Assaying protein palmitoylation in plants

<p>Abstract</p> <p>Background</p> <p>Protein S-acylation (also known as palmitoylation) is the reversible post-translational addition of acyl lipids to cysteine residues in proteins through a thioester bond. It allows strong association with membranes. Whilst prediction methods for S-acylation exist, prediction is imperfect. Existing protocols for demonstrating the S-acylation of plant proteins are either laborious and time consuming or expensive.</p> <p>Results</p> <p>We describe a biotin switch method for assaying the S-acylation of plant proteins. We demonstrate the technique by showing that the heterotrimeric G protein subunit AGG2 is S-acylated as predicted by mutagenesis experiments. We also show that a proportion of the Arabidopsis alpha-tubulin subunit pool is S-acylated <it>in planta</it>. This may account for the observed membrane association of plant microtubules. As alpha-tubulins are ubiquitously expressed they can potentially be used as a positive control for the S-acylation assay regardless of the cell type under study.</p> <p>Conclusion</p> <p>We provide a robust biotin switch protocol that allows the rapid assay of protein S-acylation state in plants, using standard laboratory techniques and without the need for expensive or specialised equipment. We propose alpha-tubulin as a useful positive control for the protocol.</p

Burkholderia thailandensis strain E555 is a surrogate for the investigation of Burkholderia pseudomallei replication and survival in macrophages

This is the final version. Available on open access from BMC via the DOI in this recordBackground: Burkholderia pseudomallei is a human pathogen causing severe infections in tropical and subtropical regions and is classified as a bio-threat agent. B. thailandensis strain E264 has been proposed as less pathogenic surrogate for understanding the interactions of B. pseudomallei with host cells. Results: We show that, unlike B. thailandensis strain E264, the pattern of growth of B. thailandensis strain E555 in macrophages is similar to that of B. pseudomallei. We have genome sequenced B. thailandensis strain E555 and using the annotated sequence identified genes and proteins up-regulated during infection. Changes in gene expression identified more of the known B. pseudomallei virulence factors than changes in protein levels and used together we identified 16% of the currently known B. pseudomallei virulence factors. These findings demonstrate the utility of B. thailandensis strain E555 to study virulence of B. pseudomallei. Conclusions: A weakness of studies using B. thailandensis as a surrogate for B. pseudomallei is that the strains used replicate at a slower rate in infected cells. We show that the pattern of growth of B. thailandensis strain E555 in macrophages closely mirrors that of B. pseudomallei. Using this infection model we have shown that virulence factors of B. pseudomallei can be identified as genes or proteins whose expression is elevated on the infection of macrophages. This finding confirms the utility of B. thailandensis strain E555 as a surrogate for B. pseudomallei and this strain should be used for future studies on virulence mechanisms.United Kingdom Ministry of Defens

Maleimide scavenging enhances determination of protein S-palmitoylation state in acyl-exchange methods.

S-palmitoylation (S-acylation) is an emerging dynamic post-translational modification of cysteine residues within proteins.Current assays for protein S-palmitoylation involve either in vivo labelling or chemical cleavage of S-palmitoyl groupsto reveal a free cysteine sulfhydryl that can be subsequently labelled with an affinity handle (acyl-exchange). Assays for protein S-palmitoylation using acylexchange chemistry therefore require blocking of non-S-palmitoylated cysteines, typically using Nethylmaleimide, to prevent non-specific detection. This in turn necessitates multiple precipitation based clean-up steps to remove reagents between stages, often leading to variable sample loss, reduced signal or protein aggregation. These combine to reduce the sensitivity, reliability and accuracy of these assays and also requires a substantial amount of time to perform. By substituting these precipitation steps with chemical scavenging of N-ethylmaleimide by 2,3-dimethyl-1,3-butadiene in an aqueous Diels-Alder 4+2 cyclo-addition reaction it is possible to greatly improve sensitivity and accuracy while reducing hands-on and overall time required for assays

Extensive genome analysis of Coxiella burnetii reveals limited evolution within genomic groups

This is the final version. Available on open access from BMC via the DOI in this recordAvailability of data and materials: Whole genome sequences were deposited in NCBI under BioProjects PRJNA430350 and PRJNA506366, as well as in the Sequence Read Archive as studies SRP130048 and SRP170036. Individual GenBank accession numbers for the WGS data are as follows: Q532 = PPFQ00000000.1 ; Q540 = PPFP00000000.1 ; Q545 = PPFO00000000.1 ; Q556 = PPFN00000000.1 ; Q559 = PPFM00000000.1 ; Cb_D1 = RQJU00000000.1; Cb_D2 = RQJT00000000.1 ; Cb_D8 = RQJS00000000.1 ; and Cb_D10 = RQJR00000000.1 .The authors declare that all other data supporting the findings of this study are available within the article and its supplementary information files.Background: Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. An improved understanding of the genetic diversity of C. burnetii is essential for the development of diagnostics, vaccines and therapeutics, but genotyping data is lacking from many parts of the world. Sporadic outbreaks of Q fever have occurred in the United Kingdom, but the local genetic make-up of C. burnetii has not been studied in detail. Results: Here, we report whole genome data for nine C. burnetii sequences obtained in the UK. All four genomes of C. burnetii from cattle, as well as one sheep sample, belonged to Multi-spacer sequence type (MST) 20, whereas the goat samples were MST33 (three genomes) and MST32 (one genome), two genotypes that have not been described to be present in the UK to date. We established the phylogenetic relationship between the UK genomes and 67 publically available genomes based on single nucleotide polymorphisms (SNPs) in the core genome, which confirmed tight clustering of strains within genomic groups, but also indicated that sub-groups exist within those groups. Variation is mainly achieved through SNPs, many of which are non-synonymous, thereby confirming that evolution of C. burnetii is based on modification of existing genes. Finally, we discovered genomic-group specific genome content, which supports a model of clonal expansion of previously established genotypes, with large scale dissemination of some of these genotypes across continents being observed. Conclusions: The genetic make-up of C. burnetii in the UK is similar to the one in neighboring European countries. As a species, C. burnetii has been considered a clonal pathogen with low genetic diversity at the nucleotide level. Here, we present evidence for significant variation at the protein level between isolates of different genomic groups, which mainly affects secreted and membrane-associated proteins. Our results thereby increase our understanding of the global genetic diversity of C. burnetii and provide new insights into the evolution of this emerging zoonotic pathogen.Defence Science and Technology Laboratory (DSTL

S-Acylation of the cellulose synthase complex is essential for its plasma membrane localization.

Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase complex (CSC) through the plane of the plasma membrane. How interactions between membranes and the CSC are regulated is currently unknown. Here, we demonstrate that all catalytic subunits of the CSC, known as cellulose synthase A (CESA) proteins, are S-acylated. Analysis of Arabidopsis CESA7 reveals four cysteines in variable region 2 (VR2) and two cysteines at the carboxy terminus (CT) as S-acylation sites. Mutating both the VR2 and CT cysteines permits CSC assembly and trafficking to the Golgi but prevents localization to the plasma membrane. Estimates suggest that a single CSC contains more than 100 S-acyl groups, which greatly increase the hydrophobic nature of the CSC and likely influence its immediate membrane environment.Biotechnology and Biological Sciences Research Council (Grant IDs: BB/H012923/1, BB/M004031/1, BB/M024911/1); Gatsby Charitable FoundationThis is the author accepted manuscript. The final version is available from the American Association for the Advancement of Science via http://dx.doi.org/10.1126/science.aaf400