A novel pathway for outer membrane protein biogenesis in Gram-negative bacteria

The understanding of the biogenesis of the outer membrane of Gram‐negative bacteria is of critical importance due to the emergence of bacteria that are becoming resistant to available antibiotics. A problem that is most serious for Gram‐negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The understanding of the Gram‐negative bacterial outer membrane is therefore critical to developing new antimicrobial agents, as this membrane makes direct contact with the external milieu, and the proteins present within this membrane are the instruments of microbial warfare, playing key roles in microbial pathogenesis, virulence and multidrug resistance. To date, a single outer membrane complex has been identified as essential for the folding and insertion of proteins into the outer membrane, this is the β‐barrel assembly machine (BAM) complex, which in some cases is supplemented by the Translocation and Assembly Module (TAM). In this issue of Molecular Microbiology, Dunstan et al. have identified a novel pathway for the insertion of a subset of integral membrane proteins into the Gram‐negative outer membrane that is independent of the BAM complex and TAM

Compound-specific amino acid ¹⁵N stable isotope probing of nitrogen assimilation by the soil microbial biomass using gas chromatography/combustion/isotope ratio mass spectrometry

RATIONALE: Organic nitrogen (N) greatly exceeds inorganic N in soils, but the complexity and heterogeneity of this important soil N pool make investigations into the fate of N‐containing additions and soil organic N cycling challenging. This paper details a novel approach to investigate the fate of applied N in soils, generating quantitative measures of microbial assimilation and of newly synthesized soil protein. METHODS: Laboratory incubation experiments applying (15)N‐ammonium, (15)N‐nitrate and (15)N‐glutamate were carried out and the high sensitivity and selectivity of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) exploited for compound‐specific (15)N stable isotope probing ((15)N‐SIP) of extracted incubation soil amino acids (AAs; as N‐acetyl, O‐isopropyl derivatives). We then describe the interpretation of these data to obtain a measure of the assimilation of the applied (15)N‐labelled substrate by the soil microbial biomass and an estimate of newly synthesised soil protein. RESULTS: The cycling of agriculturally relevant N additions is undetectable via bulk soil N content and δ (15)N values and AA concentrations. The assimilation pathways of the three substrates were revealed via patterns in AA δ (15)N values with time, reflecting known biosynthetic pathways (e.g. ammonium uptake occurs first via glutamate) and these data were used to expose differences in the rates and fluxes of the applied N substrates into the soil protein pool (glutamate > ammonium > nitrate). CONCLUSIONS: Our compound‐specific (15)N‐SIP approach using GC/C/IRMS offers a number of insights, inaccessible via existing techniques, into the fate of applied (15)N in soils and is potentially widely applicable to the study of N cycling in any soil, or indeed, in any complex ecosystem. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd

States’ Support of Non-Highway Modes of Transportation: Investigation and Synthesis

The objective of this study was to determine how Kentucky and other selected states support and assist non-highway transportation modes and to identify leading and innovative initiatives. For the purposes of this study, the non-highway modes are aviation, public transportation, rail, and waterways. This summary will briefly discuss the rationale, objectives, and methodology for this study. It also provides an overview of the outcomes of the study and the modal matrices

Development of Alditol Acetate Derivatives for the Determination of 15N-Enriched Amino Sugars by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry

Amino sugars can be used as indices to evaluate the role of soil microorganisms in active nitrogen (N) cycling in soil. This paper details the assessment of the suitability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the analysis of ¹⁵N-enriched amino sugars as alditol acetate derivatives prior to application of a novel ¹⁵N stable isotope probing (SIP) approach to amino sugars. The efficient derivatization and cleanup of alditol acetate derivatives for GC was achieved using commercially available amino sugars, including glucosamine, mannosamine, galactosamine, and muramic acid, as laboratory standards. A VF-23ms stationary phase was found to produce optimal separations of all four compounds. The structure of the alditol acetate derivatives was confirmed using gas chromatography/mass spectrometry (GC/MS). For GC–C–IRMS determinations, implementation of a two-point normalization confirmed the optimal carrier gas flow rate to be 1.7 mL min^–1. Linearity of δ¹⁵N value determinations up to δ¹⁵N_t of 469 ± 3.1‰ (where δ¹⁵N_t is the independently measured δ¹⁵N value) was confirmed when 30 nmol N was injected on-column, with the direction of deviation from δ¹⁵N_t at low sample amount dependent on the ¹⁵N abundance of the analyte. Observed between- and within-run memory effects were significant (<i>P</i> < 0.007) when a highly enriched standard (469 ± 3.1‰) was run; therefore, analytical run order and variation in ¹⁵N enrichment of analytes within the same sample must be considered. The investigated parameters have confirmed the isotopic robustness of alditol acetate derivatives of amino sugars for the GC–C–IRMS analysis of ¹⁵N-enriched amino sugars in terms of linearity over an enrichment range (natural abundance to 469 ± 3.1‰) with on-column analyte amount over 30 nmol N

Compound-specific radiocarbon, stable carbon isotope and biomarker analysis of mixed marine/terrestrial lipids preserved in archaeological pottery vessels.

At archaeological sites located on islands or near the coast, the potential exists for lipid extracts of potsherds to contain fatty acids (FA) from both aquatic and terrestrial organisms, meaning that consideration must be given to marine reservoir effects (MRE) in radiocarbon (14C) analyses. Here we studied the site of Bornais (Outer Hebrides, UK) where a local MRE, ΔR of –65 ± 45 yr was determined through the paired 14C determinations of terrestrial and marine faunal bones. Lipid analysis of 49 potsherds, revealed aquatic biomarkers in 45% of the vessels, and δ13C values of C16:0 and C18:0 FAs revealed ruminant and marine product mixing for 71% of the vessels. Compound-specific 14C analysis (CSRA) of FAs yielded intermediate 14C ages between those of terrestrial and marine bones from the same contexts, confirming an MRE existed. A database containing δ13C values for FAs from reference terrestrial and marine organisms provided endmembers for calculating the percentage marine-derived C (%marine) in FAs. We show that lipid 14C dates can be corrected using determined %marine and ΔR values, such that pottery vessels from coastal locations can be 14C dated by CSRA of FAs

An acid-compatible co-polymer for the solubilization of membranes and proteins into lipid bilayer-containing nanoparticles

The fundamental importance of membrane proteins in drug discovery has meant that membrane mimetic systems for studying membrane proteins are of increasing interest. One such system has been the amphipathic, negatively charged poly(styrene-co-maleic acid) (SMA) polymer to form “SMA Lipid Particles” (SMALPs) which have been widely adopted to solubilize membrane proteins directly from the cell membrane. However, SMALPs are only soluble under basic conditions and precipitate in the presence of divalent cations required for many downstream applications. Here, we show that the positively charged poly(styrene-co-maleimide) (SMI) forms similar nanoparticles with comparable efficiency to SMA, whilst remaining functional at acidic pH and compatible with high concentrations of divalent cations. We have performed a detailed characterization of the performance of SMI that enables a direct comparison with similar data published for SMA. We also demonstrate that SMI is capable of extracting proteins directly from the cell membrane and can solubilize functional human G-protein coupled receptors (GPCRs) expressed in cultured HEK 293T cells. “SMILPs” thus provide an alternative membrane solubilization method that successfully overcomes some of the limitations of the SMALP method

The Milky Way:Mobility and Economy at the Turn of the 3rd Millennium in Southern Central Europe

In the light of discussions surrounding the social changes attributed to the arrival of the Corded Ware culture in central Europe, here we investigate the economic strategies of one of the cultural complexes of the immediately preceding Late Neolithic. The Cham culture of southern Bavaria is characterised by a variety of economic choices but problems remain in synthesising and combining archaeozoological and archaeobotanical evidence. Using lipid residue analysis from Cham culture pottery excavated at the unenclosed settlement of Riedling, Lower Bavaria, we succeed in identifying a dairying economy at this time. Compound-specific lipid radiocarbon dates are then combined with other samples to provide a formal estimate for the duration of activity at Riedling and the first Bayesian chronological model for the Cham culture as a whole. Although data are currently not fine-grained enough to distinguish between competing models for site permanence, we suggest that the Cham culture pattern fits into a wider central European trend of greater mobility and economic flexibility in the pre-Corded Ware horizon, concluding that key economic strategies previously associated with ‘steppe invasions’ were already present in the preceding centuries. Finally, the demonstrated use of cups for milk-based products, as opposed to alcoholic drinks as previously suggested, leads us to propose possible alternative uses and users for these items

The vaccinia chondroitin sulfate binding protein drives host membrane curvature to facilitate fusion

Cellular attachment of viruses determines their cell tropism and species specificity. For entry, vaccinia, the prototypic poxvirus, relies on four binding proteins and an eleven-protein entry fusion complex. The contribution of the individual virus binding proteins to virion binding orientation and membrane fusion is unclear. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards the virus fusion machinery to facilitate fusion. Using a membrane-bleb model system together with super-resolution and electron microscopy we find that side-bound vaccinia virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins reveals that three of four contribute to binding orientation, amongst which the chondroitin sulfate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection. Our results show that viral binding proteins are active participants in successful virus membrane fusion and illustrate the importance of virus protein architecture for successful infection.</p