Kaiserslautern - Fachbereich Biologie
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The plasma membrane transporter SOS1 (SALT-OVERLY SENSITIVE1) is vital for plant survival under salt stress. SOS1 activity is tightly regulated, but little is known about the underlying mechanism. SOS1 contains a cytosolic, autoinhibitory C-terminal tail (abbreviated as SOS1 C-term), which is targeted by the protein kinase SOS2 to trigger its transport activity. Here, to identify additional binding proteins that regulate SOS1 activity, we synthesized the SOS1 C-term domain and used it as bait to probe Arabidopsis thaliana cell extracts. Several 14-3-3 proteins, which function in plant salt tolerance, specifically bound to and interacted with the SOS1 C-term. Compared to wild-type plants, when exposed to salt stress, Arabidopsis plants overexpressing SOS1 C-term showed improved salt tolerance, significantly reduced Na+ accumulation in leaves, reduced induction of the salt-responsive gene WRKY25, decreased soluble sugar, starch, and proline levels, less impaired inflorescence formation and increased biomass. It appears that overexpressing SOS1 C-term leads to the sequestration of inhibitory 14-3-3 proteins, allowing SOS1 to be more readily activated and leading to increased salt tolerance. We propose that the SOS1 C-term binds to previously unknown proteins such as 14-3-3 isoforms, thereby regulating salt tolerance. This finding uncovers another regulatory layer of the plant salt tolerance program
The number of sequenced genomes increases rapidly due to the development of faster, better and new technologies. Thus, there is a great interest in automation, and standardization of the subsequent processing and analysis stages of the generated enormous amount of data. In the current work, genomes of clones, strains and species of Streptococcus were compared, which were sequenced, annotated and analysed with several technologies and methods. For sequencing, the 454- and Illumina-technology were used. The assembly of the genomes mainly was performed by the gsAssembler (Newbler) of Roche, the annotation was performed by the annotation pipeline RAST, the transfer tool RATT or manually. Concerning analysis, sets of deduced proteins of several genomes were compared to each other and common components, the so-called core-genome, of the used genomes of one or closely related species determined. Detailed comparative analysis was performed for the genomes of isolates of two clones to gather single nucleotide variants (SNV) within genes.
This work focusses on the pathogenic organism Streptococcus pneumoniae. This species is a paradigm for transformability, virulence and pathogenicity as well as resistance mechanisms against antibiotics. Its close relatives S. mitis, S. pseudopneumoniae and S. oralis have no pathogenicity potential as high as S. pneumoniae available and are thus of high interest to understand the evolution of S. pneumoniae. Strains of two S. pneumoniae clones were chosen. One is the ST10523 clone, which is associated with patients with cystic fibrosis and is characterized by long-term persistence. This clone is lacking an active hyaluronidase, which is one of the main virulence factors. The lack of two phage clusters possibly contributed to the long persistence in the human host. The clone ST226 shows a high penicillin resistance but interestingly one strain is sensitive against penicillin. Here it could be seen that the penicillin resistance mainly arose from the presence of mosaic-PBPs, while special alleles of MurM and CiaH - both genes are associated with penicillin-resistance – were present in resistant and sensitive strains as well. Penicillin resistance of S. pneumoniae is the result of horizontal gene transfer, where DNA of closely related species, mainly S. mitis or S. oralis, served as donor. The transfer of DNA from the high-level penicillin-resistant strain S. oralis Uo5 to the sensitive strain S. pneumoniae R6 was intentioned to reveal the amount of transferred DNA and whether it is possible to reach the high resistance level of S. oralis Uo5. Altogether, about 19kb of S. oralis DNA were transferred after three successive transformation steps, about 10-fold less than during transfer from S. mitis, which is more closely related to S. pneumoniae, as donor. MurE was identified as new resistance determinant. Since the resistance level of the donor strain could not be reached, it is assumed, that further unknown factors are present which contribute to penicillin resistance. The comparison of S. pneumoniae and its close relatives was performed using deduced protein sequences. 1.041 homologous proteins are common to the four complete genomes of S. pneumoniae R6, S. pseudopneumoniae IS7493, S. mitis B6 and S. oralis Uo5. Most of the virulence and pathogenicity factors described for S. pneumoniae could also be found in commensal species. These observations were confirmed by further investigations by Kilian et al. (Kilian, et al., 2019). After adding 26 complete S. pneumoniae genomes to the analysis, only 104 gene products could be identified as specific for this species. Investigations of a larger number of related streptococci, which were isolated from human and several primates, confirmed the presence of most of the virulence factors of human pneumococci in S. oralis and S. mitis strains from primates. While NanBC is common among S. pneumoniae and is missing in all S. oralis, all S. oralis contain a ß-N-acetyl-hexosaminidase which vice versa is missing in S. pneumoniae. The occurrence of S. oralis also in free-living chimpanzees suggests the assumption, that this species is part of the commensal flora of these Old-World monkeys unlike S. pneumoniae which has evolved with its human host. Compared to S. pneumoniae, S. oralis shows an amazing variability in factors important for biosynthesis of peptidoglycan and teichoic acid (PBP, MurMN, lic-cluster). Some streptococci contain a second PGP3 homologue. Additional analyses with further isolates, especially of wild animals, are necessary to determine host-specific components.
A building-block model reveals new insights into the biogenesis of yeast mitochondrial ribosomes
(2020)
Most of the mitochondrial proteins in yeast are encoded in the nuclear genome, get synthesized by cytosolic ribosomes and are imported via TOM and TIM23 into the matrix or other subcompartments of mitochondria. The mitochondrial DNA in yeast however also encodes a small set of 8 proteins from which most are hydrophobic membrane proteins and build core components of the OXPHOS complexes. They get synthesized by mitochondrial ribosomes which are descendants of bacterial ribosomes and still have some similarities to them. On the other hand, mitochondrial ribosomes experienced various structural and functional changes during evolution that specialized them for the synthesis of the mitochondrial encoded membrane proteins. The mitoribosome contains mitochondria-specific ribosomal proteins and replaced the bacterial 5S rRNA by mitochondria-specific proteins and rRNA extensions. Furthermore, the mitoribosome is tethered to the inner mitochondrial membrane to facilitate a co-translational insertion of newly synthesized proteins. Thus, also the assembly process of mitoribosomes differs from that of bacteria and is to date not well understood.
Therefore, the biogenesis of mitochondrial ribosomes in yeast should be investigated. To this end, a strain was generated in which the gene of the mitochondrial RNA-polymerase RPO41 is under control of an inducible GAL10-promoter. Since the scaffold of ribosomes is built by ribosomal RNAs, the depletion of the RNA-polymerase subsequently leads to a loss of mitochondrial ribosomes. Reinduction of Rpo41 initiates the assembly of new mitoribosomes, which makes this strain an attractive model to study mitoribosome biogenesis.
Initially, the effects of Rpo41 depletion on cellular and mitochondrial physiology was investigated. Upon Rpo41 depletion, growth on respiratory glycerol medium was inhibited. Furthermore, mitochondrial ribosomal 21S and 15S rRNA was diminished and mitochondrial translation was almost completely absent. Also, mitochondrial DNA was strongly reduced due to the fact that mtDNA replication requires RNA primers that get synthesized by Rpo41.
Next, the effect of reinduction of Rpo41 on mitochondria was tested. Time course experiments showed that mitochondrial translation can partially recover from 48h Rpo41 depletion within a timeframe of 4.5h. Sucrose gradient sedimentation experiments further showed that the mitoribosomal constitution was comparable to wildtype control samples during the time course of 4.5h of reinduction, suggesting that the ribosome assembly is not fundamentally altered in Gal-Rpo41 mitochondria. In addition, the depletion time was found to be critical for recovery of mitochondrial translation and mitochondrial RNA levels. It was observed that after 36h of Rpo41 depletion, the rRNA levels and mitochondrial translation recovered to almost 100%, but only within a time course of 10h.
Finally, mitochondria from Gal-Rpo41 cells isolated after different timepoints of reinduction were used to perform complexome profiling and the assembly of mitochondrial protein complexes was investigated. First, the steady state conditions and the assembly process of mitochondrial respiratory chain complexes were monitored. The individual respiratory chain complexes and the super-complexes of complex III, complex IV and complex V were observed. Furthermore, it was seen that they recovered from Rpo41 depletion within 4.5h of reinduction. Complexome profiles of the mitoribosomal small and large subunit discovered subcomplexes of mitoribosomal proteins that were assumed to form prior to their incorporation into assembly intermediates. The complexome profiles after reinduction indeed showed the formation of these subcomplexes before formation of the fully assembled subunit. In the mitochondrial LSU one subcomplex builds the membrane facing protuberance and a second subcomplex forms the central protuberance. In contrast to the preassembled subcomplexes, proteins that were involved in early assembly steps were exclusively found in the fully assembled subunit. Proteins that assemble at the periphery of the mitoribosome during intermediate and late assembly steps where found in soluble form suggesting a pool of unassembled proteins that supply assembly intermediates with proteins.
Taken together, the findings of this thesis suggest a so far unknow building-block model for mitoribosome assembly in which characteristic structures of the yeast mitochondrial ribosome form preassembled subcomplexes prior to their incorporation into the mitoribosome.
Cell division and cell elongation are fundamental processes for growth. In contrast to animal cells, plant cells are surrounded by rigid walls and therefore loosening of the wall is required during elongation. On the other hand, vacuole size has been shown to correlate with cell size and inhibition of vacuolar expansion limits cell growth. However, the specific role of the vacuole during cell elongation is still not fully resolved. Especially the question whether the vacuole is the leading unit during cellular growth or just passively expands upon water uptake remains to be answered. Here, we review recent findings about the contribution of the vacuole to cell elongation. In addition, we also discuss the connection between cell wall status and vacuolar morphology. In particular, we focus on the question whether vacuolar size is dictated by cell size or vice versa and share our personnel view about the sequential steps during cell elongation.
Since plants lack specialized immune cells, each cell has to defend itself independently against a plethora of different pathogens. Therefore, successful plant defense strongly relies on precise and efficient regulation of intracellular processes in every single cell. Smooth trafficking within the plant endomembrane is a prerequisite for a diverse set of immune responses. Pathogen recognition, signaling into the nucleus, cell wall enforcement, secretion of antimicrobial proteins and compounds, as well as generation of reactive oxygen species, all heavily depend on vesicle transport. In contrast, pathogens have developed a variety of different means to manipulate vesicle trafficking to prevent detection or to inhibit specific plant responses. Intriguingly, the plant endomembrane system exhibits remarkable plasticity upon pathogen attack. Unconventional trafficking pathways such as the formation of endoplasmic reticulum (ER) bodies or fusion of the vacuole with the plasma membrane are initiated and enforced as the counteraction. Here, we review the recent findings on unconventional and defense-induced trafficking pathways as the plant´s measures in response to pathogen attack. In addition, we describe the endomembrane system manipulations by different pathogens, with a focus on tethering and fusion events during vesicle trafficking.
Cells depend on the continuous renewal of their proteome composition during the cell cycle and in order to replace aberrant proteins or to react to changing environmental conditions. In higher eukaryotes, protein synthesis is achieved by up to five million ribosomes per cell. With the fast kinetics of translation, the large number of newly made proteins generates a substantial burden for protein homeostasis and requires a highly orchestrated cascade of factors promoting folding, sorting and final maturation. Several of the involved factors directly bind to translating ribosomes for the early processing of emerging nascent polypeptides and the translocation of ribosome nascent chain complexes to target membranes. In plant cells, protein synthesis also occurs in chloroplasts serving the expression of a relatively small set of 60–100 protein-coding genes. However, most of these proteins, together with nucleus-derived subunits, form central complexes majorly involved in the essential processes of photosynthetic light reaction, carbon fixation, metabolism and gene expression. Biogenesis of these heterogenic complexes adds an additional level of complexity for protein biogenesis. In this review, we summarize the current knowledge about co-translationally binding factors in chloroplasts and discuss their role in protein folding and ribosome translocation to thylakoid membranes.
Biological soil crusts (biocrusts) have been recognized as key ecological players in arid and semiarid regions at both local and global scales. They are important biodiversity components, provide critical ecosystem services, and strongly influence soil-plant relationships, and successional trajectories via facilitative, competitive, and edaphic engineering effects. Despite these important ecological roles, very little is known about biocrusts in seasonally dry tropical forests. Here we present a first baseline study on biocrust cover and ecosystem service provision in a human-modified landscape of the Brazilian Caatinga, South America's largest tropical dry forest. More specifically, we explored (1) across a network of 34 0.1 ha permanent plots the impact of disturbance, soil, precipitation, and vegetation-related parameters on biocrust cover in different stages of forest regeneration, and (2) the effect of disturbance on species composition, growth and soil organic carbon sequestration comparing early and late successional communities in two case study sites at opposite ends of the disturbance gradient. Our findings revealed that biocrusts are a conspicuous component of the Caatinga ecosystem with at least 50 different taxa of cyanobacteria, algae, lichens and bryophytes (cyanobacteria and bryophytes dominating) covering nearly 10% of the total land surface and doubling soil organic carbon content relative to bare topsoil. High litter cover, high disturbance by goats, and low soil compaction were the leading drivers for reduced biocrust cover, while precipitation was not associated Second-growth forests supported anequally spaced biocrust cover, while in old-growth-forests biocrust cover was patchy. Disturbance reduced biocrust growth by two thirds and carbon sequestration by half. In synthesis, biocrusts increase soil organic carbon (SOC) in dry forests and as they double the SOC content in disturbed areas, may be capable of counterbalancing disturbance-induced soil degradation in this ecosystem. As they fix and fertilize depauperated soils, they may play a substantial role in vegetation regeneration in the human-modified Caatinga, and may have an extended ecological role due to the ever-increasing human encroachment on natural landscapes. Even though biocrusts benefit from human presence in dry forests, high levels of anthropogenic disturbance could threaten biocrust-provided ecosystem services, and call for further, in-depth studies to elucidate the underlying mechanisms.
Micronuclei-based model system reveals functional consequences of chromothripsis in human cells
(2019)
Cancer cells often harbor chromosomes in abnormal numbers and with aberrant structure. The consequences of these chromosomal aberrations are difficult to study in cancer, and therefore several model systems have been developed in recent years. We show that human cells with extra chromosome engineered via microcell-mediated chromosome transfer often gain massive chromosomal rearrangements. The rearrangements arose by chromosome shattering and rejoining as well as by replication-dependent mechanisms. We show that the isolated micronuclei lack functional lamin B1 and become prone to envelope rupture, which leads to DNA damage and aberrant replication. The presence of functional lamin B1 partly correlates with micronuclei size, suggesting that the proper assembly of nuclear envelope might be sensitive to membrane curvature. The chromosomal rearrangements in trisomic cells provide growth advantage compared to cells without rearrangements. Our model system enables to study mechanisms of massive chromosomal rearrangements of any chromosome and their consequences in human cells.
More than ten years ago, ER-ANT1 was shown to act as an ATP/ADP antiporter and to exist in the endoplasmic reticulum (ER) of higher plants. Because structurally different transporters generally mediate energy provision to the ER, the physiological function of ER-ANT1 was not directly evident.
Interestingly, mutant plants lacking ER-ANT1 exhibit a photorespiratory phenotype. Although many research efforts were undertaken, the possible connection between the transporter and photorespiration also remained elusive. Here, a forward genetic approach was used to decipher the role of ER-ANT1 in the plant context and its association to photorespiration.
This strategy identified that additional absence of a putative HAD-type phosphatase partially restored the photorespiratory phenotype. Localisation studies revealed that the corresponding protein is targeted to the chloroplast. Moreover, biochemical analyses demonstrate that the HAD-type phosphatase is specific for pyridoxal phosphate. These observations, together with transcriptional and metabolic data of corresponding single (ER-ANT1) and double (ER-ANT1, phosphatase) loss-of-function mutant plants revealed an unexpected connection of ER-ANT1 to vitamin B6 metabolism.
Finally, a scenario is proposed, which explains how ER-ANT1 may influence B6 vitamer phosphorylation, by this affects photorespiration and causes several other physiological alterations observed in the corresponding loss-of-function mutant plants.
The Atacama Desert is one of the driest and probably oldest deserts on Earth where only a few extremophile organisms are able to survive. This study investigated two terricolous and two epiphytic lichens from the fog oasis “Las Lomitas” within the National Park Pan de Azúcar which represents a refugium for a few vascular desert plants and many lichens that can thrive on fog and dew alone. Ecophysiological measurements and climate records were combined with molecular data of the mycobiont, their green algal photobionts and lichenicolous fungi to gain information about the ecology of lichens within the fog oasis. Phylogenetic and morphological investigations led to the identification and description of the new lichen species Acarospora conafii sp. nov. as well as the lichenicolous fungi that accompanied them and revealed the trebouxioid character of all lichen photobionts. Their photosynthetic responses were compared during natural scenarios such as reactivation by high air humidity and in situ fog events to elucidate the activation strategies of this lichen community. Epiphytic lichens showed photosynthetic activity that was rapidly induced by fog and high relative air humidity whereas terricolous lichens were only activated by fog.