ATG8 Family Proteins Act as Scaffolds for Assembly of the ULK Complex: SEQUENCE REQUIREMENTS FOR LC3-INTERACTING REGION (LIR) MOTIFS*

Autophagy is a lysosome-dependent degradation system conserved among eukaryotes. The mammalian Atg1 homologues, Unc-51 like kinase (ULK) 1 and 2, are multifunctional proteins with roles in autophagy, neurite outgrowth, and vesicle transport. The mammalian ULK complex involved in autophagy consists of ULK1, ULK2, ATG13, FIP200, and ATG101. We have used pulldown and peptide array overlay assays to study interactions between the ULK complex and six different ATG8 family proteins. Strikingly, in addition to ULK1 and ULK2, ATG13 and FIP200 interacted with human ATG8 proteins, all with strong preference for the GABARAP subfamily. Similarly, yeast and Drosophila Atg1 interacted with their respective Atg8 proteins, demonstrating the evolutionary conservation of the interaction. Use of peptide arrays allowed precise mapping of the functional LIR motifs, and two-dimensional scans of the ULK1 and ATG13 LIR motifs revealed which substitutions that were tolerated. This information, combined with an analysis of known LIR motifs, provides us with a clearer picture of sequence requirements for LIR motifs. In addition to the known requirements of the aromatic and hydrophobic residues of the core motif, we found the interactions to depend strongly on acidic residues surrounding the central core LIR motifs. A preference for either a hydrophobic residue or an acidic residue following the aromatic residue in the LIR motif is also evident. Importantly, the LIR motif is required for starvation-induced association of ULK1 with autophagosomes. Our data suggest that ATG8 proteins act as scaffolds for assembly of the ULK complex at the phagophore.     

Filter bed systems treating domestic wastewater in the Nordic countries – Performance and reuse of filter media

Nine filter beds have been constructed in the Nordic countries, Denmark, Finland, Norway and Sweden. Filter beds consist of a septic tank followed by an aerobic pre-treatment biofilter and a subsequent saturated flow grass-covered filter. Thus, filter beds are similar to subsurface flow constructed wetlands with pre-treatment biofilters, but do not have wetland plants with roots submerged into the saturated filter. All saturated filters contain Filtralite^®P, a light-weight expanded clay aggregate possessing high phosphorus sorption capacity. The filter bed systems showed stable and consistent performance during the testing period of 3 years. Removal of organic matter measured as biochemical oxygen demand (BOD) was >80%, total phosphorus (TP) >94% and total nitrogen (TN) ranged from 32 to 66%. Effluent concentrations of fecal indicator bacteria met the European bathing water quality criteria in all systems. One system was investigated for virus removal and somatic viruses were not detected in the effluent. The investigations revealed that the majority of the BOD and nitrogen removal occurred in the pre-treatment filters and the phosphorus and bacteria removal was more prominent in the saturated filters. The saturated filters could be built substantially smaller than the current design guidelines without sacrificing treatment performance. The used filter material met the Norwegian regulations for reuse in agriculture with respect to heavy metals, bacteria and parasites. When saturated with phosphorus, the light-weight aggregate, Filtralite^®P used in the saturated bed is a suitable phosphorus fertilizer and additionally has a liming effect.     

Micro ecosystems from feed industry surfaces: a survival and biofilm study of <Emphasis Type="Italic">Salmonella</Emphasis>versus host resident flora strains

Background  

The presence of Salmonella enterica serovars in feed ingredients, products and processing facilities is a well recognized problem worldwide. In Norwegian feed factories, strict control measures are implemented to avoid establishment and spreading of Salmonella throughout the processing chain. There is limited knowledge on the presence and survival of the resident microflora in feed production plants. Information on interactions between Salmonella and other bacteria in feed production plants and how they affect survival and biofilm formation of Salmonella is also limited. The aim of this study was to identify resident microbiota found in feed production environments, and to compare the survival of resident flora strains and Salmonella to stress factors typically found in feed processing environments. Moreover, the role of dominant resident flora strains in the biofilm development of Salmonella was determined.     

In vivo analysis of the regulatory genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455 reveals their differential control over antibiotic biosynthesis

Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.     

The Pseudomonas fluorescens AlgG protein,but not its mannuronan C-5-epimerase activity,is needed for alginate polymer formation

Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to alpha-L-guluronic acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.     

Manganese,copper, and zinc in cerebrospinal fluid from patients with multiple sclerosis

The concentrations of manganese, copper, and zinc in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and patients with no known neurological disease (control group) were measured. Manganese and copper levels were determined by two different analytical methods: atomic absorption spectrometry (AAS) and high-resolution inductively coupled plasma-mass spectrometry (HR-ICP-MS), whereas zinc levels were determined by HR-ICP-MS only. Manganese levels (mean±SEM) were significantly decreased in the CSF of MS patients (1.07±0.13 μg/L, ICP-MS; 1.08±0.11 μg/L, AAS) compared to the levels in the control group (1.78±0.26 μg/L, ICP-MS; 1.51±0.17 μg/L, AAS). Copper levels were significantly elevated in the CSF of MS patients (10.90±1.11 μg/L; ICP-MS, 11.53±0.83 μg/L, AAS) compared to the levels in the control group (8.67±0.49 μg/L, ICP-MS; 9.10±0.62 μg/L, AAS). There were no significant differences between the CSF zinc levels of MS and control patients. The physiological basis for the differences in manganese and copper concentrations between MS patients and controls is unknown, but could be related to alterations in the manganese-containing enzyme glutamine synthetase and the copper-containing enzyme cytochrome oxidase.