Transmembrane modulator-dependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases

Protein-tyrosine kinases regulating bacterial exopolysaccharide synthesis autophosphorylate on tyrosines located in a conserved C-terminal region. So far no other substrates have been identified for these kinases. Here we demonstrate that Bacillus subtilis YwqD not only autophosphorylates at Tyr-228, but that it also phosphorylates the two UDP-glucose dehydrogenases (UDP-glucose DHs) YwqF and TuaD at a tyrosine residue. However, phosphorylation of YwqF and TuaD occurs only in the presence of the transmembrane protein YwqC. The presumed intracellular C-terminal part of YwqC (last 50 amino acids) seems to interact with the tyrosine-kinase and to allow YwqD-catalysed phosphorylation of the two UDP-glucose DHs, which are key enzymes for the synthesis of acidic polysaccharides. However, only when phosphorylated by YwqD do the two enzymes exhibit detectable UDP-glucose DH activity. Dephosphorylation of P-Tyr-YwqF and P-Tyr-TuaD by the P-Tyr-protein phosphatase YwqE switched off their UDP-glucose DH activity. YwqE, which is encoded by the fourth gene of the B.subtilis ywqCDEF operon, also dephosphorylates P-Tyr-YwqD.     

Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones

The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.     

Membrane protein insertion regulated by bringing electrostatic and hydrophobic interactions into play. A case study with the translocation domain of diphtheria toxin

The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactions between proteins and membranes. During cell intoxication, this domain undergoes a change from a soluble and folded state at alkaline pH to a functional membrane-inserted state at acid pH. We found that hydrophobic and electrostatic interactions occur in a sequential manner between the domain and the membrane during the insertion. The first step involves hydrophobic interactions by the C-terminal region. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane. Accumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The second step, as the pH decreases, leads to the functional inserted state. It arises from the changes in the balance of electrostatic attractions and repulsions between the N-terminal part and the membrane. Our study shows how the structural changes and the interaction with membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system.     

Major changes in the cell wall during silique development in Arabidopsis thaliana

Fruit development is a highly complex process, which involves major changes in plant metabolism leading to cell growth and differentiation. Changes in cell wall composition and structure play a major role in modulating cell growth. We investigated the changes in cell wall composition and the activities of associated enzymes during the dry fruit development of the model plant Arabidopsis thaliana. Silique development is characterized by several specific phases leading to fruit dehiscence and seed dispersal. We showed that early phases of silique growth were characterized by specific changes in non-cellulosic sugar content (rhamnose, arabinose, xylose, galactose and galacturonic acid). Xyloglucan oligosaccharide mass profiling further showed a strong increase in O-acetylated xyloglucans over the course of silique development, which could suggest a decreased capacity of xyloglucans to be associated with each other or to cellulose. The degree of methylesterification, mediated by the activity of pectin methylesterases (PMEs), decreased over the course of silique growth and dehiscence. The major changes in cell wall composition revealed by our analysis suggest that it could be major determinants in modulating cell wall rheology leading to growth or growth arrest.     

Dual cardiac contractile effects of the alpha2-AMPK deletion in low-flow ischemia and reperfusion

Because the question "is AMP-activated protein kinase (AMPK) alpha(2)-isoform a friend or a foe in the protection of the myocardium against ischemia-reperfusion injury?" is still in debate, we studied the functional consequence of its deletion on the contractility, the energetics, and the respiration of the isolated perfused heart and characterized the response to low-flow ischemia and reperfusion with glucose and pyruvate as substrates. alpha(2)-AMPK deletion did not affect basal contractility, respiration, and high-energy phosphate contents but induced a twofold reduction in glycogen content and a threefold reduction in glucose uptake. Low-flow ischemia increased AMPK phosphorylation and stimulated glucose uptake and phosphorylation in both alpha(2)-knockout (alpha(2)-KO) and wild-type (WT) groups. The high sensitivity of alpha(2)-KO to the development of ischemic contracture was attributed to the constitutive impairment in glucose transport and glycogen content and not to a perturbation of the energy transfer by creatine kinase (CK). The functional coupling of MM-CK to myofibrillar ATPase and the CK fluxes were indeed similar in alpha(2)-KO and WT. Low-flow ischemia impaired CK flux by 50% in both strains, showing that alpha(2)-AMPK does not control CK activity. Despite the higher sensitivity to contracture, the postischemic contractility recovered to similar levels in both alpha(2)-KO and WT in the absence of fatty acids. In their presence, alpha(2)-AMPK deletion also accelerated the contracture but delayed postischemic contractile recovery. In conclusion, alpha(2)-AMPK is required for a normal glucose uptake and glycogen content, which protects the heart from the development of the ischemic contracture, but not for contractile recovery in the absence of fatty acids.     

Autophagic Cell Death in Dictyostelium Requires the Receptor Histidine Kinase DhkM

Dictyostelium constitutes a genetically tractable model for the analysis of autophagic cell death (ACD). During ACD, Dictyostelium cells first transform into paddle cells and then become round, synthesize cellulose, vacuolize, and die. Through random insertional mutagenesis, we identified the receptor histidine kinase DhkM as being essential for ACD. Surprisingly, different DhkM mutants showed distinct nonvacuolizing ACD phenotypes. One class of mutants arrested ACD at the paddle cell stage, perhaps through a dominant-negative effect. Other mutants, however, progressed further in the ACD program. They underwent rounding and cellulose synthesis but stopped before vacuolization. Moreover, they underwent clonogenic but not morphological cell death. Exogenous 8-bromo-cAMP restored vacuolization and death. A role for a membrane receptor at a late stage of the ACD pathway is puzzling, raising questions as to which ligand it is a receptor for and which moieties it phosphorylates. Together, DhkM is the most downstream-known molecule required for this model ACD, and its distinct mutants genetically separate previously undissociated late cell death events.