Production of Membrane Proteins in Pseudomonas stutzeri.

Functional and structural studies on membrane proteins are often hampered by insufficient yields, misfolding and aggregation during the production and purification process. Escherichia coli is the most commonly used expression host for the production of recombinant prokaryotic integral membrane proteins. However, in many cases expression hosts other than E. coli are more appropriate for certain target proteins. Here, we report a convenient, systematically developed expression system using the γ-proteobacterium Pseudomonas stutzeri as an alternative production host for over-expression of integral membrane proteins. P. stutzeri can be easily and inexpensively cultured in large quantities. The Pseudomonas expression vectors are designed for inducible expression of affinity-tagged fusion proteins controlled by the PBAD promoter. This chapter provides detailed protocols of the different steps required to successfully produce and isolate recombinant membrane proteins with high yields in P. stutzeri.

Heterohexamers Formed by CcmK3 and CcmK4 Increase the Complexity of Beta Carboxysome Shells.

Bacterial microcompartments (BMCs) encapsulate enzymes within a selectively permeable, proteinaceous shell. Carboxysomes are BMCs containing ribulose-1,5-bisphosphate carboxylase oxygenase and carbonic anhydrase that enhance carbon dioxide fixation. The carboxysome shell consists of three structurally characterized protein types, each named after the oligomer they form: BMC-H (hexamer), BMC-P (pentamer), and BMC-T (trimer). These three protein types form cyclic homooligomers with pores at the center of symmetry that enable metabolite transport across the shell. Carboxysome shells contain multiple BMC-H paralogs, each with distinctly conserved residues surrounding the pore, which are assumed to be associated with specific metabolites. We studied the regulation of ß-carboxysome shell composition by investigating the BMC-H genes ccmK3 and ccmK4 situated in a locus remote from other carboxysome genes. We made single and double deletion mutants of ccmK3 and ccmK4 in Synechococcus elongatus PCC7942 and show that, unlike CcmK3, CcmK4 is necessary for optimal growth. In contrast to other CcmK proteins, CcmK3 does not form homohexamers; instead CcmK3 forms heterohexamers with CcmK4 with a 1:2 stoichiometry. The CcmK3-CcmK4 heterohexamers form stacked dodecamers in a pH-dependent manner. Our results indicate that CcmK3-CcmK4 heterohexamers potentially expand the range of permeability properties of metabolite channels in carboxysome shells. Moreover, the observed facultative formation of dodecamers in solution suggests that carboxysome shell permeability may be dynamically attenuated by "capping" facet-embedded hexamers with a second hexamer. Because ß-carboxysomes are obligately expressed, heterohexamer formation and capping could provide a rapid and reversible means to alter metabolite flux across the shell in response to environmental/growth conditions.

Pseudomonas stutzeri as an alternative host for membrane proteins.

BACKGROUND: Studies on membrane proteins are often hampered by insufficient yields of the protein of interest. Several prokaryotic hosts have been tested for their applicability as production platform but still Escherichia coli by far is the one most commonly used. Nevertheless, it has been demonstrated that in some cases hosts other than E. coli are more appropriate for certain target proteins. RESULTS: Here we have developed an expression system for the heterologous production of membrane proteins using a single plasmid-based approach. The gammaproteobacterium Pseudomonas stutzeri was employed as a new production host. We investigated several basic microbiological features crucial for its handling in the laboratory. The organism belonging to bio-safety level one is a close relative of the human pathogen Pseudomonas aeruginosa. Pseudomonas stutzeri is comparable to E. coli regarding its growth and cultivation conditions. Several effective antibiotics were identified and a protocol for plasmid transformation was established. We present a workflow including cloning of the target proteins, small-scale screening for the best production conditions and finally large-scale production in the milligram range. The GFP folding assay was used for the rapid analysis of protein folding states. In summary, out of 36 heterologous target proteins, 20 were produced at high yields. Additionally, eight transporters derived from P. aeruginosa could be obtained with high yields. Upscaling of protein production and purification of a Gluconate:H+ Symporter (GntP) family transporter (STM2913) from Salmonella enterica to high purity was demonstrated. CONCLUSIONS: Pseudomonas stutzeri is an alternative production host for membrane proteins with success rates comparable to E. coli. However, some proteins were produced with high yields in P. stutzeri but not in E. coli and vice versa. Therefore, P. stutzeri extends the spectrum of useful production hosts for membrane proteins and increases the success rate for highly produced proteins. Using the new pL2020 vector no additional cloning is required to test both hosts in parallel.

ß-Carboxysome bioinformatics: identification and evolution of new bacterial microcompartment protein gene classes and core locus constraints.

Carboxysomes are bacterial microcompartments (BMCs) that enhance CO2 fixation in all cyanobacteria. Structurally, carboxysome shell proteins are classified according to the type of oligomer formed: hexameric (BMC-H), trimeric (BMC-T) and pentameric (BMC-P) proteins. To understand the forces driving the evolution of the carboxysome shell, we conducted a bioinformatic study of genes encoding ß-carboxysome shell proteins, taking advantage of the recent large increase in sequenced cyanobacterial genomes. In addition to the four well-established BMC-H (CcmK1-4) classes, our analysis reveals two new CcmK classes, which we name CcmK5 and CcmK6. CcmK5 is phylogenetically closest to CcmK3 and CcmK4, and the ccmK5 gene is found only in genomes lacking ccmK3 and ccmk4 genes. ccmK6 is found predominantly in heterocyst-forming cyanobacteria. The gene encoding the BMC-T homolog CcmO is associated with the main carboxysome locus (MCL) in only 60% of all species. We find five evolutionary origins of separation of ccmO from the MCL. Transcriptome analysis demonstrates that satellite ccmO genes, in contrast to MCL-associated ccmO genes, are never co-regulated with other MCL genes. The dispersal of carboxysome shell genes across the genome allows for distinct regulation of their expression, perhaps in response to changes in environmental conditions.

Hsp90 is involved in the regulation of cytosolic precursor protein abundance in tomato.

Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 (Hsp70) and 90 (Hsp90) assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins with respect to the cytosolic chaperone-dependent regulation. Some preproteins such as pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins such as pSSU is more strongly dependent on Hsp70. The E3 ligase, C-terminus of Hsp70-interacting protein (Chip), appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable with the cytosolic unfolded protein response.

Hsp90 is involved in the regulation of cytosolic precursor protein abundance in tomato.

Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the heat stress protein 70 and 90 (Hsp70 or Hsp90) families assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins in respect to the cytosolic chaperone dependent regulation. Some preproteins like pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins like pSSU is more strongly dependent on Hsp70. The E3 ligase Chip appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable to the cytosolic unfolded protein response.

Comparative transcriptome atlases reveal altered gene expression modules between two Cleomaceae C3 and C4 plant species.

C(4) photosynthesis outperforms the ancestral C(3) state in a wide range of natural and agro-ecosystems by affording higher water-use and nitrogen-use efficiencies. It therefore represents a prime target for engineering novel, high-yielding crops by introducing the trait into C(3) backgrounds. However, the genetic architecture of C(4) photosynthesis remains largely unknown. To define the divergence in gene expression modules between C(3) and C(4) photosynthesis during leaf ontogeny, we generated comprehensive transcriptome atlases of two Cleomaceae species, Gynandropsis gynandra (C(4)) and Tarenaya hassleriana (C(3)), by RNA sequencing. Overall, the gene expression profiles appear remarkably similar between the C(3) and C(4) species. We found that known C(4) genes were recruited to photosynthesis from different expression domains in C(3), including typical housekeeping gene expression patterns in various tissues as well as individual heterotrophic tissues. Furthermore, we identified a structure-related module recruited from the C(3) root. Comparison of gene expression patterns with anatomy during leaf ontogeny provided insight into genetic features of Kranz anatomy. Altered expression of developmental factors and cell cycle genes is associated with a higher degree of endoreduplication in enlarged C(4) bundle sheath cells. A delay in mesophyll differentiation apparent both in the leaf anatomy and the transcriptome allows for extended vein formation in the C(4) leaf.

self-assembling GFP: a versatile tool for plant (membrane) protein analyses.

The investigation of cellular processes on the molecular level is important to understand the functional network within plant cells. self-assembling GFP has evolved to be a versatile tool for (membrane) protein analyses. Based on the autocatalytical reassembling property of the nonfluorescent strands 1-10 and 11, protein distribution and membrane protein topology can be analyzed in vivo. Here, we provide basic protocols to determine membrane protein topology in Arabidopsis thaliana protoplasts.

Implementing an advanced waste separation step in an MBT plant: assessment of technical, economic and environmental impacts.

Heavy fractions resulting from mechanical treatment stages of mechanical-biological waste treatment plants are posing very specific demands with regard to further treatment (large portions of inert and high-caloric components). Based on the current Austrian legal situation such a waste stream cannot be landfilled and must be thermally treated. The aim of this research was to evaluate if an inert fraction generated from this waste stream with advanced separation technologies, two sensor-based [near-infrared spectroscopy (NIR), X-ray transmission (XRT)] and two mechanical systems (wet and dry) is able to be disposed of. The performance of the treatment options for separation was evaluated by characterizing the resulting product streams with respect to purity and yield. Complementing the technical evaluation of the processing options, an assessment of the economic and global warming effects of the change in waste stream routing was conducted. The separated inert fraction was evaluated with regard to landfilling. The remaining high-caloric product stream was evaluated with regard to thermal utilization. The results show that, in principal, the selected treatment technologies can be used to separate high-caloric from inert components. Limitations were identified with regard to the product qualities achieved, as well as to the economic expedience of the treatment options. One of the sensor-based sorting systems (X-ray) was able to produce the highest amount of disposeable heavy fraction (44.1%), while having the lowest content of organic (2.0% C biogenic per kg waste input) components. None of the high-caloric product streams complied with the requirements for solid recovered fuels as defined in the Austrian Ordinance on Waste Incineration. The economic evaluation illustrates the highest specific treatment costs for the XRT (€ 23.15 per t), followed by the NIR-based sorting system (€ 15.67 per t), and the lowest costs for the air separation system (€ 10.79 per t). Within the ecological evaluation it can be shown that the results depend strongly on the higher heating value of the high caloric light fraction and on the content of C biogenic of the heavy fraction. Therefore, the XRT system had the best results for the overall GWP [-14 kg carbon dioxide equivalents (CO2 eq) per t of input waste] and the NIR-based the worst (193 kg CO2 eq per t of input waste). It is concluded that three of the treatment options would be suitable under the specific conditions considered here. Of these, sensor-based sorting is preferable owing to its flexibility.

Toc33 and Toc64-III cooperate in precursor protein import into the chloroplasts of Arabidopsis thaliana.

The import of cytosolically synthesized precursor proteins into chloroplasts by the translocon at the outer envelope membrane of chloroplasts (TOC) is crucial for organelle function. The recognition of precursor proteins at the chloroplast surface precedes translocation and involves the membrane-inserted receptor subunits Toc34 and Toc159. A third receptor, Toc64, was discussed to recognize cytosolic complexes guiding precursor proteins to the membrane surface, but this function remains debated. We analysed Arabidopsis thaliana plants carrying a T-DNA insertion in the gene encoding the Toc64 homolog Toc64-III. We observed a light intensity-dependent growth phenotype, which is distinct from the phenotype of ppi1, the previously described mutant of the TOC34 homolog TOC33. Furthermore, chloroplast import of the model precursor proteins pOE33 and pSSU into chloroplasts is reduced in protoplasts isolated from plants with impaired Toc64-III function. This suggests that Toc64-III modulates the translocation efficiency in vivo. A ppi1 and toc64-III double mutant shows a significant increase in the transcript levels of HSP90 and TOC75-III, the latter coding for the pore-forming TOC component. Remarkably, the protein level of Toc75-III is significantly reduced, suggesting that Toc64-III and Toc33 cooperate in the insertion or stabilization of Toc75-III. Accordingly, the results presented support Toc64 as an import-relevant component of the TOC complex.

Energy from waste in Europe: an analysis and comparison of the EU 27.

This article focuses on analysing the development of waste-generated energy in the countries of the European Union (EU 27). Besides elaborating the relevant legal and political framework in the waste and energy sector as well as climate protection, the results from correlation analyses based on the databases of the energy statistics from Eurostat are discussed. The share of energy from waste is correlated with macro-economic, waste- and energy-sector-related data, which have been defined as potentially relevant for energy recovery from waste in the countries of the European Union. The results show that a single factor influencing the extent of waste-generated energy could not be isolated as it is being influenced not only by the state of economic development and the state of development of waste management systems in the respective countries but also by energy-sector-related factors and the individual priority settings in those countries. Nevertheless the main driving force for an increase in the utilization of waste for energy generation can be seen in the legal and political framework of the European Union leading to the consequence that market conditions influence the realization of waste management infrastructure for waste-generated energy.

An mRNA blueprint for C4 photosynthesis derived from comparative transcriptomics of closely related C3 and C4 species.

C(4) photosynthesis involves alterations to the biochemistry, cell biology, and development of leaves. Together, these modifications increase the efficiency of photosynthesis, and despite the apparent complexity of the pathway, it has evolved at least 45 times independently within the angiosperms. To provide insight into the extent to which gene expression is altered between C(3) and C(4) leaves, and to identify candidates associated with the C(4) pathway, we used massively parallel mRNA sequencing of closely related C(3) (Cleome spinosa) and C(4) (Cleome gynandra) species. Gene annotation was facilitated by the phylogenetic proximity of Cleome and Arabidopsis (Arabidopsis thaliana). Up to 603 transcripts differ in abundance between these C(3) and C(4) leaves. These include 17 transcription factors, putative transport proteins, as well as genes that in Arabidopsis are implicated in chloroplast movement and expansion, plasmodesmatal connectivity, and cell wall modification. These are all characteristics known to alter in a C(4) leaf but that previously had remained undefined at the molecular level. We also document large shifts in overall transcription profiles for selected functional classes. Our approach defines the extent to which transcript abundance in these C(3) and C(4) leaves differs, provides a blueprint for the NAD-malic enzyme C(4) pathway operating in a dicotyledon, and furthermore identifies potential regulators. We anticipate that comparative transcriptomics of closely related species will provide deep insight into the evolution of other complex traits.