BACKGROUND: Microbial expansins (EXLXs) are non-lytic proteins homologous to plant expansins involved in plant cell wall formation. Due to their non-lytic cell wall loosening properties and potential to disaggregate cellulosic structures, there is considerable interest in exploring the ability of microbial expansins (EXLX) to assist the processing of cellulosic biomass for broader biotechnological applications. Herein, EXLXs with different modular structure and from diverse phylogenetic origin were compared in terms of ability to bind cellulosic, xylosic, and chitinous substrates, to structurally modify cellulosic fibrils, and to boost enzymatic deconstruction of hardwood pulp.
RESULTS: Five heterogeneously produced EXLXs (Clavibacter michiganensis; CmiEXLX2, Dickeya aquatica; DaqEXLX1, Xanthomonas sacchari; XsaEXLX1, Nothophytophthora sp.; NspEXLX1 and Phytophthora cactorum; PcaEXLX1) were shown to bind xylan and hardwood pulp at pH 5.5 and CmiEXLX2 (harboring a family-2 carbohydrate-binding module) also bound well to crystalline cellulose. Small-angle X-ray scattering revealed a 20-25% increase in interfibrillar distance between neighboring cellulose microfibrils following treatment with CmiEXLX2, DaqEXLX1, or NspEXLX1. Correspondingly, combining xylanase with CmiEXLX2 and DaqEXLX1 increased product yield from hardwood pulp by ~ 25%, while supplementing the TrAA9A LPMO from Trichoderma reesei with CmiEXLX2, DaqEXLX1, and NspEXLX1 increased total product yield by over 35%.
CONCLUSIONS: This direct comparison of diverse EXLXs revealed consistent impacts on interfibrillar spacing of cellulose microfibers and performance of carbohydrate-active enzymes predicted to act on fiber surfaces. These findings uncover new possibilities to employ EXLXs in the creation of value-added materials from cellulosic biomass.

Antimicrobial peptides (AMPs) are a various group of molecules found in a wide range of organisms and act as a defense mechanism against different kinds of infectious pathogens (bacteria, viruses, and fungi, etc.). This study explored the antibacterial activity of nine candidates reported in the literature for their effect on human and animal bacteria, (i.e., Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa) against Erwinia amylovora (E. amylovora), the causal agent of fire blight disease on pome fruits. The antibacterial activity of these peptides against E. amylovora was evaluated in vitro using viable-quantitative PCR (v-qPCR), fluorescence microscopy (FM), optical density (OD), and transmission electron microscopy (TEM), while the in vivo control efficacy was evaluated in treating experimental fire blight on pear fruits. With a view to their safe and ecofriendly field use in the future, the study also used animal and plant eukaryotic cells to evaluate the possible toxicity of these AMPs. Results in vitro showed that KL29 was the most potent peptide in inhibiting E. amylovora cell proliferation. In addition, the results of v-qPCR, FM, and TEM showed that KL29 has a bifunctional mechanism of action (lytic and non-lytic) when used at different concentrations against E. amylovora. KL29 reduced fire blight symptoms by 85% when applied experimentally in vivo. Furthermore, it had no impact on animal or plant cells, thus demonstrating its potential for safe use as an antibacterial agent. This study sheds light on a new and potent antibacterial peptide for E. amylovora and its modes of action, which could be exploited to develop sustainable treatments for fire blight.

A highly sensitive and selective non-lytic M13 phage-based electrochemical impedance spectroscopy (EIS) cytosensor for early detection of coliforms is introduced for the first time. Gold nanoparticles were electrochemically deposited on the surface of glassy carbon electrode, and the M13 phage particles were immobilized on them using 3-mercaptopropionic acid linker and zero-length crosslinking chemistry (EDC/NHS). Next, the sensor surface was blocked to avoid non-specific binding. The M13-EIS cytosensor was tested for detection of F+ pili Escherichia coli species, using XL1-Blue and K12 strains, as examples of coliforms. The selectivity against non-host strains was demonstrated using Pseudomonas Chlororaphis. The binding of E. coli to the M13 phage on the cytosensor surface increased the charge transfer resistance, enabling detection of coliforms. The biosensor achieved a limit of detection (LOD) of 14 CFU/mL, the lowest reported to-date using EIS-phage sensors, and exhibited a high selectivity towards the tested coliforms. The SEM micrographs confirmed the successful capturing of E. coli on the M13-based EIS cytosensor. Moreover, the sensor showed almost the same sensitivity in the simulated river water samples as in phosphate buffer, reflecting its applicability to real samples. On the other hand, this sensor system exhibited high stability under harsh environmental conditions of pH (3.0-10.0) and temperature as high as 45 °C for up to two weeks. Overall, this sensor system has excellent potential for real field detection of fecal coliforms.

We report a dimerization strategy to enhance the antibacterial potency of an otherwise weak cationic amphiphilic polyproline helical (CAPH) peptide. Overall, the dimeric CAPHs were more active against Escherichia coli and Staphylococcus aureus than the monomeric counterpart, reaching up to a 60-fold increase in potency. At their minimum inhibitory concentration (MIC), the dimeric peptides demonstrated no hemolytic activity or bacterial membrane disruption as monitored by β-galactosidase release in E. coli. At higher concentrations the dimeric agents were found to induce β-galactosidase release, but maintained negligible hemolytic activity, pointing to a potential shift in the mechanism of action at higher concentrations. Thus, discontinuous dimerization of an unnatural proline-rich peptide was a successful strategy to create potent de novo antibacterial peptides without membrane lysis.