No abstract

ATP-dependent proteases translocate proteins through a narrow pore for their controlled destruction. However, how a protein substrate containing a knotted topology affects this process remains unknown. Here, we characterized the effects of the trefoil-knotted protein MJ0366 from on the operation of the ClpXP protease from ClpXP completely degrades MJ0366 when pulling from the C-terminal ssrA-tag. However, when a GFP moiety is appended to the N terminus of MJ0366, ClpXP releases intact GFP with a 47-residue tail. The extended length of this tail suggests that ClpXP tightens the trefoil knot against GFP, which prevents GFP unfolding. Interestingly, if the linker between the knot core of MJ0366 and GFP is longer than 36 residues, ClpXP tightens and translocates the knot before it reaches GFP, enabling the complete unfolding and degradation of the substrate. These observations suggest that a knot-induced stall during degradation of multidomain proteins by AAA proteases may constitute a novel mechanism to produce partially degraded products with potentially new functions.

No abstract

Osmotic stress constitutes a major bacterial stress factor in the soil and during industrial fermentation. In this paper, we quantified the metabolic response, in terms of metabolic flux redistribution, of a lysine-overproducing strain of Corynebacterium glutamicum grown under continuous culture, to gradually increasing osmolality. Oxygen and carbon dioxide evolution rates, and the changes in concentration of extracellular, as well as intracellular, metabolites were measured throughout the osmotic gradient. The metabolic fluxes were estimated from these measurements and from the mass balance constraints at each metabolite-node of the assumed metabolic reaction network. Our results show that formation rates of compatible solutes--trehalose first and proline at a later stage of the gradient--increased with osmotic stress to equilibrate the external osmotic pressure. Estimated flux distributions indicate that the observed increase in the glucose specific uptake rate with osmotic stress is channeled through the main energy generating pathways-- glycolysis and the tricarboxylic acid cycle--while the flux through the pentose phosphate pathway remains constant throughout the gradient. This results in a significant increase in the net specific ATP production rate, which may possibly be used to support the higher energy requirements required for cellular maintenance at high osmolalities. Finally, nodal analysis confirmed that the PEP/pyruvate node is essentially rigid and that the glucose-6-phosphate, oxaloacetate and alpha-ketoglutarate nodes are flexible and therefore adaptable to changes in osmotic pressure in C. glutamicum.

Atrazine sorption was studied in six Andisol and Ultisol soils. Humic and fulvic acids and humin contributions were established. Sorption on soils was well described by the Freundlich model. Kf values ranged from 2.2-15.6 μg(1-1/n)mL(1/n)g⁻¹. The relevance of humic acid and humin was deduced from isotherm and kinetics experiments. KOC values varied between 221 and 679 mLg⁻¹ for these fractions. Fulvic acid presented low binding capacity. Sorption was controlled by instantaneous equilibrium followed by a time-dependent phase. The Elovich equation, intraparticle diffusion model, and a two-site nonequilibrium model allowed us to conclude that (i) there are two rate-limited phases in Andisols related to intrasorbent diffusion in organic matter and retarded intraparticle diffusion in the organo-mineral complex and that (ii) there is one rate-limited phase in Ultisols attributed to the mineral composition. The lower organic matter content of Ultisols and the slower sorption rate and mechanisms involved must be considered to assess the leaching behavior of atrazine.