diversity, density, distribution and host range of the phasmid community (Phasmatodea) of a moist neotropical forest in Panamá, and (2) to experimentally assess bottom-up and top-down factors that may regulate populations of the phasmid Metriophasma diocles. The phasmid community of Barro Colorado Island was poor in species and low in density. Phasmids mainly occurred along forest edges and restricted host ranges of phasmid species reflected the successional status of their host plants. Only M. diocles that fed on early and late successional plants occurred regularly in the forest understory. A long generation time with a comparably low fecundity converted into a low biotic potential of M. diocles. However, modeled potential population density increased exponentially and exceeded the realized densities of this species already after one generation indicating that control factors continuously affect M. diocles natural populations. Egg hatching failure decreased potential population growth by 10 % but was of no marked effect at larger temporal scale. Interspecific differences in defensive physical and chemical leaf traits of M. diocles host plants, amongst them leaf toughness the supposedly most effective anti-herbivore defense, seemed not to affect adult female preference and nymph performance. Alternatively to these defenses, I suggest that the pattern of differential preference and performance may be based on interspecific differences in qualitative toxic compounds or in nutritive quality of leaves. The significant rejection of leaf tissue with a low artificial increase of natural phenol contents by nymphs indicated a qualitative defensive pathway in Piper evolution. In M. diocles, oviposition may not be linked to nymph performance, because the evolutionary prediction of a relation between female adult preference and nymph performance was missing. Consequently, the recruitment of nymphs into the reproductive adult phase may be crucially affected by differential performance of nymphs. Neonate M. diocles nymphs suffered strong predation pressure when exposed to natural levels of predation. Concluding from significantly increased predation-related mortality at night, I argue that arthropods may be the main predators of this nocturnal herbivore. Migratory behavior of nymphs seemed not to reflect predation avoidance. Instead, I provided first evidence that host plant quality may trigger off-plant migration. In conclusion, I suggest that predation pressure with its direct effects on nymph survival may be a stronger factor regulating M. diocles populations, compared to direct and indirect effects of host plant quality, particularly because slow growth and off-host migration both may feed back into an increase of predation related mortality.
Fragmentation of habitats, especially of tropical rainforests, ranks globally among the most pervasive man-made disturbances of ecosystems. There is growing evidence for long-term effects of forest frag-mentation and the accompanying creation of artificial edges on ecosystem functioning and forest structure, which are altered in a way that generally transforms these forests into early successional systems. Edge-induced disruption of species interactions can be among the driving mechanisms governing this transformation. These species interactions can be direct (trophic interactions, competition, etc.) or indirect (modification of the resource availability for other organisms). Such indirect interactions are called ecosystem engineering. Leaf-cutting ants of the genus Atta are dominant herbivores and keystone-species in the Neotropics and have been called ecosystem engineers. In contrast to other prominent ecosystem engineers that have been substantially decimated by human activities some species of leaf-cutting ants profit from anthropogenic landscape alterations. Thus, leaf-cutting ants are a highly suitable model to investigate the potentially cascading effects caused by herbivores and ecosystem engineers in modern anthropogenic landscapes following fragmentation. The present thesis aims to describe this interplay between consequences of forest fragmentation for leaf-cutting ants and resulting impacts of leaf-cutting ants in fragmented forests. The cumulative thesis starts out with a review of 55 published articles demonstrating that herbivores, especially generalists, profoundly benefit from forest edges, often due to (1) favourable microenviron-mental conditions, (2) an edge-induced increase in food quantity/quality, and (3; less well documented) disrupted top-down regulation of herbivores (Wirth, Meyer et al. 2008; Progress in Botany 69:423-448). Field investigations in the heavily fragmented Atlantic Forest of Northeast Brazil (Coimbra forest) were subsequently carried out to evaluate patterns and hypotheses emerging from this review using leaf-cutting ants of the genus Atta as a model system. Colony densities of both Atta species occuring in the area changed similarly with distance to the edge but the magnitude of the effect was species-specific. Colony density of A. cephalotes was low in the forest interior (0.33 ± 1.11 /ha, pooling all zones >50 m into the forest) and sharply increased by a factor of about 8.5 towards the first 50 m (2.79 ± 3.3 /ha), while A. sexdens was more uniformly distributed (Wirth, Meyer et al. 2007; Journal of Tropical Ecology 23:501-505). The accumulation of Atta colonies persisted at physically stable forest edges over a four-year interval with no significant difference in densities between years despite high rates of colony turn-over (little less than 50% in 4 years). Stable hyper-abundant populations of leaf-cutting ants accord with the constantly high availability of pioneer plants (their preferred food source) as previously demonstrated at old stabilised forest edges in the region (Meyer et al. submitted; Biotropica). In addition, plants at the forest edge might be more attractive to leaf-cutting ants because of their physiological responses to the edge environment. In bioassays with laboratory colonies I demonstrated that drought-stressed plants are more attractive to leaf-cutting ants because of an increase in leaf nutrient content induced by osmoregulation (Meyer et al. 2006; Functional Ecology 20:973-981). Since plants along forest edges are more prone to experience drought stress, this mechanism might contribute to the high resource availabil-ity for leaf-cutting ants at forest edges. In light of the hyper-abundance of leaf-cutting ants within the forest edge zone (first 50 m), their po-tentially far-reaching ecological importance in anthropogenic landscapes is apparent. Based on previous colony-level estimates, we extrapolated that herbivory by A. cephalotes removes 36% of the available foliage at forest edges (compared to 6% in the forest interior). In addition, A. cephalotes acted as ecosys-tem engineers constructing large nests (on average 55 m2: 95%-CI: 22-136) that drastically altered forest structure. The ants opened gaps in the canopy and forest understory at nest sites, which allowed three times as much light to reach the nest surface as compared to the forest understory. This was accompa-nied by an increase in soil temperatures and a reduction in water availability. Modifications of microcli-mate and forest structure greatly surpassed previously published estimates. Since higher light levels were detectable up to about 4 m away from the nest edge, an area roughly four times as big as the actual nest (about 200 and 50 m2, respectively) was impacted by every colony, amounting to roughly 6% of the total area at the forest edge (Meyer et al. in preparation; Ecology). The hypothesized impacts of high cutting pressure and microclimatic alterations at nest sites on forest regeneration were directly tested using transplanted seedlings of six species of forest trees. Nests of A. cephalotes differentially impacted survival and growth of seedlings. Survival differed highly significantly between habitats and species and was generally high in the forest, yet low on nests where it correlated strongly with seed size of the species. These results indicate that the disturbance regime created by leaf-cutting ants differs from other distur-bances, since nest conditions select for plant species that profit from additional light, yet are large-seeded and have resprouting abilities, which are best suited to tolerate repeated defoliation on a nest (Meyer et al. in preparation; Journal of Tropical Ecology). On an ecosystem scale leaf-cutting ants might amplify edge-driven microclimatic alterations by very high rates of herbivory and the maintenance of canopy gaps above frequent nests. By allowing for an increased light penetration Atta may, ultimately, contribute to a dominating, self-replacing pioneer communities at forest edges, possibly creating a positive feed-back loop. Based on the persisting hyper-abundance of leaf-cutting ants at old edges of Coimbra forest and the multifarious impacts documented, we conclude that the ecological importance of leaf-cutting ants in pristine forests, where they are commonly believed to be keystone species despite very low colony densities, is greatly surpassed in anthropogenic landscapes In fragmented forests, Atta has been identified as an essential component of a disturbance regime that causes a post-fragmentation retrogressive succession. Apparently, these forests have reached a new self-replacing secondary state. I suggest additional human interference in form of thoughtful management in order to break this cycle of self-enhancing disturbance and to enable forest regeneration along the edges of threatened forest remnants. Thereby the situation of the forest as a whole can be ameliorated and the chances for a long-term retention of biodiversity in these landscapes increased.
Fragmentation of tropical rain forests is pervasive and results in various modifications in the ecosystem functioning such as … It has long been noticed that the colony densities of a dominant herbivore in the neotropics - leaf-cutting ant (LCA) - increase in fragmentation-related habitats like forest edges and small fragments, however the reasons for this increase are not clear. The aim of the study was to test the hypothesis that bottom-up control of LCA populations is less effective in fragmented compared to continuous forests and thus explains the increase in LCA colony densities in these habitats. In order to test for less effective bottom-up control, I proposed four working hypotheses. I hypothesized that LCA colonies in fragmented habitats (1) find more palatable vegetation due to low plant defences, (2) forage on few dominant species resulting in a narrow diet breadth, (3) possess small foraging areas and (4) increase herbivory rate at the colony level. The study was conducted in the remnants of the Atlantic rainforest in NE Brazil. Two fragmentation-related forest habitats were included: the edge and a 3500-ha continuous forest and the interior of the 50-ha forest fragment. The interior of the continuous forest served as a control habitat for the study. All working hypotheses can be generally accepted. The results indicate that the abundance of LCA host plant species in the habitats created by forest fragmentation along with weaker chemical defense of those species (especially the lack of terpenoids) allow ants to forage predominantly on palatable species and thus reduce foraging costs on other species. This is supported by narrower ant diet breadth in these habitats. Similarly, small foraging areas in edge habitats and in small forest fragments indicate that there ants do not have to go far to find the suitable host species and thus they save foraging costs. Increased LCA herbivory rates indicate that the damages (i.e., amount of harvested foliage) caused by LCA are more important in fragmentation-related habitats which are more vulnerable to LCA herbivory due to the high availability of palatable plants and a low total amount of foliage (LAI). (1) Few plant defences, (2) narrower ant diet breadth, (3) reduced colony foraging areas, and (4) increased herbivory rates, clearly indicate a weaker bottom-up control for LCA in fragmented habitats. Weak bottom-up control in the fragmentation-related habitats decreases the foraging costs of a LCA colony in these habitats and the colonies might use the surplus of energy resulting from reduced foraging costs to increase the colony growth, the reproduction and turnover. If correct, this explains why fragmented habitats support more LCA colonies at a given time compared to continuous forest habitats. Further studies are urgently needed to estimate LCA colony growth and turnover rates. There are indices that edge effects of forest fragmentation might be more responsible in regulating LCA populations than area or isolation effects. This emphasizes the need to conserve big forest fragments not to fall below a critical size and retain their regular shape. Weak bottom-up control of LCA populations has various consequences on forested ecosystems. I suggest a loop between forest fragmentation and LCA population dynamics: the increased LCA colony densities, along with lower bottom-up control increase LCA herbivory pressure on the forest and thus inevitably amplify the deleterious effects of fragmentation. These effects include direct consequences of leaf removal by ants and various indirect effects on ecosystem functioning. This study contributes to our understanding of how primary fragmentation effects, via the alteration of trophic interactions, may translate into higher order effects on ecosystem functions.
Haustoria of the rust fungus pathogen Uromyces fabae deliver RTP1 (Rust Transferred Protein1) into host plant cells. In this work, different heterologous expression systems were used to study RTP1 biological function as well as RTP1 transfer mechanism. The first part of this thesis focused on the identification of the subcellular target compartment of RTP1 in plant cells. In this respect we could identify a functional bipartite nuclear localization signal within RTP1. However, stable and transient expression studies of RTP1 in different plant species, including the host plant Vicia faba, interfered with plant cell vitality but did not result in detection of RTP1 protein. These findings led us to propose that RTP1 interferes with plant gene expression. However, the molecular basis of this interference remains unclear. By deletion studies, we could localize the active region of RTP1 within a 45 amino acid central domain. In the second part of this study, two different lines of approaches were taken to study RTP1 transfer mechanism. First, transient expression of secreted RTP1 (sRTP1) also interfered with plant cell vitality. Addition of an endoplasmic reticulum retention signal abolished sRTP1 interference with plant cell vitality, suggesting that RTP1 can reenter the plant cell from the apoplast after secretion in the absence of the pathogen. We have identified a PEST-like region within RTP1, however, contribution of this region to the stability of RTP1 is not clear. Site directed mutagenesis analysis showed that the PEST-like region is likely to play a role during the transfer of RTP1 through plant plasma membrane. In the second line of approach, we established a recombinant delivery model, using Ustilago maydis/Zea mays pathosystem, to pursue RTP1 translocation into the plant cell. Our results indicate that U. maydis is capable of secreting high amounts of recombinant RTP1, showing similar glycosylation pattern as RTP1 secreted from rust haustoria. Our data propose the use of this model system to study RTP1 domains mediating its entry into the plant cell. Haustoria of the rust fungus pathogen Uromyces fabae deliver RTP1 (Rust Transferred Protein1) into host plant cells. In this work, different heterologous expression systems were used to study RTP1 biological function as well as RTP1 transfer mechanism. The first part of this thesis focused on the identification of the subcellular target compartment of RTP1 in plant cells. In this respect we could identify a functional bipartite nuclear localization signal within RTP1. However, stable and transient expression studies of RTP1 in different plant species, including the host plant Vicia faba, interfered with plant cell vitality but did not result in detection of RTP1 protein. These findings led us to propose that RTP1 interferes with plant gene expression. However, the molecular basis of this interference remains unclear. By deletion studies, we could localize the active region of RTP1 within a 45 amino acid central domain. In the second part of this study, two different lines of approaches were taken to study RTP1 transfer mechanism. First, transient expression of secreted RTP1 (sRTP1) also interfered with plant cell vitality. Addition of an endoplasmic reticulum retention signal abolished sRTP1 interference with plant cell vitality, suggesting that RTP1 can reenter the plant cell from the apoplast after secretion in the absence of the pathogen. We have identified a PEST-like region within RTP1, however, contribution of this region to the stability of RTP1 is not clear. Site directed mutagenesis analysis showed that the PEST-like region is likely to play a role during the transfer of RTP1 through plant plasma membrane. In the second line of approach, we established a recombinant delivery model, using Ustilago maydis/Zea mays pathosystem, to pursue RTP1 translocation into the plant cell. Our results indicate that U. maydis is capable of secreting high amounts of recombinant RTP1, showing similar glycosylation pattern as RTP1 secreted from rust haustoria. Our data propose the use of this model system to study RTP1 domains mediating its entry into the plant cell.
The study provides insights into the dynamic processes of vascular epiphyte vegetation in two host tree species of lowland forest in Panama. Further, a novel approach is presented to examine the possible role of host tree identity in the structuring of vascular epiphyte communities: For three locally common host tree species (Socratea exorrhiza, Marila laxiflora, Perebea xanthochyma) we created null models of the expected epiphyte assemblages assuming that epiphyte colonization reflected random distribution of epiphytes in the forest. In all three tree species, abundances of the majority of epiphyte species (69 – 81 %) were indistinguishable from random, while the remaining species were about equally over- or underrepresented compared to their occurrence in the entire forest plot. Permutations based on the number of colonized trees (reflecting observed spatial patchiness) yielded similar results. Finally, a Canonical Correspondence Analysis also confirmed host-specific differences in epiphyte assemblages. In spite of pronounced preferences of some epiphytes for particular host trees, no epiphyte species was restricted to a single host. We conclude that the epiphytes on a given tree species are not simply a random sample of the local species pool, but there are no indications of host specificity either. To determine the qualitative and quantitative long-term changes in the vascular epiphyte assemblage of the host tree Socratea exorrhiza, in the lowland forest of the San Lorenzo Crane Plot, we followed the fate of the vascular epiphyte assemblage on 99 individuals of this palm species, in three censuses over the course of five years. The composition of the epiphyte assemblage changed little during the course of the study. While the similarity of epiphyte vegetation decreased on single palm individuals through time, the similarity analyzed over all palms increased. Even well-established epiphyte individuals experienced high mortality with only 46 % of the originally mapped individuals surviving the following five years. We found a positive correlation between host tree size and epiphyte richness and detected higher colonization rates of epiphytes per surface area on larger trees. Epiphyte assemblages on single Socratea exorrhiza trees were highly dynamic while the overall composition of the epiphyte vegetation on the host tree species in the study plot was rather stable. We suggest that higher recruitment rates due to localized seed dispersal by already established epiphytes on larger palms promote the colonization of epiphytes on larger palms. Given the known growth rates and mortality rates of the host tree species, the maximum time available for colonization and reproduction of epiphytes on a given Socratea exorrhiza tree is estimated to be about 60 years. Changes in the epiphyte vegetation of c. 1000 individuals of the host tree species Annona glabra at Barro Colorado Island over the course of eight year were documented by means of repeated censuses. Considerable increase in the abundance of the dominating epiphyte species and ongoing colonization of the host tree species suggests that the epiphyte vegetation has not reached a steady state in the maximal 80 years since the establishment of the host tree. Epiphyte species composition as a whole was rather stable. We disentangled the relationship between epiphyte colonization and tree size/available time for colonization with the finding that tree size explained only a low proportion of colonization while other factors like connectivity to dispersal source and time explain may explain a larger part. Epiphyte populations are patchily distributed and examined species exhibit properties of a metapopulation with asynchronous local population growth, high local population turnover, a positive relationship between regional occurrence and patch population size, and negatively correlated relationship between extinction and patch occupancy. The documented metapopulation processes highlight the importance of not colonized suitable habitat for the conservation of epiphytes.
In this study, 27 marine bacteria were screened for production of bioactive metabolites. Two strains from the surface of the soft coral Sinularia polydactyla, collected from the Red Sea, and three strains from different habitats in the North Sea were selected as a promising candidates for isolation of antimicrobial substances. A total of 50 compounds were isolated from the selected bacterial strains. From these metabolites 25 substances were known from natural sources, 10 substances were known as synthetic chemical and herein are reported as new natural products, and 13 metabolites are new. Two substances are still under elucidation. All new compounds were chemically and biologically characterized. Pseudoalteromonas sp. T268 produced simple phenol and oxindole derivatives. Production of homogentisic acid and WZ 268S-6 from this bacteria was affected by the salinity stress. WZ 268S-6 shows antimicrobial and cytotoxic activities. Its target is still unclear. Isolation of isatin from this strain points out for the possibility of using this substance as a chemotaxonomical marker for Alteromonas-like bacteria. A large number of nitro-substituted aromatic compounds were isolated from both Salegentibacter sp. T436 and Vibrio sp. WMBA1-4. They may be derived from metabolism of phenylalanine or tyrosine. From Salegentibacter sp. T436, 24 compounds were isolated, of which four compounds are new and six compounds were known as synthetic chemicals. WZ 436S-16 (dinitro-β-styrene) is the most potent antimicrobial and cytotoxic compound. It inhibits the oxygen uptake by N. coryli and causes apoptosis in the human promyelocytic leukaemia (HL-60 cells). From Vibrio sp. WMBA1-4, 13 new alkaloids were isolated, of which four were known as synthetic products and herein are reported as new substances from natural sources. The majority of these compounds show antimicrobial and cytotoxic activities. The cytotoxic activity of WMB4S-11 against the mouse lymphocytic leukaemia (L1210 cells) is due to the inhibition in the protein biosynthesis, while the remaining cytotoxic alkaloids have no effect on the synthesis of macromolecules in this cell line. The antibacterial activity of WMB4S-2, -11, -12, -13 and the antifungal activity of WMB4S-9 are not due to the inhibition in the macromolecules biosynthesis or in the oxygen uptake by the microorganisms. The biological activity of these nitro-aromatic compounds from Salegentibacter sp. T436 and Vibrio sp. WMBA1-4 is influenced by the presence of a nitro group and its position in respect to the hydroxyl group, number of the nitro groups, and the type of substitutions on the side chain. In diaryl-maleimide derivatives, types and position of substitution on the aryl rings, on the maleimide moity, and the hydrophobicity of the aryl ring itself lead to variations in the extent of the bioactivity of these derivatives. This is the first time that vibrindole (WMB4S-14) and turbomycin B or its noncationic form (WMB4S-15), isolated from Vibrio sp., are reported as cytotoxic compounds. WMB4S-15 inhibits the biosynthesis of macromolecules in L1210 cells. The structural similarity between some of the metabolites in this study and previously reported compounds from sponges, ascidians, and bryozoan indicates that the microbial origin of these compounds must be considered.