An integrated model system to gain mechanistic insights into biofilm-associated antimicrobial resistance in Pseudomonas aeruginosa MPAO1.

Pseudomonas aeruginosa MPAO1 is the parental strain of the widely utilized transposon mutant collection for this important clinical pathogen. Here, we validate a model system to identify genes involved in biofilm growth and biofilm-associated antibiotic resistance. Our model employs a genomics-driven workflow to assemble the complete MPAO1 genome, identify unique and conserved genes by comparative genomics with the PAO1 reference strain and genes missed within existing assemblies by proteogenomics. Among over 200 unique MPAO1 genes, we identified six general essential genes that were overlooked when mapping public Tn-seq data sets against PAO1, including an antitoxin. Genomic data were integrated with phenotypic data from an experimental workflow using a user-friendly, soft lithography-based microfluidic flow chamber for biofilm growth and a screen with the Tn-mutant library in microtiter plates. The screen identified hitherto unknown genes involved in biofilm growth and antibiotic resistance. Experiments conducted with the flow chamber across three laboratories delivered reproducible data on P. aeruginosa biofilms and validated the function of both known genes and genes identified in the Tn-mutant screens. Differential protein abundance data from planktonic cells versus biofilm confirmed the upregulation of candidates known to affect biofilm formation, of structural and secreted proteins of type VI secretion systems, and provided proteogenomic evidence for some missed MPAO1 genes. This integrated, broadly applicable model promises to improve the mechanistic understanding of biofilm formation, antimicrobial tolerance, and resistance evolution in biofilms.

Solid-State Protein Junctions: Cross- Laboratory Study Shows Preservation of Mechanism at Varying Electronic Coupling.

Successful integration of proteins in solid-state electronics requires contacting them in a non-invasive fashion, with a solid conducting surface for immobilization as one such contact. The contacts can affect and even dominate the measured electronic transport. Often substrates, substrate treatments, protein immobilization, and device geometries differ between laboratories. Thus the question arises how far results from different laboratories and platforms are comparable and how to distinguish genuine protein electronic transport properties from platform-induced ones. We report a systematic comparison of electronic transport measurements between different laboratories, using all commonly used large-area schemes to contact a set of three proteins of largely different types. Altogether we study eight different combinations of molecular junction configurations, designed so that Ageoof junctions varies from 105 to 10-3 µm2. Although for the same protein, measured with similar device geometry, results compare reasonably well, there are significant differences in current densities (an intensive variable) between different device geometries. Likely, these originate in the critical contact-protein coupling (∼contact resistance), in addition to the actual number of proteins involved, because the effective junction contact area depends on the nanometric roughness of the electrodes and at times, even the proteins may increase this roughness. On the positive side, our results show that understanding what controls the coupling can make the coupling a design knob. In terms of extensive variables, such as temperature, our comparison unanimously shows the transport to be independent of temperature for all studied configurations and proteins. Our study places coupling and lack of temperature activation as key aspects to be considered in both modeling and practice of protein electronic transport experiments.

Mechanism of Orientation-Dependent Asymmetric Charge Transport in Tunneling Junctions Comprising Photosystem I.

Recently, photoactive proteins have gained a lot of attention due to their incorporation into bioinspired (photo)electrochemical and solar cells. This paper describes the measurement of the asymmetry of current transport of self-assembled monolayers (SAMs) of the entire photosystem I (PSI) protein complex (not the isolated reaction center, RCI), on two different "director SAMs" supported by ultraflat Au substrates. The director SAMs induce the preferential orientation of PSI, which manifest as asymmetry in tunneling charge-transport. We measured the oriented SAMs of PSI using eutectic Ga-In (EGaIn), a large-area technique, and conducting probe atomic force microscopy (CP-AFM), a single-complex technique, and determined that the transport properties are comparable. By varying the temperatures at which the measurements were performed, we found that there is no measurable dependence of the current on temperature from ±0.1 to ±1.0 V bias, and thus, we suggest tunneling as the mechanism for transport; there are no thermally activated (e.g., hopping) processes. Therefore, it is likely that relaxation in the electron transport chain is not responsible for the asymmetry in the conductance of SAMs of PSI complexes in these junctions, which we ascribe instead to the presence of a large, net dipole moment present in PSI.