Post-translational Claisen Condensation and Decarboxylation en Route to the Bicyclic Core of Pantocin A

Pantocin A (PA) is a member of the growing family of ribosomally encoded and post-translationally modified peptide natural products (RiPPs). PA is much smaller than most known RiPPs, a tripeptide with a tight bicyclic core that appears to be cleaved from the middle of a larger 30-residue precursor peptide. We show here that the enzyme PaaA catalyzes the double dehydration and decarboxylation of two glutamic acid residues in the 30-residue precursor PaaP. Further truncates of PaaP leader and follower peptide sequences demonstrate the different impacts of these two regions on PaaA-mediated tailoring and delineate an essential role for the follower sequence in the decarboxylation step. The crystal structure of apo PaaA is reported, allowing identification of structural features that set PaaA apart from other homologous enzymes that typically do not catalyze such extended post-translational chemistry. Together, these data reveal how additional chemistry can be extracted from a ubiquitous enzyme family toward ribosomally derived peptide natural product biosynthesis and suggest that more examples of such enzymes likely exist in untapped genomic space

Dithiolopyrrolones: biosynthesis, synthesis, and activity of a unique class of disulfide-containing antibiotics

Dithiolopyrrolone natural products have unique structures and exhibit a broad-spectrum of antimicrobial and anticancer activities. Isolated in the late 1940s, these molecules have attracted an increasing interest towards their biosynthesis, synthesis and mechanisms of action

Critical behavior of the planar magnet model in three dimensions

We use a hybrid Monte Carlo algorithm in which a single-cluster update is combined with the over-relaxation and Metropolis spin re-orientation algorithm. Periodic boundary conditions were applied in all directions. We have calculated the fourth-order cumulant in finite size lattices using the single-histogram re-weighting method. Using finite-size scaling theory, we obtained the critical temperature which is very different from that of the usual XY model. At the critical temperature, we calculated the susceptibility and the magnetization on lattices of size up to $42^3$. Using finite-size scaling theory we accurately determine the critical exponents of the model and find that $\nu$=0.670(7), $\gamma/\nu$=1.9696(37), and $\beta/\nu$=0.515(2). Thus, we conclude that the model belongs to the same universality class with the XY model, as expected.Comment: 11 pages, 5 figure

SABRE hyperpolarisation of vitamin B3 as a function of pH

In this work we describe how the signal enhancements obtained through the SABRE process in methanol-d4 solution are significantly affected by pH. Nicotinic acid (vitamin B3, NA) is used as the agent, and changing pH is shown to modify the level of polarisation transfer by over an order of magnitude, with significant improvements being seen in terms of the signal amplitude and relaxation rate at high pH values. These observations reveal that manipulating pH to improve SABRE enhancements levels may improve the potential of this method to quantify low concentrations of analytes in mixtures.1H NMR spectroscopy results link this change to the form of the SABRE catalyst, which changes with pH, resulting in dramatic changes in the magnitude of the ligand exchange rates. The presented data also uses the fact that the chemical shifts of the nicotinic acids NMR resonances are affected by pH to establish that hyperpolarised 1H-based pH mapping with SABRE is possible. Moreover, the strong polarisation transfer field dependence shown in the amplitudes of the associated higher order longitudinal terms offers significant opportunities for the rapid detection of hyperpolarised NA in H2O itself without solvent suppression. 1H and 13C MRI images of hyperpolarised vitamin B3 in a series of test phantoms are presented that show pH dependent intensity and contrast. This study therefore establishes that when the pH sensitivity of NA is combined with the increase in signal gain provided for by SABRE hyperpolarisation, a versatile pH probe results

Inhibition of cell differentiation in Bacillus subtilis by Pseudomonas protegens

Interspecies interactions have been described for numerous bacterial systems, leading to the identification of chemical compounds that impact bacterial physiology and differentiation for processes such as biofilm formation. Here, we identified soil microbes that inhibit biofilm formation and sporulation in the common soil bacterium Bacillus subtilis. We did so by creating a reporter strain that fluoresces when the transcription of a biofilm-specific gene is repressed. Using this reporter in a coculture screen, we identified Pseudomonas putida and Pseudomonas protegens as bacteria that secrete compounds that inhibit biofilm gene expression in B. subtilis. The active compound produced by P. protegens was identified as the antibiotic and antifungal molecule 2,4-diacetylphloroglucinol (DAPG). Colonies of B. subtilis grown adjacent to a DAPG-producing P. protegens strain had altered colony morphologies relative to B. subtilis colonies grown next to a DAPG-null P. protegens strain (phlD strain). Using a subinhibitory concentration of purified DAPG in a pellicle assay, we saw that biofilm-specific gene transcription was delayed relative to transcription in untreated samples. These transcriptional changes also corresponded to phenotypic alterations: both biofilm biomass and spore formation were reduced in B. subtilis liquid cultures treated with subinhibitory concentrations of DAPG. Our results add DAPG to the growing list of antibiotics that impact bacterial development and physiology at subinhibitory concentrations. These findings also demonstrate the utility of using coculture as a means to uncover chemically mediated interspecies interactions between bacteria

VprBP/DCAF1 Regulates the Degradation and Nonproteolytic Activation of the Cell Cycle Transcription Factor FoxM1

The oncogenic transcription factor FoxM1 plays a vital role in cell cycle progression, is activated in numerous human malignancies, and is linked to chromosome instability. We characterize here a cullin 4-based E3 ubiquitin ligase and its substrate receptor, VprBP/DCAF1 (CRL4VprBP), which we show regulate FoxM1 ubiquitylation and degradation. Paradoxically, we also found that the substrate receptor VprBP is a potent FoxM1 activator. VprBP depletion reduces expression of FoxM1 target genes and impairs mitotic entry, whereas ectopic VprBP expression strongly activates a FoxM1 transcriptional reporter. VprBP binding to CRL4 is reduced during mitosis, and our data suggest that VprBP activation of FoxM1 is ligase independent. This implies a nonproteolytic activation mechanism that is reminiscent of, yet distinct from, the ubiquitin-dependent transactivation of the oncoprotein Myc by other E3s. Significantly, VprBP protein levels were upregulated in high-grade serous ovarian patient tumors, where the FoxM1 signature is amplified. These data suggest that FoxM1 abundance and activity are controlled by VprBP and highlight the functional repurposing of E3 ligase substrate receptors independent of the ubiquitin system

Large-Scale Bioinformatics Analysis of Bacillus Genomes Uncovers Conserved Roles of Natural Products in Bacterial Physiology

ABSTRACT Bacteria possess an amazing capacity to synthesize a diverse range of structurally complex, bioactive natural products known as specialized (or secondary) metabolites. Many of these specialized metabolites are used as clinical therapeutics, while others have important ecological roles in microbial communities. The biosynthetic gene clusters (BGCs) that generate these metabolites can be identified in bacterial genome sequences using their highly conserved genetic features. We analyzed an unprecedented 1,566 bacterial genomes from Bacillus species and identified nearly 20,000 BGCs. By comparing these BGCs to one another as well as a curated set of known specialized metabolite BGCs, we discovered that the majority of Bacillus natural products are comprised of a small set of highly conserved, well-distributed, known natural product compounds. Most of these metabolites have important roles influencing the physiology and development of Bacillus species. We identified, in addition to these characterized compounds, many unique, weakly conserved BGCs scattered across the genus that are predicted to encode unknown natural products. Many of these “singleton” BGCs appear to have been acquired via horizontal gene transfer. Based on this large-scale characterization of metabolite production in the Bacilli , we go on to connect the alkylpyrones, natural products that are highly conserved but previously biologically uncharacterized, to a role in Bacillus physiology: inhibiting spore development. IMPORTANCE Bacilli are capable of producing a diverse array of specialized metabolites, many of which have gained attention for their roles as signals that affect bacterial physiology and development. Up to this point, however, the Bacillus genus’s metabolic capacity has been underexplored. We undertook a deep genomic analysis of 1,566 Bacillus genomes to understand the full spectrum of metabolites that this bacterial group can make. We discovered that the majority of the specialized metabolites produced by Bacillus species are highly conserved, known compounds with important signaling roles in the physiology and development of this bacterium. Additionally, there is significant unique biosynthetic machinery distributed across the genus that might lead to new, unknown metabolites with diverse biological functions. Inspired by the findings of our genomic analysis, we speculate that the highly conserved alkylpyrones might have an important biological activity within this genus. We go on to validate this prediction by demonstrating that these natural products are developmental signals in Bacillus and act by inhibiting sporulation

Thiopeptide antibiotics stimulate biofilm formation in Bacillus subtilis

Thiazolyl peptides are known antibiotics produced by diverse bacterial taxa. It has been believed that antibiotics are deployed by bacteria as weapons, providing them with an evolutionary advantage over other microbes. We show here that these weapons can also act as chemical tools that elicit biofilm production in the model bacterium Bacillus subtilis. Importantly, the biofilm-inducing (and therefore signaling) properties of these compounds are independent of their killing activity. We go on to use this biofilm-inducing activity to identify and confirm the presence of thiazolyl peptide gene clusters in other bacteria. These results indicate that thiazolyl peptides, and potentially other antibiotics, have the ability to alter bacterial behavior in ways important both to the environment and to human health

The substrate lends a hand

Duramycin is a small post-translationally modified peptide with antibody-like affinity for phosphatidylethanolamine. As it turns out, the same functionality that is essential for duramycin activity helps to catalyze the formation of its conformationally constrained and compact polycyclic architecture

The genome-wide distribution of non-B DNA motifs is shaped by operon structure and suggests the transcriptional importance of non-B DNA structures in Escherichia coli

Although the right-handed double helical B-form DNA is most common under physiological conditions, DNA is dynamic and can adopt a number of alternative structures, such as the four-stranded G-quadruplex, left-handed Z-DNA, cruciform and others. Active transcription necessitates strand separation and can induce such non-canonical forms at susceptible genomic sequences. Therefore, it has been speculated that these non-B DNA motifs can play regulatory roles in gene transcription. Such conjecture has been supported in higher eukaryotes by direct studies of several individual genes, as well as a number of large-scale analyses. However, the role of non-B DNA structures in many lower organisms, in particular proteobacteria, remains poorly understood and incompletely documented. In this study, we performed the first comprehensive study of the occurrence of B DNA–non-B DNA transition-susceptible sites (non-B DNA motifs) within the context of the operon structure of the Escherichia coli genome. We compared the distributions of non-B DNA motifs in the regulatory regions of operons with those from internal regions. We found an enrichment of some non-B DNA motifs in regulatory regions, and we show that this enrichment cannot be simply explained by base composition bias in these regions. We also showed that the distribution of several non-B DNA motifs within intergenic regions separating divergently oriented operons differs from the distribution found between convergent ones. In particular, we found a strong enrichment of cruciforms in the termination region of operons; this enrichment was observed for operons with Rho-dependent, as well as Rho-independent terminators. Finally, a preference for some non-B DNA motifs was observed near transcription factor-binding sites. Overall, the conspicuous enrichment of transition-susceptible sites in these specific regulatory regions suggests that non-B DNA structures may have roles in the transcriptional regulation of specific operons within the E. coli genome