A. James Link

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides

The development of a robust and portable biosensor for the detection of pathogenic bacteria could impact areas ranging from water-quality monitoring to testing of pharmaceutical products for bacterial contamination. Of particular interest are detectors that combine the natural specificity of biological recognition with sensitive, label-free sensors providing electronic readout. Evolution has tailored antimicrobial peptides to exhibit broad-spectrum activity against pathogenic bacteria, while retaining a high degree of robustness. Here, we report selective and sensitive detection of infectious agents via electronic detection based on antimicrobial peptide-functionalized microcapacitive electrode arrays. The semiselective antimicrobial peptide magainin I—which occurs naturally on the skin of African clawed frogs—was immobilized on gold microelectrodes via a C-terminal cysteine residue. Significantly, exposing the sensor to various concentrations of pathogenic Escherichia coli revealed detection limits of approximately 1 bacterium/μL, a clinically useful detection range. The peptide-microcapacitive hybrid device was further able to demonstrate both Gram-selective detection as well as interbacterial strain differentiation, while maintaining recognition capabilities toward pathogenic strains of E. coli and Salmonella. Finally, we report a simulated “water-sampling” chip, consisting of a microfluidic flow cell integrated onto the hybrid sensor, which demonstrates real-time on-chip monitoring of the interaction of E. coli cells with the antimicrobial peptides. The combination of robust, evolutionarily tailored peptides with electronic read-out monitoring electrodes may open exciting avenues in both fundamental studies of the interactions of bacteria with antimicrobial peptides, as well as the practical use of these devices as portable pathogen detectors.

Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids

The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 → Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.

Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging.

A major aim of proteomics is the identification of proteins in a given proteome at a given metabolic state. This protocol describes the step-by-step labeling, purification and detection of newly synthesized proteins in mammalian cells using the non-canonical amino acid azidohomoalanine (AHA). In this method, metabolic labeling of newly synthesized proteins with AHA endows them with the unique chemical functionality of the azide group. In the subsequent click chemistry tagging reaction, azide-labeled proteins are covalently coupled to an alkyne-bearing affinity tag. After avidin-based affinity purification and on-resin trypsinization, the resulting peptide mixture is subjected to tandem mass spectrometry for identification. In combination with deuterated leucine-based metabolic colabeling, candidate proteins can be immediately validated. Bioorthogonal non-canonical amino-acid tagging can be combined with any subcellular fractionation, immunopurification or other proteomic method to identify specific subproteomes, thereby reducing sample complexity and enabling the identification of subtle changes in a proteome. This protocol can be completed in 5 days.

Unnatural Amino Acid Incorporation into Virus-Like Particles

Virus-like particles composed of hepatitis B virus (HBV) or bacteriophage Qbeta capsid proteins have been labeled with azide- or alkyne-containing unnatural amino acids by expression in a methionine auxotrophic strain of E. coli. The substitution does not affect the ability of the particles to self-assemble into icosahedral structures indistinguishable from native forms. The azide and alkyne groups were addressed by Cu(I)-catalyzed [3 + 2] cycloaddition: HBV particles were decomposed by the formation of more than 120 triazole linkages per capsid in a location-dependent manner, whereas Qbeta suffered no such instability. The marriage of these well-known techniques of sense-codon reassignment and bioorthogonal chemical coupling provides the capability to construct polyvalent particles displaying a wide variety of functional groups with near-perfect control of spacing.

Lasso peptides: structure, function, biosynthesis, and engineering

Lasso peptides are a class of ribosomally-synthesized and posttranslationally-modified natural products with diverse bioactivities. This review describes the structure and function of all known lasso peptides (as of mid-2012) and covers our current knowledge about the biosynthesis of those molecules. The isolation and characterization of lasso peptides are also covered as are bioinformatics strategies for the discovery of new lasso peptides from genomic sequence data. Several studies on the engineering of new or improved function into lasso peptides are highlighted, and unanswered questions in the field are also described.

Evolution of a fluorinated green fluorescent protein

The fluorescence of bacterial cells expressing a variant (GFPm) of the green fluorescent protein (GFP) was reduced to background levels by global replacement of the leucine residues of GFPm by 5,5,5-trifluoroleucine. Eleven rounds of random mutagenesis and screening via fluorescence-activated cell sorting yielded a GFP mutant containing 20 amino acid substitutions. The mutant protein in fluorinated form showed improved folding efficiency both in vivo and in vitro, and the median fluorescence of cells expressing the fluorinated protein was improved ≈650-fold in comparison to that of cells expressing fluorinated GFPm. The success of this approach demonstrates the feasibility of engineering functional proteins containing many copies of abiological amino acid constituents.

Efficient production of membrane‐integrated and detergent‐soluble G protein‐coupled receptors in Escherichia coli

G protein‐coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane‐integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane‐bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent‐solubilized and purified full‐length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.

Beyond toothpicks: new methods for isolating mutant bacteria

Over the past 50 years genetic analysis in microbiology has relied predominantly on selections and plate assays using chromogenic enzyme substrates — for example, X-gal assays for the detection of β-galactosidase activity. Recent advances in fluorescent assays and high throughput screening technologies have paved the way for the rapid isolation of mutants that confer complex phenotypes and for the quantitative analysis of the evolution of new traits in bacterial populations. This Review highlights the power of novel single-cell screening technologies and their applications to genetics, evolution and the biotechnological uses of bacteria.

Much of the Microcin J25 Leader Peptide is Dispensable

The antimicrobial peptide microcin J25 (MccJ25) is matured by two enzymes, McjB and McjC, from a 58 amino acid (aa) preprotein, McjA, into its final 21 aa lasso topology. Herein we have investigated the role of the leader peptide of McjA and found that only the eight C-terminal amino acids of this leader peptide are required for maturation of MccJ25. There is a high content of lysine residues in the McjA leader peptide, but herein we also demonstrate that these charged amino acids do not play a major role in the maturation of MccJ25. Alanine scanning mutagenesis studies revealed that the Thr-35 residue in the leader peptide is critical for correct processing of McjA into mature MccJ25. In the absence of detailed structural and biochemical data about McjB and McjC, these studies allow us to propose a putative role for the leader peptide as a simple motif for docking of the McjA preprotein in the maturation enzymes.