Amber-codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase.

The 11 lytic transglycosylases of Pseudomonas aeruginosa have overlapping activities in the turnover of the cell-wall peptidoglycan. Rare lipoprotein A (RlpA) is distinct among the 11 by its use of only peptidoglycan lacking peptide stems. The spatial localization of RlpA and its interactome within P. aeruginosa are unknown. We employed suppression of introduced amber codons at sites in the rlpA gene for the introduction of the unnatural-amino-acids Νζ -[(2-azidoethoxy)carbonyl]-l-lysine (compound 1) and Nζ -[[[3-(3-methyl-3H-diazirin-3-yl)propyl]amino]carbonyl]-l-lysine (compound 2). In live P. aeruginosa, full-length RlpA incorporating compound 1 into its sequence was fluorescently tagged using strained-promoted alkyne-azide cycloaddition and examined by fluorescence microscopy. RlpA is present at low levels along the sidewall length of the bacterium, and at higher levels at the nascent septa of replicating bacteria. In intact P. aeruginosa, UV photolysis of full-length RlpA having compound 2 within its sequence generated a transient reactive carbene, which engaged in photoaffinity capture of neighboring proteins. Thirteen proteins were identified. Three of these proteins-PBP1a, PBP5, and MreB-are members of the bacterial divisome. The use of the complementary methodologies of non-canonical amino-acid incorporation, photoaffinity proximity analysis, and fluorescent microscopy confirm a dominant septal location for the RlpA enzyme of P. aeruginosa, as a divisome-associated activity. This accomplishment adds to the emerging recognition of the value of these methodologies for identification of the intracellular localization of bacterial proteins.

The DataHarmonizer: a tool for faster data harmonization, validation, aggregation and analysis of pathogen genomics contextual information.

Pathogen genomics is a critical tool for public health surveillance, infection control, outbreak investigations as well as research. In order to make use of pathogen genomics data, they must be interpreted using contextual data (metadata). Contextual data include sample metadata, laboratory methods, patient demographics, clinical outcomes and epidemiological information. However, the variability in how contextual information is captured by different authorities and how it is encoded in different databases poses challenges for data interpretation, integration and their use/re-use. The DataHarmonizer is a template-driven spreadsheet application for harmonizing, validating and transforming genomics contextual data into submission-ready formats for public or private repositories. The tool's web browser-based JavaScript environment enables validation and its offline functionality and local installation increases data security. The DataHarmonizer was developed to address the data sharing needs that arose during the COVID-19 pandemic, and was used by members of the Canadian COVID Genomics Network (CanCOGeN) to harmonize SARS-CoV-2 contextual data for national surveillance and for public repository submission. In order to support coordination of international surveillance efforts, we have partnered with the Public Health Alliance for Genomic Epidemiology to also provide a template conforming to its SARS-CoV-2 contextual data specification for use worldwide. Templates are also being developed for One Health and foodborne pathogens. Overall, the DataHarmonizer tool improves the effectiveness and fidelity of contextual data capture as well as its subsequent usability. Harmonization of contextual information across authorities, platforms and systems globally improves interoperability and reusability of data for concerted public health and research initiatives to fight the current pandemic and future public health emergencies. While initially developed for the COVID-19 pandemic, its expansion to other data management applications and pathogens is already underway.

Chemoenzymatic macrocycle synthesis using resorcylic acid lactone thioesterase domains.

A key missing tool in the chemist's toolbox is an effective biocatalyst for macrocyclization. Macrocycles limit the conformational flexibility of small molecules, often improving their ability to bind selectively and with high affinity to a target, making them a privileged structure in drug discovery. Macrocyclic natural product biosynthesis offers an obvious starting point for biocatalyst discovery via the native macrocycle forming biosynthetic mechanism. Herein we demonstrate that the thioesterase domains (TEs) responsible for macrocyclization of resorcylic acid lactones are promising catalysts for the chemoenzymatic synthesis of 12- to 18-member ring macrolactones and macrolactams. The TE domains responsible for zearalenone and radicicol biosynthesis successfully generate resorcylate-like 12- to 18-member macrolactones and a 14-member macrolactam. In addition these enzymes can also macrolactonize a non-resorcylate containing depsipeptide, suggesting they are versatile biocatalysts. Simple saturated omega-hydroxy acyl chains are not macrocyclized, nor are the alpha-beta unsaturated derivatives, clearly outlining the scope of the substrate tolerance. These data dramatically expand our understanding of substrate tolerance of these enzymes and are consistent with our understanding of the role of TEs in iterative polyketide biosynthesis. In addition this work shows these TEs to be the most substrate tolerant polyketide macrocyclizing enzymes known, accessing resorcylate lactone and lactams as well as cyclicdepsipeptides, which are highly biologically relevant frameworks.

Whole-Genome Shotgun Sequencing of Two ß-Proteobacterial Species in Search of the Bulgecin Biosynthetic Cluster.

We have produced draft whole-genome sequences for two bacterial strains reported to produce the bulgecins as well as NRPS-derived monobactam ß-lactam antibiotics. We propose classification of ATCC 31363 as Paraburkholderia acidophila. We further reaffirm that ATCC 31433 (Burkholderia ubonensis subsp. mesacidophila) is a taxonomically distinct producer of bulgecins with notable gene regions shared with Paraburkholderia acidophila. We use RAST multiple-gene comparison and MASH distancing with published genomes to order the draft contigs and identify unique gene regions for characterization. Forty-eight natural-product gene clusters are presented from PATRIC (RASTtk) and antiSMASH annotations. We present evidence that the 10 genes that follow the sulfazecin and isosulfazecin pathways in both species are likely involved in bulgecin A biosynthesis.

Polyketide synthase and non-ribosomal peptide synthetase thioesterase selectivity: logic gate or a victim of fate?

Type 1, α/ß hydrolase-like thioesterase (TE) domains are essential offloading enzymes, releasing covalently bound products from fatty acid, polyketide, and non-ribosomal peptide biosynthetic complexes. The release step can occur by attack of an exogenous nucleophile effecting hydrolysis or transesterification or by an intramolecular O-, N-, or C-nucleophile, effecting macrolactonization, macrolactamization or Claisen-like condensation of the product. Thus in addition to ensuring turnover of the pathway, TEs provide access to increased chemical diversity. We review the diversity, structure, and mechanism of PKS and NRPS TEs and discuss recent works that highlight the role of TEs as potential arbitrators in offloading. In particular, we examine cases where TEs act as logic gates that ask a particular question about the substrate and use this information to determine the substrate's fate. As the TE mechanism occurs via two steps, we analyze both the loading and release steps independently as logic gates. The use of logic gates provides an important perspective when evaluating the evolution of TEs within a pathway, as well as highlighting work towards the goal of predicting TE function in unknown and engineered pathways.

Practice effects reveal visuomotor vulnerability in school and university rugby players.

This article reports on three pre- versus post-season prospective studies in which male university and high school contact sport players predominantly of Rugby Union (hereafter rugby) were compared with age, education, and IQ equivalent non-contact sport controls on the ImPACT (Immediate Postconcussion Assessment and Cognitive Testing) test. All analyses revealed a relative absence of practice effects on the Visual Motor Speed (VMS) composite for contact sport groups compared with controls. The VMS data for rugby players from each study were pooled and subjected to additional analysis (Rugby, n = 145; Controls, n = 106). Controls revealed significant improvement over the season (p < .001), whereas no learning effect was in evidence for rugby players whose performance remained the same (interaction effect, p = .028). It is apparent that practice effects have diagnostic potential in this context, implicating vulnerability on speeded visuomotor processing in association with participation in rugby. Pointers for further research and concussion management in the individual case are explored.

6-Deoxyerythronolide B synthase thioesterase-catalyzed macrocyclization is highly stereoselective.

Macrocyclic polyketide natural products are an indispensable source of therapeutic agents. The final stage of their biosynthesis, macrocyclization, is catalyzed regio- and stereoselectively by a thioesterase. A panel of substrates were synthesized to test their specificity for macrocyclization by the erythromycin polyketide synthase TE (DEBS TE) in vitro. It was shown that DEBS TE is highly stereospecific, successfully macrocyclizing a 14-member ring substrate with an R configured O-nucleophile, and highly regioselective, generating exclusively the 14-member lactone over the 12-member lactone.

Closed photobioreactors for production of microalgal biomasses.

Microalgal biomasses have been produced industrially for a long history for application in a variety of different fields. Most recently, microalgae are established as the most promising species for biofuel production and CO(2) bio-sequestration owing to their high photosynthesis efficiency. Nevertheless, design of photobioreactors that maximize solar energy capture and conversion has been one of the major challenges in commercial microalga biomass production. In this review, we systematically survey the recent developments in this field.

Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans.

Microalgal lipids are the oils of future for sustainable biodiesel production. However, relatively high production costs due to low lipid productivity have been one of the major obstacles impeding their commercial production. We studied the effects of nitrogen sources and their concentrations on cell growth and lipid accumulation of Neochloris oleoabundans, one of the most promising oil-rich microalgal species. While the highest lipid cell content of 0.40 g/g was obtained at the lowest sodium nitrate concentration (3 mM), a remarkable lipid productivity of 0.133 g l(-1) day(-1) was achieved at 5 mM with a lipid cell content of 0.34 g/g and a biomass productivity of 0.40 g l(-1) day(-1). The highest biomass productivity was obtained at 10 mM sodium nitrate, with a biomass concentration of 3.2 g/l and a biomass productivity of 0.63 g l(-1) day(-1). It was observed that cell growth continued after the exhaustion of external nitrogen pool, hypothetically supported by the consumption of intracellular nitrogen pools such as chlorophyll molecules. The relationship among nitrate depletion, cell growth, lipid cell content, and cell chlorophyll content are discussed.

Biofuels from microalgae.

Microalgae are a diverse group of prokaryotic and eukaryotic photosynthetic microorganisms that grow rapidly due to their simple structure. They can potentially be employed for the production of biofuels in an economically effective and environmentally sustainable manner. Microalgae have been investigated for the production of a number of different biofuels including biodiesel, bio-oil, bio-syngas, and bio-hydrogen. The production of these biofuels can be coupled with flue gas CO2 mitigation, wastewater treatment, and the production of high-value chemicals. Microalgal farming can also be carried out with seawater using marine microalgal species as the producers. Developments in microalgal cultivation and downstream processing (e.g., harvesting, drying, and thermochemical processing) are expected to further enhance the cost-effectiveness of the biofuel from microalgae strategy.