The Bristol Sponge Microbiome Collection: A Unique Repository of Deep-Sea Microorganisms and Associated Natural Products.

The deep ocean is the largest habitat for life on Earth, though the microorganisms that occupy this unique environmental niche remain largely unexplored. Due to the significant logistical and operational challenges associated with accessing the deep ocean, bioprospecting programmes that seek to generate novel products from marine organisms have, to date, focused predominantly on samples recovered from shallow seas. For this reason, the deep ocean remains a largely untapped resource of novel microbiological life and associated natural products.

Imaging rRNA Methylation in Bacteria by MR-FISH.

Methylation of RNA is normally monitored in purified cell lysates using next-generation sequencing, gel electrophoresis, or mass spectrometry as readouts. These bulk methods require the RNA from ~10 to 10 cells to be pooled to generate sufficient material for analysis. Here we describe a method-methylation-sensitive RNA in situ hybridization (MR-FISH)-that assays rRNA methylation in bacteria on a cell-by-cell basis, using methylation-sensitive hybridization probes and fluorescence microscopy.

Detecting RNA base methylations in single cells by in situ hybridization.

Methylated bases in tRNA, rRNA and mRNA control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression. Currently, bulk methods that report the average methylation state of ~10-10 cells are used to detect these modifications, obscuring potentially important biological information. Here, we use in situ hybridization of Molecular Beacons for single-cell detection of three methylations (mA, mG and mU) that destabilize Watson-Crick base pairs.

Cysteine methylation controls radical generation in the Cfr radical AdoMet rRNA methyltransferase.

The 'radical S-adenosyl-L-methionine (AdoMet)' enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), from numerous antibiotics, including agents (e.g.

High-level expression and reconstitution of active Cfr, a radical-SAM rRNA methyltransferase that confers resistance to ribosome-acting antibiotics.

Cfr is a radical-SAM (S-adenosyl-L-methionine) enzyme that methylates the 8 position of 23S rRNA residue A2503 to confer resistance to multiple antibiotic classes acting upon the large subunit of the bacterial ribosome. Radical-SAM enzymes use an Fe-S cluster to generate the 5'-deoxyadenosyl (DOA) radical from SAM, enabling them to modify intrinsically unreactive centres such as adenosine C8. However, despite its mechanistic interest and clinical relevance, until recently Cfr remained little characterised.

[FeFe]-hydrogenase maturation: HydG-catalyzed synthesis of carbon monoxide.

Biosynthesis of the unusual organometallic H-cluster at the active site of the [FeFe]-hydrogenase requires three accessory proteins, two of which are radical AdoMet enzymes (HydE, HydG) and one of which is a GTPase (HydF). We demonstrate here that HydG catalyzes the synthesis of CO using tyrosine as a substrate. CO production was detected by using deoxyhemoglobin as a reporter and monitoring the appearance of the characteristic visible spectroscopic features of carboxyhemoglobin.

Catalytic activity of the anaerobic tyrosine lyase required for thiamine biosynthesis in Escherichia coli.

Thiazole synthase in Escherichia coli is an alphabeta heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the C alpha-C beta bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. This reactive intermediate acts as one of three substrates for the thiazole cyclization reaction catalyzed by ThiG.

Product inhibition in the radical S-adenosylmethionine family.

Members of the radical S-adenosylmethionine (AdoMet) superfamily reductively cleave AdoMet to generate the highly reactive 5'-deoxyadenosyl radical (DOA()) which initiates biological transformations by abstraction of a hydrogen atom. We demonstrate that three members of the family: biotin synthase (BioB), lipoyl synthase (LipA) and tyrosine lyase (ThiH) are inhibited in vitro by a combination of the products 5'-deoxyadenosine (DOA) and methionine. These results suggest the observed inhibition is a common feature of the radical AdoMet proteins that form DOA and methionine as products.