Proteomics,phylogenetics, and coexpression analyses indicate novel interactions in the plastid CLP chaperone-protease system

The chloroplast chaperone CLPC1 unfolds and delivers substrates to the stromal CLPPRT protease complex for degradation. We previously used an in vivo trapping approach to identify interactors with CLPC1 in Arabidopsis thaliana by expressing a STREPII-tagged copy of CLPC1 mutated in its Walker B domains (CLPC1-TRAP) followed by affinity purification and mass spectrometry. To create a larger pool of candidate substrates, adaptors, or regulators, we carried out a far more sensitive and comprehensive in vivo protein trapping analysis. We identified 59 highly enriched CLPC1 protein interactors, in particular proteins belonging to families of unknown functions (DUF760, DUF179, DUF3143, UVR-DUF151, HugZ/DUF2470), as well as the UVR domain proteins EXE1 and EXE2 implicated in singlet oxygen damage and signaling. Phylogenetic and functional domain analyses identified other members of these families that appear to localize (nearly) exclusively to plastids. In addition, several of these DUF proteins are of very low abundance as determined through the Arabidopsis PeptideAtlas http://www.peptideatlas.org/builds/arabidopsis/ showing that enrichment in the CLPC1-TRAP was extremely selective. Evolutionary rate covariation indicated that the HugZ/DUF2470 family coevolved with the plastid CLP machinery suggesting functional and/or physical interactions. Finally, mRNA-based coexpression networks showed that all 12 CLP protease subunits tightly coexpressed as a single cluster with deep connections to DUF760-3. Coexpression modules for other trapped proteins suggested specific functions in biological processes, e.g., UVR2 and UVR3 were associated with extraplastidic degradation, whereas DUF760-6 is likely involved in senescence. This study provides a strong foundation for discovery of substrate selection by the chloroplast CLP protease system.     

A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.     

Analysis of the Requirement for a pUB110 mob Region during Tn916-Dependent Mobilization

Tn916-dependent mobilization of nonconjugative plasmids pUB110 and its derivative pUB110Deltam was compared. Deleting a 787-bp fragment from the pUB110 mob region created plasmid pUB110Deltam. Deletion of the mob region of pUB110 rendered the plasmid nontransferable by the conjugative plasmids of Bacillus thuringiensis subsp. israelensis. During matings between Bacillus subtilis (Tn916) and B. thuringiensis subsp. israelensis, however, Tn916-dependent mobilization of plasmids pUB110 and pUB110Deltam was observed at a frequency of approximately 2 x 10(-6) transconjugants per donor. The results show that Tn916-mediated conjugal transfer of plasmids is a mob-independent event. Jaworski and Clewell (J. Bacteriol 177; 6644-6651) recently demonstrated the presence of an IncP-like nicking site in the oriT of Tn916. These data suggest that a IncP-like nickling site is essential for Tn916-mediated plasmid transfer.     

Habitat preferences of the urban wall spider Oecobius navus (Araneae, Oecobiidae)

Abstract  To assess the relative impact of a range of habitat variables on spider abundance, field and laboratory experiments were conducted on populations of the urban wall spider Oecobius navus in suburban Perth, Western Australia. Habitat characteristics investigated were: substrate type, wind speed, rainfall, sunlight exposure, relative humidity, air temperature, substrate temperature, artificial lighting and prey type/abundance. In the field, O. navus was found to be associated with high humidity, low air temperature and shelter from sunlight and rainfall. Oecobius navus was more abundant at sites with greater prey abundance. The most common prey item was the red meat ant Iridomyrmex chasei . Juvenile spiders were more abundant than adult spiders; however, patterns between spider abundance and habitat variables were similar for both adults and juveniles. Laboratory experiments showed that O. navus preferred to build webs on wooden substrates, and pitted limestone walls. These findings indicate that O. navus may be vulnerable to desiccation and/or thermal stress, and thus survives better on sheltered walls.     

Evolutionary adaptation of a reflex system: sensory hysteresis counters muscle ‘catch’ tension

Summary The metathoracic femoral chordotonal organ is a receptor of the locust,Schistocerca, hindleg that encodes the angle of the femoro-tibial joint. However, the discharge of the organ shows considerable hysteresis, in that there is a substantial decline in the level of afferent firing when the tibia is moved and then returned to its initial position. Similar hysteresis is also seen in some joint receptors and interneurons of other invertebrates and vertebrates. When the chordotonal organ is stimulated in freely moving locusts, mimicking sudden changes in joint angle, reflex discharges can be elicited in the tibial extensor muscle that resist apparent joint movement and also show similar hysteresis. This pattern of motoneuron activity is demonstrated to potentially function to eliminate residual, catch muscle tensions that result from increases in motoneuron firing frequency. This adaptation could also serve to produce accurate load compensation.     

Identification,purification, and partial characterization of vitellin from the eggs of Eurygaster integriceps putton (heteroptera,pentatomidae)

A single vitellin was identified in the eggs of the wheat bug, Eurygaster integriceps, and purified by a two-step procedure including ion exchange and gel permeation chromatography. Its nondenatured molecular mass is estimated to be 385 kDa. Under reducing conditions—SDS-PAGE—the wheat bug vitellin gives five polypeptides at 110 kDa, 80 kDa, 69 kDa, 53 kDa, and 38 kDa. Its amino acid composition is characterized by high content of aspartic acid/asparagine and glutamic acid/glutamine and low content of methionine and histidine. The lipid moiety (5.65% by weight) includes diacylglycerols, cholesterol, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin. Carbohydrate content amounts to 4.54% by weight. A part of the wheat bug vitellin is dimerized during the course of deposition into the yolk granules. © 1995 Wiley-Liss, Inc.     

γ-Butyrobetaine hydroxylase: A unique protective effect of catalase

Catalase stimulates the activity of homogeneous γ-butyrobetaine hydroxylase by approximately 300-fold. The stimulation of the hydroxylation reaction elicited by catalase is saturable, and although a number of proteins may be substituted for catalase, none is as effective. γ-Butyrobetaine hydroxylase is also irreversibly inactivated in the presence of one of its substrates, oxygen, and its cofactor, ascorbate. This inactivation of the hydroxylase activity may be prevented by (i) the presence of high concentrations (2 mg/ml) of various proteins, (ii) the presence of catalytic concentrations (20 μg/ml) of catalase, or (iii) the presence of 10 mm histidine or dithiothreitol. Oxidized species of ascorbate do not appear to be responsible for the inactivation process. Time-dependent inactivation is also observed when γ-butyrobetaine hydroxylase is preincubated with hydrogen peroxide generated by the glucose oxidase-catalyzed oxidation of glucose. At low concentrations, superoxide dismutase was not as effective as an equivalent protein concentration of catalase in protecting against inactivation, and hydroxyl radical scavengers were completely ineffective. In measurements of γ-butyrobetaine hydroxylase activity, the presence of catalase both stimulates the catalytic activity of the hydroxylase and protects the enzyme from inactivation by a product of the interaction of components in the assay mixture, presumably hydrogen peroxide.     

High-throughput phenotypic characterization of Pseudomonas aeruginosa membrane transport genes

The deluge of data generated by genome sequencing has led to an increasing reliance on bioinformatic predictions, since the traditional experimental approach of characterizing gene function one at a time cannot possibly keep pace with the sequence-based discovery of novel genes. We have utilized Biolog phenotype MicroArrays to identify phenotypes of gene knockout mutants in the opportunistic pathogen and versatile soil bacterium Pseudomonas aeruginosa in a relatively high-throughput fashion. Seventy-eight P. aeruginosa mutants defective in predicted sugar and amino acid membrane transporter genes were screened and clear phenotypes were identified for 27 of these. In all cases, these phenotypes were confirmed by independent growth assays on minimal media. Using qRT-PCR, we demonstrate that the expression levels of 11 of these transporter genes were induced from 4- to 90-fold by their substrates identified via phenotype analysis. Overall, the experimental data showed the bioinformatic predictions to be largely correct in 22 out of 27 cases, and led to the identification of novel transporter genes and a potentially new histamine catabolic pathway. Thus, rapid phenotype identification assays are an invaluable tool for confirming and extending bioinformatic predictions.     

Replacement of the antifreeze-like domain of human N-acetylneuraminic acid phosphate synthase with the mouse antifreeze-like domain impacts both N-acetylneuraminic acid 9-phosphate synthase and 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activities

Human NeuNAc-9-P synthase is a two-domain protein with ability to synthesize both NeuNAc-9-P and KDN-9-P. Its mouse counterpart differs by only 20 out of 359 amino acids but does not produce KDN-9-P. By replacing the AFL domain of the human NeuNAc-9-P synthase which accommodates 12 of these differences, with the mouse AFL domain we examined its importance for the secondary KDN-9-P synthetic activity. The chimeric protein retained almost half of the ability of the human enzyme for KDN-9-P synthesis while the NeuNAc-9-P production was reduced to less than 10%. Data from the homology modeling and the effect of divalent ions and temperature on the enzyme activities suggest conformational differences between the human and mouse AFL domains that alter the shape of the cavity accommodating the substrates. Therefore, although the AFL domain itself does not define the ability of the human enzyme for KDN-9-P synthesis, it is important for both activities by aiding optimal positioning of the substrates.     

A phylogenomic analysis of the shikimate dehydrogenases reveals broadscale functional diversification and identifies one functionally distinct subclass

The shikimate dehydrogenases (SDH) represent a widely distributed enzyme family with an essential role in secondary metabolism. This superfamily had been previously subdivided into 4 enzyme groups (AroE, YdiB, SdhL, and RifI), which show clear biochemical and functional differences ranging from amino acid biosynthesis to antibiotic production. Despite the importance of this group, little is known about how such essential enzymatic functions can evolve and diversify. We dissected the enzyme superfamily with a phylogenomic analysis of approximately 250 fully sequenced genomes, making use of previously characterized representatives from each enzyme class, and the key substrate-binding residues known to distinguish substrate specificity. We identified 5 major evolutionary and functional SDH subgroups and several other potentially unique functional classes within this complex enzyme family and then validated the functional distinctiveness of each group by characterizing the 5 SDH homologs found in Pseudomonas putida KT2440 biochemically. We identified an entirely novel functionally distinct subgroup, which we designated Ael1 (AroE-like1) and also delineated a new group of shikimate/quinate dehydrogenases (YdiB2), which is phylogenetically distinct from the previously described Escherichia coli YdiB. The combination of biochemical, phylogenetic, and genomic approaches has revealed the broad extent to which the SDH enzyme superfamily has diversified. Five functional groups were validated with the potential for at least 5 additional subgroups. Our analysis also identified a new SDH functional group, which appears to have evolved recently from an ancestral AroE, illustrating a very prominent role of horizontal transmission and neofunctionalizaton in the evolutionary and functional diversification of this enzyme family.