RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences

Escherichia coli RseP (formerly YaeL) is believed to function as a 'regulated intramembrane proteolysis' (RIP) protease that introduces the second cleavage into anti-sigma(E) protein RseA at a position within or close to the transmembrane segment. However, neither its enzymatic activity nor the substrate cleavage position has been established. Here, we show that RseP-dependent cleavage indeed occurs within predicted transmembrane sequences of membrane proteins in vivo. Moreover, RseP catalyzed the same specificity proteolysis in an in vitro reaction system using purified components. Our in vivo and in vitro results show that RseP can cleave transmembrane sequences of some model membrane proteins that are unrelated to RseA, provided that the transmembrane region contains residues of low helical propensity. These results show that RseP has potential ability to cut a broad range of membrane protein sequences. Intriguingly, it is nevertheless recruited to the sigma(E) stress-response cascade as a specific player of RIP.     

细菌Cpx双组分信号转导系统应对外界环境变化的响应调节机制研究进展

细菌能够在其他微生物无法生存的环境中生长,必然具有更加强大的适应外界环境的能力.细胞信号转导的效率决定了细菌对外界刺激做出应答反应的速率和能力.双组分调控系统是维持细菌在压力环境中存活的重要结构.Cpx双组分信号转导系统是革兰氏阴性菌中普遍存在的双组分调控系统之一,在响应外界环境变化并做出适应性反应的过程中起着主要作用...     

Extracytoplasmic phosphatases of selected species of Saccharomyces, Kluyveromyces and Rhodotorula

Phosphatase activities of yeasts belonging to the genera Saccharomyces, Kluyveromyces and Rhodotorula were studied. Rhodotorula rubra exhibited activities at acid, neutral and alkaline pH; the other yeasts only had activity at acid pH. Growing yeasts in a constant pH (4.5) medium decreased phosphatase activities in Saccharomyces and Kluyveromyces, while neutral activity was enhanced in Rhodotorula rubra which excreted more enzyme under these conditions. Washing cells with sucrose solutions lowered phosphatase activities in all yeasts, due to enzyme liberation. Acid phosphatase activities in isolated and purified cell walls were very small. Phosphatases thus appear not to be strongly bound to yeast cell walls.     

Cell envelope stress response in Gram-positive bacteria

The bacterial cell envelope is the first and major line of defence against threats from the environment. It is an essential and yet vulnerable structure that gives the cell its shape and counteracts the high internal osmotic pressure. It also provides an important sensory interface and molecular sieve, mediating both information flow and the controlled transport of solutes. The cell envelope is also the target for numerous antibiotics. Therefore, the monitoring and maintenance of cell envelope integrity in the presence of envelope perturbating agents and conditions is crucial for survival. The underlying signal transduction is mediated by two regulatory principles, two-component systems and extracytoplasmic function sigma factors, in both the Firmicutes (low-GC) and Actinobacteria (high-GC) branches of Gram-positive bacteria. This study presents a comprehensive overview of cell envelope stress-sensing regulatory systems. This knowledge will then be applied for in-depth comparative genomics analyses to emphasize the distribution and conservation of cell envelope stress-sensing systems. Finally, the cell envelope stress response will be placed in the context of the overall cellular physiology, demonstrating that its regulatory systems are linked not only to other stress responses but also to the overall homeostasis and lifestyle of Gram-positive bacteria.     

A bacterial virulence factor with a dual role as an adhesin and a solute-binding protein: the crystal structure at 1.5 A resolution of the PEB1a protein from the food-borne human pathogen Campylobacter jejuni

The PEB1a protein is an antigenic factor exposed on the surface of the food-borne human pathogen Campylobacter jejuni, which has a major role in adherence and host colonisation. PEB1a is also the periplasmic binding protein component of an aspartate/glutamate ABC transporter essential for optimal microaerobic growth on these dicarboxylic amino acids. Here, we report the crystal structure of PEB1a at 1.5 A resolution. The protein has a typical two-domain alpha/beta structure, characteristic of periplasmic extracytoplasmic solute receptors and a chain topology related to the type II subfamily. An aspartate ligand, clearly defined by electron density in the interdomain cleft, forms extensive polar interactions with the protein, the majority of which are made with the larger domain. Arg89 and Asp174 form ion-pairing interactions with the main chain alpha-carboxyl and alpha-amino-groups, respectively, of the ligand, while Arg67, Thr82, Lys19 and Tyr156 co-ordinate the ligand side-chain carboxyl group. Lys19 and Arg67 line a positively charged groove, which favours binding of Asp over the neutral Asn. The ligand-binding cleft is of sufficient depth to accommodate a glutamate. This is the first structure of an ABC-type aspartate-binding protein, and explains the high affinity of the protein for aspartate and glutamate, and its much weaker binding of asparagine and glutamine. Stopped-flow fluorescence spectroscopy indicates a simple bimolecular mechanism of ligand binding, with high association rate constants. Sequence alignments and phylogenetic analyses revealed PEB1a homologues in some Gram-positive bacteria. The alignments suggest a more distant homology with GltI from Escherichia coli, a known glutamate and aspartate-binding protein, but Lys19 and Tyr156 are not conserved in GltI. Our results provide a structural basis for understanding both the solute transport and adhesin/virulence functions of PEB1a.     

A Function of 40 kDa Outer Membrane Protein in Serratia marcescens

The function of one of the outer membrane proteins of Serratia marcescens was investigated. S. marcescens with an abundant 40 kDa outer membrane protein was induced to form spheroplast at a high rate in an isotonic medium in the presence of calcium, although the spheroplasts were generally fragile in the isotonic environment. The degree of spheroplast induction was correlated to the amount of the 40 kDa protein present in the membrane. In the 40 kDa proteinless mutant strains, the spheroplast induction rate was remarkably decreased. Autoradiography of the outer membrane revealed the presence of a calcium-binding protein as a radioactive band whose position coincided with the 40 kDa protein. These results suggest that the 40 kDa protein has an important role in maintaining the structural integrity of the cell wall against osmotic shock.     

Crystallographic structure and biochemical analysis of the Thermus thermophilus osmotically inducible protein C

The X-ray crystallographic structure of osmotically inducible Protein C from the thermophilic bacterium, Thermus thermophilus HB8, was solved to 1.6A using the multiple wavelength anomalous dispersion method and a selenomethionine incorporated protein (Se-MAD). The crystal space group was P1 with cell dimensions of a=37.58 A, b=40.95 A, c=48.14 A, alpha=76.9 degrees, beta=74.0 degrees and gamma=64.1 degrees. The two tightly interacting monomers in the asymmetric unit are related by a non-crystallographic 2-fold. The dimer structure is defined primarily by two very long anti-parallel, over-lapping alpha-helices at the core, with a further six-stranded anti-parallel beta-sheet on the outside of the structure. With respect to the beta-sheets, both A and B monomers contribute three strands each resulting in an intertwining of the structure. The active site consists of two cysteine residues from one monomer and an arginine and glutamic acid from the other. Enzymatic assays have revealed that T.thermophilus OsmC has a hydroperoxide peroxidase activity.