Histidine Folding Switch In Effector Protein Avrpto Regulates Its Transport Through The Bacterial Type Iii Secretion System, As Determined By Nmr, Fluorescence, Circular Dichroism Spectroscopies And Translocation Assays

Bacteria have evolved several secretion systems as direct conduits into host cell cytoplasm for bacterial effector protein trafficking to further their symbiotic or pathogenic goals, while avoiding detection and host immune responses. Gram-negative bacterium Pseudomonas syringae uses the type III secretion system that spans bacterial membranes and extends with a needlelike protrusion to open up through the host cell membrane. There are structural constraints to passage through this conduit, where the inner channel diameter of 2-3 nm prevents effector protein export in the folded form. The requirement for transient unfolding places unique restrictions on the stability thresholds for effector structure and function, essential for bacterial pathogenicity. Effector tertiary structure is typically needed for function in the host, as has been shown for one of the most widely studied effectors, AvrPto. AvrPto is an 18-kDa protein with a helical core of low stability and disordered N- and C-termini, secreted by the plant pathogen Pseudomonas syringae. AvrPto features a single amino acid folding switch governed by histidine 87 that acts in the moderately acidic pH range expected in the biological environment of the bacterium. At neutral plant host cytosol of pH ~7.4 AvrPto is predominantly in the folded form. We disabled the His 87encoded pH switch by the H87Y mutation and observed a slight increase in stability and a dramatic shift in the acid denaturation into the more acidic pH. This mutation significantly affected the translocation efficiency of AvrPto during the initial hours of Pseudomonas syringae infection at different temperatures. Through direct comparison of in vivo data with in vitro AvrPto stability trends we conclude that the amount of available cytoplasmic unfolded AvrPto is a factor regulating AvrPto translocation, and favors an acidic bacterial cytosol. Preliminary ratiometric GFP fluorescence data predicts bacterial intracellular pH of < 6, in agreement with this prediction. Taken together, a mechanism for effector transport emerges that depends on destabilized effector pools for the T3SS as a significant factor in effector translocation hierarchy and the overall infection process

Local Embeddedness in International Professional Service Firm Networks

Networks have been recognized as an alternative internationalization approach for Professional Service Firms (PSFs). However, the exploration of tensions, which result from attempts to unite network members and at the same time support their diversity, has been rather neglected in the past. Furthermore, the role of local embeddedness in the presented setting seems to have been overlooked. We have implemented a qualitative research addressing the unity-diversity tension that occurs in cross-border interfirm networks from the perspective of managers in locally embedded PSFs. Through the lens of agency theory, we assess these kinds of networks specifically from two angles which shape the conflicts within them. First, local knowledge asymmetries among the network members hinder the practicality of formal outcome-based contracts. Second, professional knowledge asymmetries result in monitoring problems among the members and thus make formal behavior-based contracts inapplicable. Through our research, we have further identified cultural differences among the members as a third factor contributing to conflicts. The members of these specific networks approach the examined tensions with creating unity by implementing informal, relational contracts based on social mechanisms such as trust, solidarity, information exchange and flexibility. This helps them to align the interests of their members as well as reduce professional knowledge asymmetries. At the same time, they seek to preserve their diversity in the form of their local embeddedness, i.e. their local knowledge, cultural background and their local networks

Ethenoguanines undergo glycosylation by nucleoside 2'-deoxyribosyltransferases at non-natural sites.

Deoxyribosyl transferases and functionally related purine nucleoside phosphorylases are used extensively for synthesis of non-natural deoxynucleosides as pharmaceuticals or standards for characterizing and quantitating DNA adducts. Hence exploring the conformational tolerance of the active sites of these enzymes is of considerable practical interest. We have determined the crystal structure at 2.1 Å resolution of Lactobacillus helveticus purine deoxyribosyl transferase (PDT) with the tricyclic purine 8,9-dihydro-9-oxoimidazo[2,1-b]purine (N2,3-ethenoguanine) at the active site. The active site electron density map was compatible with four orientations, two consistent with sites for deoxyribosylation and two appearing to be unproductive. In accord with the crystal structure, Lactobacillus helveticus PDT glycosylates the 8,9-dihydro-9-oxoimidazo[2,1-b]purine at N7 and N1, with a marked preference for N7. The activity of Lactobacillus helveticus PDT was compared with that of the nucleoside 2'-deoxyribosyltransferase enzymes (DRT Type II) from Lactobacillus leichmannii and Lactobacillus fermentum, which were somewhat more effective in the deoxyribosylation than Lactobacillus helveticus PDT, glycosylating the substrate with product profiles dependent on the pH of the incubation. The purine nucleoside phosphorylase of Escherichia coli, also commonly used in ribosylation of non-natural bases, was an order of magnitude less efficient than the transferase enzymes. Modeling based on published active-site structures as templates suggests that in all cases, an active site Phe is critical in orienting the molecular plane of the purine derivative. Adventitious hydrogen bonding with additional active site residues appears to result in presentation of multiple nucleophilic sites on the periphery of the acceptor base for ribosylation to give a distribution of nucleosides. Chemical glycosylation of O9-benzylated 8,9-dihydro-9-oxoimidazo[2,1-b]purine also resulted in N7 and N1 ribosylation. Absent from the enzymatic and chemical glycosylations is the natural pattern of N3 ribosylation, verified by comparison of spectroscopic and chromatographic properties with an authentic standard synthesized by an unambiguous route

Final refinement statistics for PDT crystallized with 8,9-dihydro-9-oxoimidazo[2,1-<i>b</i>]purine.

a<p><i>R</i> factor  = <i>Σ</i>_hkl||F_obs |−k|F_cal||/<i>Σ</i>_hkl|F_obs| where F_obs and F_cal are observed and calculated structure factors, respectively.</p>b<p>For <i>R</i>_free the sum is extended over a subset of reflections (5%) excluded from all stages of refinement.</p><p>Final refinement statistics for PDT crystallized with 8,9-dihydro-9-oxoimidazo[2,1-<i>b</i>]purine.</p