Oligomeric behavior of the RND transporters CusA and AcrB in micellar solution of detergent

AbstractWe have used analytical ultracentrifugation to explore the oligomeric states of AcrB and CusA in micellar solution of detergent. These two proteins belong to the resistance, nodulation and cell division (RND) family of efflux proteins that are involved in multiple drug and heavy metal resistance. Only the structure of AcrB has been determined so far. Although functional RND proteins should assemble as trimers as AcrB does, both AcrB and CusA form a mixture of quaternary structures (from monomer to heavy oligomer) in detergent solution. The distribution of the oligomeric states was studied as a function of different parameters: nature and concentration of the detergent, ionic strength, pH, protein concentration. This pseudo-heterogeneity does not hamper the crystallization of AcrB as a homotrimer

Chemical forms of selenium in the metal-resistant bacterium Ralstonia metallidurans CH34 exposed to selenite and selenate

International audienceRalstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se0). We have studied the kinetics of selenite (SeIV) and selenate (SeVI) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se0 and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide, and a slow detoxification pathway leading to Se0. Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins), and Se0 was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration and the bacteria accumulated approximately 25 fold less Se than when exposed to selenite. SeIV was detected as transient species in the first 12 hours after selenate introduction, Se0 also occurred as minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions selenate mostly follows an assimilatory pathway, and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite - but not selenate -contaminated environments

Characterization of a small tRNA-binding protein that interacts with the archaeal proteasome complex

Authors acknowledge financial support from the French Agence Nationale de la Recherche (grant [ANR-18-CE11-0018-01] to B.F. and [ANR-16-CE12-0016-01] to B.C.O). This work used the platforms of the Grenoble Instruct-ERIC Centre (ISBG: UMS3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) within the Grenoble Partnership for Structural Biology. The IBS Electron Microscope facility is supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA.The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with the translational machinery remains to be described. Here, we characterised a small orphan protein, Q9UZY3 (Uniprot ID) conserved in Thermococcales. The protein was identified in native pull-down experiments using the proteasome regulatory complex (PAN) as bait. X-ray crystallography and SAXS experiments revealed that the protein is monomeric and adopts a β-barrel core structure with an Oligonucleotide/oligosaccharide-Binding (OB) fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays tRNA binding properties. Pull-downs, co-immunoprecipitation and ITC studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull-downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN-20S proteasome machinery in Pyrococcus abyssi cellular extracts. The protein was therefore named Pbp11, for Proteasome Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA processing enzymes and exosome subunits dependent on Pbp11's N-terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery.Publisher PDFPeer reviewe

The Rhubarb Connection and Other Revelations: The Everyday World of Metal Ions

International audiencePink warships that vanish at dusk, urinary maladies of an emperor, and a gold test for cocaine – behold the chemistry of metal ions as never before.In this book you will learn about the sarcophagus molecule, the Chen-Kao test, and how murderers can be caught blue-handed with the wonders of glowing luminol. You will also meet the hidden chemistry of metal ions in everyday life, from the clever modern devices that measure blood-sugar levels, to the leather on your shoes and chewing gum stuck to their soles.Expect to encounter a fair share of heroes and villains, real and fictional, scientist and layperson. Such characters include an ex-MI5 employee running a hospital ward in London amid falling German V1 rockets, a notorious racing cyclist, a proud butler and the lady who first proposed nuclear fission (it’s not who you think it is).With engaging, humorous and intelligent prose, the reader will discover the fascinating back-stories of chemical discoveries and inventions where metal ions have played a major role

Cross-Talk Between Nickel and Other Metals in Microbial Systems

International audienceNickel is one of several transition metal cations that have an important function in bacterial cells. Owing to similar sizes and chemical properties, all transition metal cations interact with each other’s metabolism in biological systems. The task of a cell is to acquire sufficient nickel ions from its environment and to allocate this metal ion efficiently to nickel-dependent proteins. This must be done without causing too much collateral damage due to the potential negative effects of nickel ions on the metabolism of other transition metal cations, and vice versa. This chapter describes how this can be accomplished from the point of view of the bacterial cell

Differences between CusA and AcrB Crystallisation Highlighted by Protein Flexibility

Background: Until very recently, AcrB was the only Resistance Nodulation and cell Division transporter for which the structure has been elucidated. Towards a general understanding of this protein family, CusA and AcrB were compared. Methodology/Principal Findings: In dodecylmaltoside, AcrB crystallised in many different conditions, while CusA does not. This could be due to the difference in dynamic between these proteins as judged from limited proteolysis assays. Addition of various compounds, in particular heavy metal cations, stabilises CusA. Conclusion/Significance: This approach could constitute a first step towards CusA crystallisation

Side Chain Orientation from Methyl 1 H− 1 H Residual Dipolar Couplings Measured in Highly Deuterated Proteins

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Seleno-L-Methionine Is the Predominant Organic Form of Selenium in Cupriavidus metallidurans CH34 Exposed to Selenite or Selenate

International audienceThe accumulated organic form of selenium previously detected by X-ray absorption near-edge structure (XANES) analyses in Cupriavidus metallidurans CH34 exposed to selenite or selenate was identified as seleno-l-methionine by coupling high-performance liquid chromatography to inductively coupled plasma-mass spectrometry