Mechanistic Insight into the Early Stages of Toroidal Pore Formation by the Antimicrobial Peptide Smp24.

The antimicrobial peptide Smp24, originally derived from the venom of Scorpio maurus palmatus, is a promising candidate for further drug development. However, before doing so, greater insight into the mechanism of action is needed to construct a reliable structure-activity relationship. The aim of this study was to specifically investigate the critical early stages of peptide-induced membrane disruption. Single-channel current traces were obtained via planar patch-clamp electrophysiology, with multiple types of pore-forming events observed, unlike those expected from the traditional, more rigid mechanistic models. To better understand the molecular-level structures of the peptide-pore assemblies underlying these observed conductance events, molecular dynamics simulations were used to investigate the peptide structure and orientation both before and during pore formation. The transition of the peptides to transmembrane-like states within disordered toroidal pores occurred due to a peptide-induced bilayer-leaflet asymmetry, explaining why pore stabilization does not always follow pore nucleation in the experimental observations. To fully grasp the structure-activity relationship of antimicrobial peptides, a more nuanced view of the complex and dynamic mechanistic behaviour must be adopted.

Recombinant expression and subcellular targeting of the particulate methane monooxygenase (pMMO) protein components in plants.

Methane is a potent greenhouse gas, which has contributed to approximately a fifth of global warming since pre-industrial times. The agricultural sector produces significant methane emissions, especially from livestock, waste management and rice cultivation. Rice fields alone generate around 9% of total anthropogenic emissions. Methane is produced in waterlogged paddy fields by methanogenic archaea, and transported to the atmosphere through the aerenchyma tissue of rice plants. Thus, bioengineering rice with catalysts to detoxify methane en route could contribute to an efficient emission mitigation strategy. Particulate methane monooxygenase (pMMO) is the predominant methane catalyst found in nature, and is an enzyme complex expressed by methanotrophic bacteria. Recombinant expression of pMMO has been challenging, potentially due to its membrane localization, multimeric structure, and polycistronic operon. Here we show the first steps towards the engineering of plants for methane detoxification with the three pMMO subunits expressed in the model systems tobacco and Arabidopsis. Membrane topology and protein-protein interactions were consistent with correct folding and assembly of the pMMO subunits on the plant ER. Moreover, a synthetic self-cleaving polypeptide resulted in simultaneous expression of all three subunits, although low expression levels precluded more detailed structural investigation. The work presents plant cells as a novel heterologous system for pMMO allowing for protein expression and modification.

Peering down the sink: A review of isoprene metabolism by bacteria.

Isoprene (2-methyl-1,3-butadiene) is emitted to the atmosphere each year in sufficient quantities to rival methane (>500 Tg C yr-1 ), primarily due to emission by trees and other plants. Chemical reactions of isoprene with other atmospheric compounds, such as hydroxyl radicals and inorganic nitrogen species (NOx ), have implications for global warming and local air quality, respectively. For many years, it has been estimated that soil-dwelling bacteria consume a significant amount of isoprene (~20 Tg C yr-1 ), but the mechanisms underlying the biological sink for isoprene have been poorly understood. Studies have indicated or confirmed the ability of diverse bacterial genera to degrade isoprene, whether by the canonical iso-type isoprene degradation pathway or through other less well-characterized mechanisms. Here, we review current knowledge of isoprene metabolism and highlight key areas for further research. In particular, examples of isoprene-degraders that do not utilize the isoprene monooxygenase have been identified in recent years. This has fascinating implications both for the mechanism of isoprene uptake by bacteria, and also for the ecology of isoprene-degraders in the environments.

Metal(loid) speciation and transformation by aerobic methanotrophs.

Manufacturing and resource industries are the key drivers for economic growth with a huge environmental cost (e.g. discharge of industrial effluents and post-mining substrates). Pollutants from waste streams, either organic or inorganic (e.g. heavy metals), are prone to interact with their physical environment that not only affects the ecosystem health but also the livelihood of local communities. Unlike organic pollutants, heavy metals or trace metals (e.g. chromium, mercury) are non-biodegradable, bioaccumulate through food-web interactions and are likely to have a long-term impact on ecosystem health. Microorganisms provide varied ecosystem services including climate regulation, purification of groundwater, rehabilitation of contaminated sites by detoxifying pollutants. Recent studies have highlighted the potential of methanotrophs, a group of bacteria that can use methane as a sole carbon and energy source, to transform toxic metal (loids) such as chromium, mercury and selenium. In this review, we synthesise recent advances in the role of essential metals (e.g. copper) for methanotroph activity, uptake mechanisms alongside their potential to transform toxic heavy metal (loids). Case studies are presented on chromium, selenium and mercury pollution from the tanneries, coal burning and artisanal gold mining, respectively, which are particular problems in the developing economy that we propose may be suitable for remediation by methanotrophs. Video Abstract.

The antimicrobial activity and biocompatibility of a controlled gentamicin-releasing single-layer sol-gel coating on hydroxyapatite-coated titanium.

AIMS: The aim of this study was to develop a single-layer hybrid organic-inorganic sol-gel coating that is capable of a controlled antibiotic release for cementless hydroxyapatite (HA)-coated titanium orthopaedic prostheses. METHODS: Coatings containing gentamicin at a concentration of 1.25% weight/volume (wt/vol), similar to that found in commercially available antibiotic-loaded bone cement, were prepared and tested in the laboratory for: kinetics of antibiotic release; activity against planktonic and biofilm bacterial cultures; biocompatibility with cultured mammalian cells; and physical bonding to the material (n = 3 in all tests). The sol-gel coatings and controls were then tested in vivo in a small animal healing model (four materials tested; n = 6 per material), and applied to the surface of commercially pure HA-coated titanium rods. RESULTS: The coating released gentamicin at > 10 × minimum inhibitory concentration (MIC) for sensitive staphylococcal strains within one hour thereby potentially giving effective prophylaxis for arthroplasty surgery, and showed > 99% elution of the antibiotic within the coating after 48 hours. There was total eradication of both planktonic bacteria and established bacterial biofilms of a panel of clinically relevant staphylococci. Mesenchymal stem cells adhered to the coated surfaces and differentiated towards osteoblasts, depositing calcium and expressing the bone marker protein, osteopontin. In the in vivo small animal bone healing model, the antibiotic sol-gel coated titanium (Ti)/HA rod led to osseointegration equivalent to that of the conventional HA-coated surface. CONCLUSION: In this study we report a new sol-gel technology that can release gentamicin from a bioceramic-coated cementless arthroplasty material. In vitro, local gentamicin levels are in excess of what can be achieved by antibiotic-loaded bone cement. In vivo, bone healing in an animal model is not impaired. This, thus, represents a biomaterial modification that may have the potential to protect at-risk patients from implant-related deep infection. Cite this article: Bone Joint J 2021;103-B(3):522-529.

Gemini surfactant as multifunctional corrosion and biocorrosion inhibitors for mild steel.

Biocorrosion is an important type of corrosion which leads to economic losses across oil and gas industries, due to increased monitoring, maintenance, and a reduction in platform availability. Ideally, a chemical compound engineered to mitigate against biocorrosion would possess both antimicrobial properties, as well as efficient corrosion inhibition. Gemini surfactants have shown efficacy in both of these properties, however there still remains a lack of electrochemical information regarding biocorrosion inhibition. The inhibition of corrosion and biocorrosion, by cationic gemini surfactants, of carbon steel was investigated. The results showed that the inhibition efficiency of the gemini surfactants was high (consistently >95%), even at low concentrations. Gemini surfactants also showed strong antimicrobial activity, with a minimum inhibitory concentration (0.018 mM). Corrosion inhibition was investigated by electrochemical impedance spectroscopy (EIS) and linear polarisation resistance (LPR), with biocorrosion experiments carried out in an anaerobic environment. Surface morphology was analysed using scanning electron microscopy (SEM).

Mutagenesis and expression of methane monooxygenase to alter regioselectivity with aromatic substrates.

Soluble methane monooxygenase (sMMO) from methane-oxidising bacteria can oxygenate more than 100 hydrocarbons and is one of the most catalytically versatile biological oxidation catalysts. Expression of recombinant sMMO has to date not been achieved in Escherichia coli and so an alternative expression system must be used to manipulate it genetically. Here we report substantial improvements to the previously described system for mutagenesis of sMMO and expression of recombinant enzymes in a methanotroph (Methylosinus trichosporium OB3b) expression system. This system has been utilised to make a number of new mutants and to engineer sMMO to increase its catalytic precision with a specific substrate whilst increasing activity by up to 6-fold. These results are the first 'proof-of-principle' experiments illustrating the feasibility of developing sMMO-derived catalysts for diverse applications.

Temocillin: a new candidate antibiotic for local antimicrobial delivery in orthopaedic surgery?

OBJECTIVES: To assess the performance of the Gram-negative-specific antibiotic temocillin in polymethylmethacrylate bone cement pre-loaded with gentamicin, as a strategy for local antibiotic delivery. METHODS: Temocillin was added at varying concentrations to commercial gentamicin-loaded bone cement. The elution of the antibiotic from cement samples over a 2 week period was quantified by LC-MS. The eluted temocillin was purified by fast protein liquid chromatography and the MICs for a number of antibiotic-resistant Escherichia coli were determined. The impact strength of antibiotic-loaded samples was determined using a Charpy-type impact testing apparatus. RESULTS: LC-MS data showed temocillin eluted to clinically significant concentrations within 1 h in this laboratory system and the eluted temocillin retained antimicrobial activity against all organisms tested. Impact strength analysis showed no significant difference between cement samples with or without temocillin. CONCLUSIONS: Temocillin can be added to bone cement and retains its antimicrobial activity after elution. The addition of up to 10% temocillin did not affect the impact strength of the cement. The results show that temocillin is a promising candidate for use in antibiotic-loaded bone cement.

Mutagenesis of the "leucine gate" to explore the basis of catalytic versatility in soluble methane monooxygenase.

Soluble methane monooxygenase (sMMO) from methane-oxidizing bacteria is a multicomponent nonheme oxygenase that naturally oxidizes methane to methanol and can also cooxidize a wide range of adventitious substrates, including mono- and diaromatic hydrocarbons. Leucine 110, at the mouth of the active site in the alpha subunit of the hydroxylase component of sMMO, has been suggested to act as a gate to control the access of substrates to the active site. Previous crystallography of the wild-type sMMO has indicated at least two conformations of the enzyme that have the "leucine gate" open to different extents, and mutagenesis of homologous enzymes has indicated a role for this residue in the control of substrate range and regioselectivity with aromatic substrates. By further refinement of the system for homologous expression of sMMO that we developed previously, we have been able to prepare a range of site-directed mutations at position 110 in the alpha subunit of sMMO. All the mutants (with Gly, Cys, Arg, and Tyr, respectively, at this position) showed relaxations of regioselectivity compared to the wild type with monoaromatic substrates and biphenyl, including the appearance of new products arising from hydroxylation at the 2- and 3- positions on the benzene ring. Mutants with the larger Arg and Trp residues at position 110 also showed shifts in regioselectivity during naphthalene hydroxylation from the 2- to the 1- position. No evidence that mutagenesis of Leu 110 could allow very large substrates to enter the active site was found, however, since the mutants (like the wild type) were inactive toward the triaromatic hydrocarbons anthracene and phenanthrene. Thus, our results indicate that the "leucine gate" in sMMO is more important in controlling the precision of regioselectivity than the sizes of substrates that can enter the active site.