Molecular Engineering of E. coli Bacterioferritin: A Versatile Nanodimensional Protein Cage.

Currently, intense interest is focused on the discovery and application of new multisubunit cage proteins and spherical virus capsids to the fields of bionanotechnology, drug delivery, and diagnostic imaging as their internal cavities can serve as hosts for fluorophores or bioactive molecular cargo. Bacterioferritin is unusual in the ferritin protein superfamily of iron-storage cage proteins in that it contains twelve heme cofactors and is homomeric. The goal of the present study is to expand the capabilities of ferritins by developing new approaches to molecular cargo encapsulation employing bacterioferritin. Two strategies were explored to control the encapsulation of a diverse range of molecular guests compared to random entrapment, a predominant strategy employed in this area. The first was the inclusion of histidine-tag peptide fusion sequences within the internal cavity of bacterioferritin. This approach allowed for the successful and controlled encapsulation of a fluorescent dye, a protein (fluorescently labeled streptavidin), or a 5 nm gold nanoparticle. The second strategy, termed the heme-dependent cassette strategy, involved the substitution of the native heme with heme analogs attached to (i) fluorescent dyes or (ii) nickel-nitrilotriacetate (NTA) groups (which allowed for controllable encapsulation of a histidine-tagged green fluorescent protein). An in silico docking approach identified several small molecules able to replace the heme and capable of controlling the quaternary structure of the protein. A transglutaminase-based chemoenzymatic approach to surface modification of this cage protein was also accomplished, allowing for future nanoparticle targeting. This research presents novel strategies to control a diverse set of molecular encapsulations and adds a further level of sophistication to internal protein cavity engineering.

Stretching Peptides to Generate Small Molecule ß-Strand Mimics.

Advances in the modulation of protein-protein interactions (PPIs) enable both characterization of PPI networks that govern diseases and design of therapeutics and probes. The shallow protein surfaces that dominate PPIs are challenging to target using standard methods, and approaches for accessing extended backbone structures are limited. Here, we incorporate a rigid, linear, diyne brace between side chains at the i to i+2 positions to generate a family of low-molecular-weight, extended-backbone peptide macrocycles. NMR and density functional theory studies show that these stretched peptides adopt stable, rigid conformations in solution and can be tuned to explore extended peptide conformational space. The diyne brace is formed in excellent conversions (>95%) and amenable to high-throughput synthesis. The minimalist structure-inducing tripeptide core (<300 Da) is amenable to further synthetic elaboration. Diyne-braced inhibitors of bacterial type 1 signal peptidase demonstrate the utility of the technique.

SPI "sandwich": Combined SUMO-Peptide-Intein expression system and isolation procedure for improved stability and yield of peptides.

Recombinant peptide production in Escherichia coli is often accomplished through cloning and expression of a fusion protein. The fusion protein partner generally has two requirements: (a) it contains an affinity tag to assist with purification and (b) it can be cleaved off to leave only the desired peptide sequence behind. Common soluble fusion partners include small ubiquitin-like modifier protein (SUMO), maltose-binding protein (MBP), glutathione S-transferase (GST), or intein proteins. However, heterologously expressed peptides can suffer from proteolytic degradation or instability. This degradation can pose a major issue for applications requiring a large amount of purified peptide, such as NMR structural assignments or biochemical assays. Improving peptide yield by testing various expression and isolation conditions requires a significant amount of effort and may not lead to improved results. Here, we cloned and expressed four different peptides as SUMO fusion proteins. These peptides (lactococcin A, leucocin A, faerocin MK, neopetrosiamide A) were truncated during expression and isolation as SUMO fusions, resulting in low yields of purified peptide. To prevent this degradation and improve yield, we designed a new expression system to create a "sandwiched" fusion protein of the form: His6 -SUMO-peptide-intein (SPI). These sandwiched peptides were more stable and protected against degradation, resulting in improved yields (up to 17-fold) under a set of standard expression and isolation procedures. This SPI expression system uses only two commercially available vectors and standard protein purification techniques, and therefore may offer an economical and facile route to improve yields for peptides that undergo degradation.

Draft genome sequence of Staphylococcus agnetis 4244, a strain with gene clusters encoding distinct post-translationally modified antimicrobial peptides.

OBJECTIVES: Here we report the draft genome sequence of Staphylococcus agnetis 4244, a strain involved in bovine mastitis, and its ability to inhibit different species of antibiotic-resistant Gram-positive bacteria owing to bacteriocin production. METHODS: An Illumina MiSeq platform was used for genome sequencing. De novo genome assembly was done using the A5-miseq pipeline. Genome annotation was performed by the RAST server, and mining of bacteriocinogenic gene clusters was done using the BAGEL4 and antiSMASH v.5.0 platforms. Investigation of the spectrum of activity of S. agnetis 4244 was performed on BHI agar by deferred antagonism assay. RESULTS: The total scaffold size was determined to be 2 511 708 bp featuring a G+C content of 35.6%. The genome contains 2431 protein-coding sequences and 80 RNA sequences. Genome analyses revealed three prophage sequences inserted in the genome as well as several genes involved in drug resistance and two bacteriocin gene clusters (encoding a thiopeptide and a sactipeptide) encoded on the bacterial chromosome. Staphylococcus agnetis 4244 was able to inhibit all 44 strains of antibiotic-resistant Gram-positive bacteria tested in this study, including vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA) and other antibiotic-resistant staphylococcal strains. CONCLUSION: This study emphasises the potential biotechnological application of this strain for production of bacteriocins that could be used in the food industry as biopreservatives and/or in medicine as alternative therapeutic options against VRE, MRSA, vancomycin-intermediate S. aureus and other antibiotic-resistant Gram-positive bacteria, including biofilm-forming isolates. It also provides some genetic features of the draft genome of S. agnetis 4244.

Draft Genome Sequence of the Thermophilic Bacterium Bacillus licheniformis SMIA-2, an Antimicrobial- and Thermostable Enzyme-Producing Isolate from Brazilian Soil.

Bacillus licheniformis SMIA-2, a thermophilic and thermostable enzyme-producing bacterium, is found to be active against several strains of Staphylococcus aureus and several Bacillus species. Here, we report the 4.30-Mbp draft genome and bioinformatic predictions supporting gene inventories for amylase, protease, cellulase, xylanase, and antimicrobial compound biosynthesis.

Unveiling the active isomer of cycloalanopine, a cyclic opine from Lactobacillus rhamnosus LS8, through synthesis and analog production.

Opines are widely distributed natural products formed by the reductive condensation of amino acids with α-keto acids or carbonyls of carbohydrates. They have important biological roles in bacteria, higher plants, fungi, invertebrates and mammals, including humans. An unusual cyclic opine of undefined stereochemistry, cycloalanopine, was previously isolated from Lactobacillus rhamnosus LS8 and reported to have antimicrobial activity against both Gram-negative and Gram-positive bacteria. In this work, we report a three-step strategy to synthetically access pure isomers of this cyclic compound and analogs thereof. In the key step, acyclic bis-hydrazides can be oxidized with (diacetoxyiodo) benzene to corresponding cyclic N,N'-diacylhydrazides. The three cycloalanopine isomers, along with several analogs, were synthesized and tested against a panel of Gram-positive and Gram-negative bacteria. We identified the active isomer as the meso compound: (4R,6S)-4,6-dimethyl-1,2,5-triazepan-3,7-dione. Additionally, a glycine derivative, (R)-4-methyl-1,2,5-triazepan-3,7-dione, was ascertained to be more potent. This compound was active against both Gram-positive and Gram-negative organisms with the strongest potency against Escherichia coli and Acinetobacter baumannii, an opportunistic pathogen found in hospital-derived infections.

Dissecting the Binding Interactions of Teixobactin with the Bacterial Cell-Wall Precursor Lipid†II.

The prevalence of life-threatening, drug-resistant microbial infections has challenged researchers to consider alternatives to currently available antibiotics. Teixobactin is a recently discovered "resistance-proof" antimicrobial peptide that targets the bacterial cell wall precursor lipid II. In doing so, teixobactin exhibits potent antimicrobial activity against a wide range of Gram-positive organisms. Herein we demonstrate that teixobactin and several structural analogues are capable of binding lipid II from both Gram-positive and Gram-negative bacteria. Furthermore, we show that when combined with known outer membrane-disrupting peptides, teixobactin is active against Gram-negative organisms.

The expanding structural variety among bacteriocins from Gram-positive bacteria.

Bacteria use various strategies to compete in an ecological niche, including the production of bacteriocins. Bacteriocins are ribosomally synthesized antibacterial peptides, and it has been postulated that the majority of Gram-positive bacteria produce one or more of these natural products. Bacteriocins can be used in food preservation and are also considered as potential alternatives to antibiotics. The majority of bacteriocins from Gram-positive bacteria had been traditionally divided into two major classes, namely lantibiotics, which are post-translationally modified bacteriocins, and unmodified bacteriocins. The last decade has seen an expanding number of ribosomally synthesized and post-translationally modified peptides (RiPPs) in Gram-positive bacteria that have antibacterial activity. These include linear azol(in)e-containing peptides, thiopeptides, bottromycins, glycocins, lasso peptides and lipolanthines. In addition, the three-dimensional (3D) structures of a number of modified and unmodified bacteriocins have been elucidated in recent years. This review gives an overview on the structural variety of bacteriocins from Gram-positive bacteria. It will focus on the chemical and 3D structures of these peptides, and their interactions with receptors and membranes, structure-function relationships and possible modes of action.

Identification and Heterologous Expression of the sec-Dependent Bacteriocin Faerocin MK from Enterococcus faecium M3K31.

Genome sequencing of Enterococcus faecium M3K31, a strain isolated from griffon vultures, previously revealed the presence of three sequences encoding for bacteriocins, namely enterocin P, enterocin HF, and a SRCAM 602-like bacteriocin. In this work, we describe the SRCAM 602-like bacteriocin that we named faerocin MK. Mature faerocin MK consists of 43 residues and contains an YGNGV-motif and two cysteine residues at positions 10 and 15, consistent with other class IIa bacteriocins. Faerocin MK and SRCAM 602 were chemically synthesized and their scope of activity was tested. Faerocin MK is active against a wide range of Gram-positive organisms while SRCAM 602 was not active against any tested organism. Although both peptides are more structured in trifluoroethanol, faerocin MK has an α-helical character nearly twice that of SRCAM 602. Nucleotide sequence analysis revealed that the faerocin MK precursor is produced with a 28-amino acid signal peptide and that an immunity gene follows the structural faerocin MK gene. Heterologous expression of the faerocin MK operon showed that faerocin MK and its immunity protein were successfully expressed in other organisms. This indicates that the bacteriocin is secreted through the sec pathway.

Selecting optimal eggs and embryonic developmental stages of fathead minnow (Pimephales promelas) for early life-stage toxicity tests.

Aquaculture research has indicated that fish embryo hatching success and larval survival can sometimes be predicted by embryo characteristics, such as blastomere cleavage patterns. An analogous strategy of individual assessment of spawned eggs could also be used to improve the quality of toxicity tests using early life-stages of fish where control-group survival determines experimental validity. Here we explored whether a simple method of assessing fathead minnow eggs and embryos for abnormalities could predict hatch success, and larval size at hatch, as indicators of embryo larval quality. Embryos were classified according to both their developmental stage and the presence of any abnormalities: uneven blastomere cleavage, atypical embryo size or shape, and the presence of inclusions in the yolk. Clutch size and fertilization rate did not predict embryo larval quality. Fewer abnormalities in embryos with ≤32 cells correlated with longer larvae at hatch. Normal embryos were more likely to hatch successfully than abnormal embryos of the same clutch, but because abnormality rates were generally low, much of the variation in hatch success could not be attributed to visible embryo malformations. Blastomere symmetry may be a useful selection criterion in embryos <3 h postfertilization. Where toxicant exposures early in embryonic development are not required or possible, hatch success could be increased by using older embryos that have survived gastrulation. Purposeful selection of embryos with at least two blastomeres, blastomere symmetry, and few inclusions can improve control survival and improve the quality of any generated (sub)lethality data. In our laboratory, application of the egg-selection criteria significantly improved control group hatch success increasing it from a mean of 84.4 to 94.2%.