Enantioselective synthesis of all stereoisomers of geosmin and of biosynthetically related natural products.

Synthetic routes to geosmin and its enantiomer are well established, but the enantioselective synthesis of stereoisomers of geosmin is unknown. Here a stereoselective synthesis of all stereoisomers of geosmin is reported, yielding all compounds in high enantiomeric purity. Furthermore, the stereoselective synthesis of a geosmin derivative isolated from a mangrove associated streptomycete was performed, establishing the absolute configuration of the natural product. Finally, a new side product of the geosmin synthase from Streptomyces ambofaciens was isolated and its structure was elucidated by NMR spectroscopy. The absolute configuration of this new compound was determined through a stereoselective synthesis.

Genomic Evolution of the Marine Bacterium Phaeobacter inhibens during Biofilm Growth.

Phaeobacter inhibens 2.10 is an effective biofilm former on marine surfaces and has the ability to outcompete other microorganisms, possibly due to the production of the plasmid-encoded secondary metabolite tropodithietic acid (TDA). P. inhibens 2.10 biofilms produce phenotypic variants with reduced competitiveness compared to the wild type. In the present study, we used longitudinal, genome-wide deep sequencing to uncover the genetic foundation that contributes to the emergent phenotypic diversity in P. inhibens 2.10 biofilm dispersants. Our results show that phenotypic variation is not due to the loss of the plasmid that carries the genes for TDA synthesis but instead show that P. inhibens 2.10 biofilm populations become rapidly enriched in single nucleotide variations in genes involved in the synthesis of TDA. While variants in genes previously linked to other phenotypes, such as lipopolysaccharide production (i.e., rfbA) and cellular persistence (i.e., metG), also appear to be selected for during biofilm dispersal, the number and consistency of variations found for genes involved in TDA production suggest that this metabolite imposes a burden on P. inhibens 2.10 cells. Our results indicate a strong selection pressure for the loss of TDA in monospecies biofilm populations and provide insight into how competition (or a lack thereof) in biofilms might shape genome evolution in bacteria. IMPORTANCE Biofilm formation and dispersal are important survival strategies for environmental bacteria. During biofilm dispersal, cells often display stable and heritable variants from the parental biofilm. Phaeobacter inhibens is an effective colonizer of marine surfaces, in which a subpopulation of its biofilm dispersal cells displays a noncompetitive phenotype. This study aimed to elucidate the genetic basis of these phenotypic changes. Despite the progress made to date in characterizing the dispersal variants in P. inhibens, little is understood about the underlying genetic changes that result in the development of the specific variants. Here, P. inhibens phenotypic variation was linked to single nucleotide polymorphisms (SNPs), in particular in genes affecting the competitive ability of P. inhibens, including genes related to the production of the antibiotic tropodithietic acid (TDA) and bacterial cell-cell communication (e.g., quorum sensing). This work is significant as it reveals how the biofilm lifestyle might shape genome evolution in a cosmopolitan bacterium.

Sigillins from Springtails Are Potent Natural Insecticides.

Sigillins are highly chlorinated natural products from the springtail Ceratophysella sigillata (Collembola) that are used to deter arthropod predators. We report here the isolation and structure elucidation of sigillin F, a hydrogenated benzopyranone compound bearing two trichloromethyl groups, and the synthesis of trideoxysigillin (8), a non-natural compound representing the basic scaffold of the sigillins. Sigillins A and F showed insecticidal activity toward various insects, similar to the commercial insecticide imidacloprid, whereas 8 was inactive. The highest mortality was observed for the aphids Megoura viciae and Myzus persicae, but other insect species were also susceptible. Sigillins act as noncompetitive antagonists of the GABA receptor. This mode of action is identical to that of known insecticides with high chlorine content such as dieldrin or endosulfan. The high content of sigillins in C. sigillata, more than 4 mM in concentration, indicates self-resistance. Strikingly, the Collembola and humans have both arrived at the same target with related types of compounds to combat insects.

How to fight multiple enemies: target-specific chemical defences in an aposematic moth.

Animals have evolved different defensive strategies to survive predation, among which chemical defences are particularly widespread and diverse. Here we investigate the function of chemical defence diversity, hypothesizing that such diversity has evolved as a response to multiple enemies. The aposematic wood tiger moth (Arctia plantaginis) displays conspicuous hindwing coloration and secretes distinct defensive fluids from its thoracic glands and abdomen. We presented the two defensive fluids from laboratory-reared moths to two biologically relevant predators, birds and ants, and measured their reaction in controlled bioassays (no information on colour was provided). We found that defensive fluids are target-specific: thoracic fluids, and particularly 2-sec-butyl-3-methoxypyrazine, which they contain, deterred birds, but caused no aversive response in ants. By contrast, abdominal fluids were particularly deterrent to ants, while birds did not find them repellent. Our study, to our knowledge, is the first to show evidence of a single species producing separate chemical defences targeted to different predator types, highlighting the importance of taking into account complex predator communities in studies on the evolution of prey defence diversity.

Metabolism of 2,3-dihydroxypropane-1-sulfonate by marine bacteria.

Both enantiomers of the sulfoquinovose breakdown product 2,3-dihydroxypropane-1-sulfonate, an important sulfur metabolite produced by marine algae, were synthesised in a 34S-labelled form and used in feeding experiments with marine bacteria. The labelling was efficiently incorporated into the sulfur-containing antibiotic tropodithietic acid and sulfur volatiles by the algal symbiont Phaeobacter inhibens, but not into sulfur volatiles released by marine bacteria associated with crustaceans. The ecological implications and the relevance of these findings for the global sulfur cycle are discussed.

Sigillin†A, a unique polychlorinated arthropod deterrent from the snow flea Ceratophysella sigillata.

The snow flea Ceratophysella sigillata, a winter-active species of springtail, produces unique polychlorinated octahydroisocoumarins to repel predators. The structure of the major compound, sigillin A, was elucidated through isolation, spectroscopic analysis, and X-ray crystallography. Sigillin A showed high repellent activity in a bioassay with predatory ants. A promising approach for the total synthesis of members of this new class of natural compounds was also developed.

Streptopyridines, volatile pyridine alkaloids produced by Streptomyces sp. FORM5.

Streptomyces sp. FORM5 is a bacterium that is known to produce the antibiotic streptazolin and related compounds. We investigated the strain for the production of volatiles using the CLSA (closed-loop stripping analysis) method. Liquid and agar plate cultures revealed the formation of new 2-alkylpyridines (streptopyridines), structurally closely related to the already known 2-pentadienylpiperidines. The structures of the streptopyridines A to E were confirmed by total synthesis. The analysis of the liquid phase by solvent extraction or extraction with an Oasis adsorbent showed that streptazolin and 2-pentadienylpiperidine are the major compounds, while the streptopyridines are only minor components. In the gas phase, only the streptopyridines could be detected. Therefore, an orthogonal set of analysis is needed to assess the metabolic profile of bacteria, because volatile compounds are obviously overlooked by traditional analytical methods. The streptopyridines are strain specific volatiles that are accompanied by a broad range of headspace constituents that occur in many actinomycetes. Volatiles might be of ecological importance for the producing organism, and, as biosynthetic intermediates or shunt products, they can be useful as indicators of antibiotic production in a bacterium.

Nickel-Catalyzed [2+2+2] Cycloaddition of Diynes and Cyanamides.

A variety of bicyclic N,N-disubstituted 2-aminopyridines have been prepared from diynes and cyanamides by nickel-catalyzed [2+2+2] cycloaddition reactions. The reactions proceeded at room temperature with low catalyst loading to afford 2-aminopyridines in good to excellent yields. The method is amenable to both internal and terminal diynes and proceeds in a regioselective manner. A number of cyanamides with diverse functional group tolerance were used. The intermolecular version employing 3-hexyne and N-cyanopyrrolidine also afforded the desired N,N-disubstituted 2-aminopyridine in good yield.

Global Response of Phaeobacter inhibens DSM 17395 to Deletion of Its 262-kb Chromid Encoding Antibiotic Synthesis.

The marine alphaproteobacterium Phaeobacter inhibens DSM 17395, a member of the Roseobacter group, was recently shown to markedly enhance growth upon deletion of its 262-kb chromid encoding biosynthesis of tropodithietic acid (TDA). To scrutinize the metabolic/regulatory adaptations that underlie enhanced growth of the Δ262 mutant, its transcriptome and proteome compared to the wild type were investigated in process-controlled bioreactors with Casamino Acids as growth substrate. Genome resequencing revealed only few additional genetic changes (a heterogenic insertion, prophage activation, and several point mutations) between wild type and Δ262 mutant, albeit with no conceivable effect on the studied growth physiology. The abundances of the vast majority of transcripts and proteins involved in the catabolic network for complete substrate oxidation to CO2 were found to be unchanged, suggesting that the enhanced amino acid utilization of the Δ262 mutant did not require elevated synthesis of most enzymes of the catabolic network. Similarly, constituents of genetic information processing and cellular processes remained mostly unchanged. In contrast, 426 genes displayed differential expression, of which 410 were localized on the 3.2-Mb chromosome, 5 on the 65-kb chromid, and 11 on the 78-kb chromid. Notably, the branched-chain amino transferase IlvE acting on rapidly utilized Val, Ile, and Leu was upregulated. Moreover, the transportome was reconfigured, as evidenced from increased abundances of transcripts and proteins of several uptake systems for amino acids and inorganic nutrients (e.g., phosphate). Some components of the respiratory chain were also upregulated, which correlates with the higher respiration rates of the Δ262 mutant. Furthermore, chromosomally encoded transcripts and proteins that are peripherally related to TDA biosynthesis (e.g., the serine acyl transferase CysE) were strongly downregulated in the Δ262 mutant. Taken together, these observations reflect adaptations to enhanced growth as well as the functional interconnectivity of the replicons of P. inhibens DSM 17395.

Causes and Consequences of a Variant Strain of Phaeobacter inhibens With Reduced Competition.

Phaeobacter inhibens 2.10 is an effective biofilm former and colonizer of marine surfaces and has the ability to outcompete other microbiota. During biofilm dispersal P. inhibens 2.10 produces heritable phenotypic variants, including those that have a reduced ability to inhibit the co-occurring bacterium Pseudoalteromonas tunicata. However, the genetic changes that underpin the phenotypic variation and what the ecological consequences are for variants within the population are unclear. To answer these questions we sequenced the genomes of strain NCV12a1, a biofilm variant of P. inhibens 2.10 with reduced inhibitory activity and the P. inhibens 2.10 WT parental strain. Genome wide analysis revealed point mutations in genes involved in synthesis of the antibacterial compound tropodithietic acid (TDA) and indirectly in extracellular polymeric substances (EPS) production. However, confocal laser scanning microscopy analyses found little differences in biofilm growth between P. inhibens 2.10 WT (parental) and NCV12a1. P. inhibens NCV12a1 was also not outcompeted in co-cultured biofilms with P. tunicata, despite its reduced inhibitory activity, rather these biofilms were thicker than those produced when the WT strain was co-cultured with P. tunicata. Notably, dispersal populations from biofilms of P. inhibens NCV12a1 had a higher proportion of WT-like morphotypes when co-cultured with P. tunicata. These observations may explain why the otherwise non-inhibiting variant persists in the presence of a natural competitor, adding to our understanding of the relative importance of genetic diversification in microbial biofilms.

Non-Redfield, nutrient synergy and flexible internal elemental stoichiometry in a marine bacterium.

The stoichiometric constraints of algal growth are well understood, whereas there is less knowledge for heterotrophic bacterioplankton. Growth of the marine bacterium Phaeobacter inhibens DSM 17395, belonging to the globally distributed Roseobacter group, was studied across a wide concentration range of NH4+ and PO43-. The unique dataset covers 415 different concentration pairs, corresponding to 207 different molar N:P ratios (from 10-2 to 105). Maximal growth (by growth rate and biomass yield) was observed within a restricted concentration range at N:P ratios (∼50-120) markedly above Redfield. Experimentally determined growth parameters deviated to a large part from model predictions based on Liebig's law of the minimum, thus implicating synergistic co-limitation due to biochemical dependence of resources. Internal elemental ratios of P. inhibens varied with external nutrient supply within physiological constraints, thus adding to the growing evidence that aquatic bacteria can be flexible in their internal elemental composition. Taken together, the findings reported here revealed that P. inhibens is well adapted to fluctuating availability of inorganic N and P, expected to occur in its natural habitat (e.g. colonized algae, coastal areas). Moreover, this study suggests that elemental variability in bacterioplankton needs to be considered in the ecological stoichiometry of the oceans.