Helge B. Bode

Complete genome sequence of the myxobacterium Sorangium cellulosum.

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strains complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase–like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.

Entomopathogenic bacteria as a source of secondary metabolites.

Insects are not only the most diverse group of animals on our planet, but also a huge reservoir for unusual microorganism which are a rich source of pharmaceutically interesting natural products. This review focuses on recent advances in the understanding of secondary metabolism of Bacillus thuringiensis, Pseudomonas entomophila, and Xenorhabdus and Photorhabdus bacteria all of which are entomopathogenic. Genome-sequencing projects revealed the capacity of these bacteria to produce several different secondary metabolites including peptides, polyketides, and hybrids of both. This richness for interesting compounds is reflected by an increasing number of compounds that have been identified from these bacteria as discussed in this review.

Steroid biosynthesis in prokaryotes: identification of myxobacterial steroids and cloning of the first bacterial 2,3(S)‐oxidosqualene cyclase from the myxobacterium Stigmatella aurantiaca

Steroids, such as cholesterol, are synthesized in almost all eukaryotic cells, which use these triterpenoid lipids to control the fluidity and flexibility of their cell membranes. Bacteria rarely synthesize such tetracyclic compounds but frequently replace them with a different class of triterpenoids, the pentacyclic hopanoids. The intriguing mechanisms involved in triterpene biosynthesis have attracted much attention, resulting in extensive studies of squalene‐hopene cyclase in bacteria and (S)‐2,3‐oxidosqualene cyclases in eukarya. Nevertheless, almost nothing is known about steroid biosynthesis in bacteria. Only three steroid‐synthesizing bacterial species have been identified before this study. Here, we report on a variety of sterol‐producing myxobacteria. Stigmatella aurantiaca is shown to produce cycloartenol, the well‐known first cyclization product of steroid biosynthesis in plants and algae. Additionally, we describe the cloning of the first bacterial steroid biosynthesis gene, cas, encoding the cycloartenol synthase (Cas) of S. aurantiaca. Mutants of cas generated via site‐directed mutagenesis do not produce the compound. They show neither growth retardation in comparison with wild type nor any increase in ethanol sensitivity. The protein encoded by cas is most similar to the Cas proteins from several plant species, indicating a close evolutionary relationship between myxobacterial and eukaryotic steroid biosynthesis.

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.

Optical mapping as a routine tool for bacterial genome sequence finishing.

BackgroundIn sequencing the genomes of two Xenorhabdus species, we encountered a large number of sequence repeats and assembly anomalies that stalled finishing efforts. This included a stretch of about 12 Kb that is over 99.9% identical between the plasmid and chromosome of X. nematophila.ResultsWhole genome restriction maps of the sequenced strains were produced through optical mapping technology. These maps allowed rapid resolution of sequence assembly problems, permitted closing of the genome, and allowed correction of a large inversion in a genome assembly that we had considered finished.ConclusionOur experience suggests that routine use of optical mapping in bacterial genome sequence finishing is warranted. When combined with data produced through 454 sequencing, an optical map can rapidly and inexpensively generate an ordered and oriented set of contigs to produce a nearly complete genome sequence assembly.

Biosynthesis of volatiles by the myxobacterium Myxococcus xanthus

The volatiles emitted from cell cultures of myxobacterium Myxococcus xanthus were collected by use of a closed‐loop stripping apparatus (CLSA) and analyzed by GC‐MS. Two new natural products, (S)‐9‐methyldecan‐3‐ol ((S)‐1) and 9‐methyldecan‐3‐one (2), were identified and synthesized, together with other aliphatic ketones and alcohols, and terpenes. Biosynthesis of the two main components (S)‐1 and 2 was examined in feeding experiments carried out with the wild‐type strain DK1622 and two mutant strains JD300 and DK11017, which are impaired in the degradation pathway from leucine to isovaleryl‐SCoA. Isovaleryl‐SCoA is used as a starter, followed by chain elongation with two malonate units. Subsequent use of methyl malonate and decarboxylation leads to (S)‐1 and 2. Furthermore, 3,3‐dimethylacrylic acid (DMAA) can be used by the mutant strain to form isovaleryl‐SCoA, which corroborates recent data on the detection of a novel variety of the mevalonate pathway giving rise to isovaleryl‐SCoA from HMGCoA.

Cytosolic re-localization and optimization of valine synthesis and catabolism enables increased isobutanol production with the yeast Saccharomyces cerevisiae

BackgroundThe branched chain alcohol isobutanol exhibits superior physicochemical properties as an alternative biofuel. The yeast Saccharomyces cerevisiae naturally produces low amounts of isobutanol as a by-product during fermentations, resulting from the catabolism of valine. As S. cerevisiae is widely used in industrial applications and can easily be modified by genetic engineering, this microorganism is a promising host for the fermentative production of higher amounts of isobutanol.ResultsIsobutanol production could be improved by re-locating the valine biosynthesis enzymes Ilv2, Ilv5 and Ilv3 from the mitochondrial matrix into the cytosol. To prevent the import of the three enzymes into yeast mitochondria, N-terminally shortened Ilv2, Ilv5 and Ilv3 versions were constructed lacking their mitochondrial targeting sequences. SDS-PAGE and immunofluorescence analyses confirmed expression and re-localization of the truncated enzymes. Growth tests or enzyme assays confirmed enzymatic activities. Isobutanol production was only increased in the absence of valine and the simultaneous blockage of the mitochondrial valine synthesis pathway. Isobutanol production could be even more enhanced after adapting the codon usage of the truncated valine biosynthesis genes to the codon usage of highly expressed glycolytic genes. Finally, a suitable ketoisovalerate decarboxylase, Aro10, and alcohol dehydrogenase, Adh2, were selected and overexpressed. The highest isobutanol titer was 0.63 g/L at a yield of nearly 15 mg per g glucose.ConclusionA cytosolic isobutanol production pathway was successfully established in yeast by re-localization and optimization of mitochondrial valine synthesis enzymes together with overexpression of Aro10 decarboxylase and Adh2 alcohol dehydrogenase. Driving forces were generated by blocking competition with the mitochondrial valine pathway and by omitting valine from the fermentation medium. Additional deletion of pyruvate decarboxylase genes and engineering of co-factor imbalances should lead to even higher isobutanol production.

A natural prodrug activation mechanism in nonribosomal peptide synthesis

We have identified a new mechanism for the cleavage and activation of nonribosomally made peptides and peptide-polyketide hybrids that are apparently operational in several different bacteria. This process includes the cleavage of a precursor molecule by a membrane-bound and D-asparagine-specific peptidase, as shown here in the biosynthesis of the antibiotic xenocoumacin from Xenorhabdus nematophila.

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.