Dual Binding Mode of the Nascent Polypeptide-associated Complex Reveals a Novel Universal Adapter Site on the Ribosome

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of βNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of βNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, αNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.     

Catalytic activity of cytochrome oxidase and cytochrome c in apolar solvents containing phospholipids and low amounts of water

Cytochrome c and cytochrome oxidase, in bovine heart submitochondrial particles and in their purified forms, were transferred to a ternary system that contained phospholipids (10 mg/ml toluene), the apolar solvent toluene, and water at concentrations of 13-15 microliters (high water) and 3 microliters (low water) per milliliter of toluene. When the enzymes were transferred back to an all water system, they exhibited full catalytic capacity. In the low water ternary system, cytochrome c could be reduced by ascorbate introduced via inverted micelles. Also in this system, cytochrome oxidase was reduced by ascorbate and cytochrome c but its oxidation was highly impaired. Data on the kinetics of reduction by ascorbate of cytochrome c and cytochrome oxidase under these conditions are presented. Cytochrome oxidase reduced in the organic solvent by ascorbate failed to form a complex with CO, but formed a complex with cyanide introduced via inverted micelles. The oxidized and the ascorbate-reduced cytochrome oxidase-cyanide complex exhibited a trough at 415 nm and a peak at 433 nm. The extent and rate of formation of the cyanide complex were higher with the reduced form of cytochrome oxidase. To achieve protein-protein interactions (cytochrome c-cytochrome oxidase) in the ternary system, it was necessary to extract the two proteins together. There was no functional interaction when they were extracted separately and mixed. In the high water ternary system reduced cytochrome oxidase was not detected, and it oxidized ascorbate at a higher rate than in the low water system; however, this rate was several orders of magnitude lower than in aqueous media.     

Metabolism of indole-3-acetic acid and indole-3-methanol in wheat leaf segments

Indole-3-methanol is a product of indole-3-acetic acid metabolism in wheat leaves ( Triticum compactum Host., cv. Little Club). It leads either to the production of the corresponding aldehyde and carboxylic acid, to the production of a polar glucoside which releases indole-3-methanol on β-glucosidase treatment, or to an unidentified apolar product on mild alkaline hydrolysis in aqueous methanol. With reference to a published pathway of indole-3-acetic acid degradation, the results provide evidence for a prominent role of indole-3-methanol and also for the occurrence of co-oxidation processes in wheat leaves involving indole-3-acetic acid and phenolic cosubstrates.     

Anaerobic degradation of 1,3-propanediol by sulfate-reducing and by fermenting bacteria

Three strains of strictly anaerobic Gram-negative, non-sporeforming, motile bacteria were enriched and isolated from freshwater sediments with 1,3-propanediol as sole energy and carbon source. Strain OttPdl was a sulfate-reducing bacterium which grew also with lactate, ethanol, propanol, butanol, 1,4-butanediol, formate or hydrogen plus CO₂, the latter only in the presence of acetate. In the absence of sulfate, most of these substrates were fermented to the respective fatty acids in syntrophic cooperation with Methanospirillum hungatei. Sulfur, thiosulfate, or sulfite were reduced, nitrate not. The other two isolates degraded propanediol only in coculture with Methanospirillum hungatei. Strain OttGlycl grew in pure culture with acetoin and with glycerol in the presence of acetate. Strain WoAcl grew in pure culture only with acetoin. Both strains did not grow with other substrates, and did not reduce nitrate, sulfate, sulfur, thiosulfate or sulfite. The isolates were affiliated with the genera Desulfovibrio and Pelobacter. The pathways of propanediol degradation and the ecological importance of this process are discussed.     

Autochthonous fungi are central components in microbial community structure in raw fermented sausages

Raw meat sausage represents a unique ecological niche rich in nutrients for microbial consumption, making it particularly vulnerable to microbial spoilage. Starter cultures are applied to improve product stability and safety as well as flavour characteristics. However, the influence of starter cultures on microbial community assembly and succession throughout the fermentation process is largely unknown. In particular the effect on the fungal community has not yet been explored. We evaluate the microbiological status of four different raw meat sausages using high-throughput 16S rRNA gene and ITS2 gene sequencing. The objective was to study temporal changes of microbial composition during the fermentation process and to identify potential keystone species that play an important role within the microbial community. Our results suggest that fungi assigned to the species Debaryomyces hansenii and Alternaria alternata play a key role in microbial community dynamics during fermentation. In addition, bacteria related to the starter culture Lactobacillus sakei and the spoilage-associated genera Acinetobacter, Pseudomonas and Psychrobacter are central components of the microbial ecosystem in raw fermented sausages. Elucidating the exact role and interactions of these microorganisms has the potential to have direct impacts on the quality and safety of fermented foods.     

Using functional traits to investigate the determinants of crustacean zooplankton community structure

Understanding the various processes contributing to community assembly is among the central aims of ecology. As a means of exploring this topic we quantified the relative influences of habitat filtering and competition in establishing patterns of community functional trait diversity across a landscape of lakes. Habitat filtering has been invoked in shaping community structure when co‐occurring taxa are more similar in their traits than expected by chance (under‐dispersion), and competition has been inferred as a structuring agent when co‐occurring taxa are less similar (over‐dispersion). We tested these hypotheses in crustacean zooplankton communities using a functional trait‐based approach based on five traits defining zooplankton feeding and habitat preferences across 51 lakes spanning several large limnological gradients. In general, zooplankton communities were functionally less diverse than random assemblages created from the same regional species pool. Furthermore, functional diversity was strongly correlated with variables related to lake productivity, suggesting that access to resources was the chief habitat filtering process constraining zooplankton functional diversity. This pattern was driven by the predominantly herbivorous cladocerans as opposed to the more commonly omnivorous, and sometimes carnivorous, copepods.     

Glutaminyl‐tRNA Synthetase from Pseudomonas aeruginosa: Characterization,structure, and development as a screening platform

Pseudomonas aeruginosa has a high potential for developing resistance to multiple antibiotics. The gene (glnS) encoding glutaminyl‐tRNA synthetase (GlnRS) from P. aeruginosa was cloned and the resulting protein characterized. GlnRS was kinetically evaluated and the K_M and k_cat^obs, governing interactions with tRNA, were 1.0 μM and 0.15 s^?1, respectively. The crystal structure of the α₂ form of P. aeruginosa GlnRS was solved to 1.9 Å resolution. The amino acid sequence and structure of P. aeruginosa GlnRS were analyzed and compared to that of GlnRS from Escherichia coli. Amino acids that interact with ATP, glutamine, and tRNA are well conserved and structure overlays indicate that both GlnRS proteins conform to a similar three‐dimensional structure. GlnRS was developed into a screening platform using scintillation proximity assay technology and used to screen ~2,000 chemical compounds. Three inhibitory compounds were identified and analyzed for enzymatic inhibition as well as minimum inhibitory concentrations against clinically relevant bacterial strains. Two of the compounds, BM02E04 and BM04H03, were selected for further studies. These compounds displayed broad‐spectrum antibacterial activity and exhibited moderate inhibitory activity against mutant efflux deficient strains of P. aeruginosa and E. coli. Growth of wild‐type strains was unaffected, indicating that efflux was likely responsible for the lack of sensitivity. The global mode of action was determined using time‐kill kinetics. BM04H03 did not inhibit the growth of human cell cultures at any concentration and BM02E04 only inhibit cultures at the highest concentration tested (400 μg/ml). In conclusion, GlnRS from P. aeruginosa is shown to have a structure similar to that of E. coli GlnRS and two natural product compounds were identified as inhibitors of P. aeruginosa GlnRS with the potential for utility as lead candidates in antibacterial drug development in a time of increased antibiotic resistance.     

Fungal siderophore biosynthesis is partially localized in peroxisomes

Siderophores play a central role in iron metabolism and virulence of most fungi. Both Aspergillus fumigatus and Aspergillus nidulans excrete the siderophore triacetylfusarinine C (TAFC) for iron acquisition. In A. fumigatus, green fluorescence protein‐tagging revealed peroxisomal localization of the TAFC biosynthetic enzymes SidI (mevalonyl‐CoA ligase), SidH (mevalonyl‐CoA hydratase) and SidF (anhydromevalonyl‐CoA transferase), while elimination of the peroxisomal targeting signal (PTS) impaired both, peroxisomal SidH‐targeting and TAFC biosynthesis. The analysis of A. nidulans mutants deficient in peroxisomal biogenesis, ATP import or protein import revealed that cytosolic mislocalization of one or two but, interestingly, not all three enzymes impairs TAFC production during iron starvation. The PTS motifs are conserved in fungal orthologues of SidF, SidH and SidI. In agreement with the evolutionary conservation of the partial peroxisomal compartmentalization of fungal siderophore biosynthesis, the SidI orthologue of coprogen‐type siderophore‐producing Neurospora crassa was confirmed to be peroxisomal. Taken together, this study identified and characterized a novel, evolutionary conserved metabolic function of peroxisomes.