Alexander O. Brachmann

An environmental bacterial taxon with a large and distinct metabolic repertoire

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such ‘talented’ producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus ‘Entotheonella’ with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. ‘Entotheonella’ spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum ‘Tectomicrobia’. The pronounced bioactivities and chemical uniqueness of ‘Entotheonella’ compounds provide significant opportunities for ecological studies and drug discovery.

Pyrones as bacterial signaling molecules

Bacteria communicate via small diffusible molecules and thereby mediate group-coordinated behavior, a process referred to as quorum sensing. The prototypical quorum sensing system found in Gram-negative bacteria consists of a LuxI-type autoinducer synthase that produces N-acyl homoserine lactones (AHLs) as signals and a LuxR-type receptor that detects the AHLs to control expression of specific genes. However, many proteobacteria have proteins with homology to LuxR receptors yet lack any cognate LuxI-like AHL synthase. Here we show that in the insect pathogen Photorhabdus luminescens the orphan LuxR-type receptor PluR detects endogenously produced α-pyrones that serve as signaling molecules at low nanomolar concentrations. Additionally, the ketosynthase PpyS was identified as pyrone synthase. Reconstitution of the entire system containing PluR, the PluR-target operon we termed pcf and PpyS in Escherichia coli demonstrated that the cell-cell communication circuit is portable. Our research thus deorphanizes a signaling system and suggests that additional modes of bacterial communication may await discovery.

The Entomopathogenic Bacterial Endosymbionts Xenorhabdus and Photorhabdus: Convergent Lifestyles from Divergent Genomes

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.

A Type II Polyketide Synthase is Responsible for Anthraquinone Biosynthesis in Photorhabdus luminescens

Type II polyketide synthases are involved in the biosynthesis of numerous clinically relevant secondary metabolites with potent antibiotic or anticancer activity. Until recently the only known producers of type II PKSs were members of the Gram‐positive actimomycetes, well‐known producers of secondary metabolites in general. Here we present the second example of a type II PKS from Gram‐negative bacteria. We have identified the biosynthesis gene cluster responsible for the production of anthraquinones (AQs) from the entomopathogenic bacterium Photorhabdus luminescens. This is the first example of AQ production in Gram‐negative bacteria, and their heptaketide origin was confirmed by feeding experiments. Deletion of a cyclase/aromatase involved in AQ biosynthesis resulted in accumulation of mutactin and dehydromutactin, which have been described as shunt products of typical octaketide compounds from streptomycetes, and a pathway for AQ formation from octaketide intermediates is discussed.

Determination of the Absolute Configuration of Peptide Natural Products by Using Stable Isotope Labeling and Mass Spectrometry

Structure elucidation of natural products including the absolute configuration is a complex task that involves different analytical methods like mass spectrometry, NMR spectroscopy, and chemical derivation, which are usually performed after the isolation of the compound of interest. Here, a combination of stable isotope labeling of Photorhabdus and Xenorhabdus strains and their transaminase mutants followed by detailed MS analysis enabled the structure elucidation of novel cyclopeptides named GameXPeptides including their absolute configuration in crude extracts without their actual isolation.

Formation of 1,3‐Cyclohexanediones and Resorcinols Catalyzed by a Widely Occuring Ketosynthase

Overlooked, but widespread! A new class of ketosynthases (DarB) involved in the biosynthesis of 1,3-cyclohexanediones and dialkylresorcinols has been identified and characterized in detail. The presence of homologues in 89 different bacteria, including several pathogens, reveals that DarB as well as the corresponding natural products might be widespread, thus presenting a new but so far overlooked pathway to natural products.

A New Type of Pyrrolidine Biosynthesis Is Involved in the Late Steps of Xenocoumacin Production in Xenorhabdus nematophila

Bacteria of the genus Xenorhabdus live in symbiosis with nematodes of the genus Steinernema, and both form an entomopathogenic complex that is used commercially to kill several different insect larvae. Briefly, the nematode carries the bacteria in the gut of its free-living state called infective juvenile (IJ). IJs actively search the soil for insect larvae. After an insect is identified, the nematode infects the insect and regurgitates the bacteria, which kill the insect within 24 h post-infection. The insect cadaver is then digested by the bacteria and the nematodes, and after several cycles of nematode development, new IJs are formed, which carry the bacteria in the gut and leave the now empty insect carcass to find new prey. As there have been hints in the literature that small molecules (e.g. , secondary metabolites) produced by the bacterium are either involved in the pathogenesis against the insect or the symbiosis towards the nematode, we have started to search for ACHTUNGTRENNUNGsecondary metabolites produced by different Xenorhabdus ACHTUNGTRENNUNGspecies. During this work, we could identify xenocoumacins (XCNs) I and II (1 and 2, respectively, in Scheme 1), which were isolated several years ago from different strains of Xenorhabdus nematophila. Although both compounds show antibiotic activity, 1 is much more active and additionally shows good activity against different fungi. Currently, both compounds are thought to be involved in killing bacteria living inside the insect gut, where these bacteria would compete with XenoACHTUNGTRENNUNGrhabdus for food in the dead insect. We could identify 1 and 2 in several Xenorhabdus strains by their characteristic fragmentation pattern in HRESI-MS (see below). Moreover, a detailed analysis of two different XCN producer strains, namely X. nematophila AN6/1 and X. kozodoii DSM 17907, under different cultivation conditions led to the identification of four new XCN derivatives named XCN III–VI (3–6). Whereas only traces of 3 and 4 are observed throughout the cultivation process in strain AN6/1, 5 and 6 start to accumulate in significant amounts after 1 and 2 have been formed (after 8 h); this indicates a structural relationship (Figure 1). Interestingly, only traces of 5 and 6 were observed in cultures grown with the adsorber resin Amberlite XAD-16, which seems to protect 1 and 2 from their transformation or degradation, as has also been observed for other secondary metabolites.

Simple "on-demand" production of bioactive natural products

Exchange of the native promoter to the arabinose‐inducible promoter PBAD was established in entomopathogenic bacteria to silence and/or activate gene clusters involved in natural product biosynthesis. This allowed the “on‐demand” production of GameXPeptides, xenoamicins, and the blue pigment indigoidine. The gene clusters for the novel “mevalagmapeptides” and the highly toxic xenorhabdins were identified by this approach.

Structure Elucidation and Activity of Kolossin A, the D‐/L‐Pentadecapeptide Product of a Giant Nonribosomal Peptide Synthetase

The largest continuous bacterial nonribosomal peptide synthetase discovered so far is described. It consists of 15 consecutive modules arising from an uninterrupted, fully functional gene in the entomopathogenic bacterium Photorhabdus luminescens. The identification of its cryptic biosynthesis product was achieved by using a combination of genome analysis, promoter exchange, isotopic labeling experiments, and total synthesis of a focused collection of peptide candidates. Although it belongs to the growing class of D-/ L-peptide natural products, the encoded metabolite kolossin A was found to be largely devoid of antibiotic activity and is likely involved in interspecies communication. A stereoisomer of this peculiar natural product displayed high activity against Trypanosoma brucei rhodesiense, a recalcitrant parasite that causes the deadly disease African sleeping sickness.

Identification and Bioanalysis of Natural Products from Insect Symbionts and Pathogens

: With the development of several novel methods in genome sequencing, molecular biology, and analytical chemistry a new area of natural product chemistry is currently starting that allows the analysis of minute amounts of complex biological samples. The combination of these methods, as discussed in this review, also enables the analysis of bacteria living in symbiosis or being pathogenic to insects, which might be the largest reservoir for novel microbes associated with higher organisms due to the huge number of insect species.