Full-length single-molecule protein fingerprinting.

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis.

Self-replication of DNA by its encoded proteins in liposome-based synthetic cells.

Replication of DNA-encoded information and its conversion into functional proteins are universal properties of life. In an effort toward the construction of a synthetic minimal cell, we implement here the DNA replication machinery of the Φ29 virus in a cell-free gene expression system. Amplification of a linear DNA template by self-encoded, de novo synthesized Φ29 proteins is demonstrated. Complete information transfer is confirmed as the copied DNA can serve as a functional template for gene expression, which can be seen as an autocatalytic DNA replication cycle. These results show how the central dogma of molecular biology can be reconstituted and form a cycle in vitro. Finally, coupled DNA replication and gene expression is compartmentalized inside phospholipid vesicles providing the chassis for evolving functions in a prospective synthetic cell relying on the extant biology.

Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes.

The goal of bottom-up synthetic biology culminates in the assembly of an entire cell from separate biological building blocks. One major challenge resides in the in vitro production and implementation of complex genetic and metabolic pathways that can support essential cellular functions. Here, we show that phospholipid biosynthesis, a multiple-step process involved in cell membrane homeostasis, can be reconstituted starting from the genes encoding for all necessary proteins. A total of eight E. coli enzymes for acyl transfer and headgroup modifications were produced in a cell-free gene expression system and were co-translationally reconstituted in liposomes. Acyl-coenzyme A and glycerol-3-phosphate were used as canonical precursors to generate a variety of important bacterial lipids. Moreover, this study demonstrates that two-step acyl transfer can occur from enzymes synthesized inside vesicles. Besides clear implications for growth and potentially division of a synthetic cell, we postulate that gene-based lipid biosynthesis can become instrumental for ex vivo and protein purification-free production of natural and non-natural lipids.

The DNA-Binding Protein from Starved Cells (Dps) Utilizes Dual Functions To Defend Cells against Multiple Stresses.

UNLABELLED: Bacteria deficient in the DNA-binding protein from starved cells (Dps) are viable under controlled conditions but show dramatically increased mortality rates when exposed to any of a wide range of stresses, including starvation, oxidative stress, metal toxicity, or thermal stress. It remains unclear whether the protective action of Dps against specific stresses derives from its DNA-binding activity, which may exclude destructive agents from the chromosomal region, or its ferroxidase activity, which neutralizes and sequesters potentially damaging chemical species. To resolve this question, we have identified the critical residues of Escherichia coli Dps that bind to DNA and modulate iron oxidation. We uncoupled the biochemical activities of Dps, creating Dps variants and mutant E. coli strains that are defective in either DNA-binding or ferroxidase activity. Quantification of the contribution of each activity to the protection of DNA integrity and cellular viability revealed that both activities of Dps are required in order to counteract many differing stresses. These findings demonstrate that Dps plays a multipurpose role in stress protection via its dual activities, explaining how Dps can be of vital importance to bacterial viability over a wide range of stresses. IMPORTANCE: The DNA-binding protein from starved cells (Dps) protects bacterial cells against many different types of stressors. We find that DNA binding and iron oxidation by Dps are performed completely independently of each other. Both biochemical activities are required to protect E. coli against stressors, as well as to protect DNA from oxidative damage in vitro. These results suggest that many stressors may cause both oxidative stress and direct DNA damage.

DNA recognition by Escherichia coli CbpA protein requires a conserved arginine-minor-groove interaction.

Curved DNA binding protein A (CbpA) is a co-chaperone and nucleoid associated DNA binding protein conserved in most γ-proteobacteria. Best studied in Escherichia coli, CbpA accumulates to >2500 copies per cell during periods of starvation and forms aggregates with DNA. However, the molecular basis for DNA binding is unknown; CbpA lacks motifs found in other bacterial DNA binding proteins. Here, we have used a combination of genetics and biochemistry to elucidate the mechanism of DNA recognition by CbpA. We show that CbpA interacts with the DNA minor groove. This interaction requires a highly conserved arginine side chain. Substitution of this residue, R116, with alanine, specifically disrupts DNA binding by CbpA, and its homologues from other bacteria, whilst not affecting other CbpA activities. The intracellular distribution of CbpA alters dramatically when DNA binding is negated. Hence, we provide a direct link between DNA binding and the behaviour of CbpA in cells.

Unbiased tracking of the progression of mRNA and protein synthesis in bulk and in liposome-confined reactions.

The compartmentalization of a cell-free gene expression system inside a self-assembled lipid vesicle is envisioned as the simplest chassis for the construction of a minimal cell. Although crucial for its realization, quantitative understanding of the dynamics of gene expression in bulk and liposome-confined reactions is scarce. Here, we used two orthogonal fluorescence labeling tools to report the amounts of mRNA and protein produced in a reconstituted biosynthesis system, simultaneously and in real-time. The Spinach RNA aptamer and its fluorogenic probe were used for mRNA detection. Applying this dual-reporter assay to the analysis of transcript and protein production inside lipid vesicles revealed that their levels are uncorrelated, most probably a consequence of the low copy-number of some components in liposome-confined reactions. We believe that the stochastic nature of gene expression should be appreciated as a design principle for the assembly of a minimal cell.

Application of an in vitro DNA protection assay to visualize stress mediation properties of the Dps protein.

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage. The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Engineering of Penicillium chrysogenum for fermentative production of a novel carbamoylated cephem antibiotic precursor.

Penicillium chrysogenum was successfully engineered to produce a novel carbamoylated cephalosporin that can be used as a synthon for semi-synthetic cephalosporins. To this end, genes for Acremonium chrysogenum expandase/hydroxylase and Streptomyces clavuligerus carbamoyltransferase were expressed in a penicillinG high-producing strain of P.chrysogenum. Growth of the engineered strain in the presence of adipic acid resulted in production of adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA) and of several adipoylated pathway intermediates. A combinatorial chemostat-based transcriptome study, in which the ad7-ACCCA-producing strain and a strain lacking key genes in beta-lactam synthesis were grown in the presence and absence of adipic acid, enabled the dissection of transcriptional responses to adipic acid per se and to ad7-ACCCA production. Transcriptome analysis revealed that adipate catabolism in P.chrysogenum occurs via beta-oxidation and enabled the identification of putative genes for enzymes involved in mitochondrial and peroxisomal beta-oxidation pathways. Several of the genes that showed a specifically altered transcript level in ad7-ACCCA-producing cultures were previously implicated in oxidative stress responses.

Functional characterization of the penicillin biosynthetic gene cluster of Penicillium chrysogenum Wisconsin54-1255.

Industrial strain improvement via classical mutagenesis is a black box approach. In an attempt to learn from and understand the mutations introduced, we cloned and characterized the amplified region of industrial penicillin production strains. Upon amplification of this region Penicillium chrysogenum is capable of producing an increased amount of antibiotics, as was previously reported [Barredo, J.L., Diez, B., Alvarez, E., Martín, J.F., 1989a. Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high yielding strains of Penicillium chrysogenum. Curr. Genet. 16, 453-459; Newbert, R.W., Barton, B., Greaves, P., Harper, J., Turner, G., 1997. Analysis of a commercially improved Penicillium chrysogenum strain series, involvement of recombinogenic regions in amplification and deletion of the penicillin gene cluster. J. Ind. Microbiol. 19, 18-27]. Bioinformatic analysis of the central 56.9kb, present as six direct repeats in the strains analyzed in this study, predicted 15 Open Reading Frames (ORFs). Besides the three penicillin biosynthetic genes (pcbAB, pcbC and penDE) only one ORF has an orthologue of known function in the database: the Saccharomyces cerevisiae gene ERG25. Surprisingly, many genes known to encode direct or indirect steps beta-lactam biosynthesis like phenyl acetic acid CoA ligase and transporters are not present. Detailed analyses reveal a detectable transcript for most of the predicted ORFs under the conditions tested. We have studied the role of these in relation to penicillin production and amplification of the biosynthetic gene cluster. In contrast to what was expected, the genes encoding the three penicillin biosynthetic enzymes alone are sufficient to restore full beta-lactam synthesis in a mutant lacking the complete region. Therefore, the role of the other 12 ORFs in this region seems irrelevant for penicillin biosynthesis.