A bacterial source for mollusk pyrone polyketides

Abstract

In the oceans, secondary metabolites often protect otherwise poorly defended invertebrates, such as shell-less mollusks, from predation. The origins of these metabolites are largely unknown, but many of them are thought to be made by symbiotic bacteria. In contrast, mollusks with thick shells and toxic venoms are thought to lack these secondary metabolites because of reduced defensive needs. Here, we show that heavily defended cone snails also occasionally contain abundant secondary metabolites, γ-pyrones known as nocapyrones, which are synthesized by symbiotic bacteria. The bacteria, Nocardiopsis alba CR167, are related to widespread actinomycetes that we propose to be casual symbionts of invertebrates on land and in the sea. The natural roles of nocapyrones are unknown, but they are active in neurological assays, revealing that mollusks with external shells are an overlooked source of secondary metabolite diversity.

Figure 1. Actinobacteria in C. rolani

A) Live sample of C. rolani used in this study. B) Cultivated N. alba CR167 from C. rolani.

Figure 2

Structures of nocapyrones H-Q (1–10) and A-C (11–13).

Figure 3. Organization of the nocapyrone biosynthetic gene clusters and model for nocapyrone biosynthesis

KS, β-keto acyl synthase; AT, acyl transferase; ACP, acyl carrier protein; mMCoA, methyl malonyl CoA; MT, methyltransferase; Ox, oxidoreductase. This scheme shows the hypothetical biogenesis (see text).

Figure 4. NcpB is the nocapyrone O-methyltransferase

HPLC traces are shown with detection at 228 nm. A) Standards of pure 9 and 9a. B) Enzyme assay containing 9a, NcpB, and SAM. C) Control enzyme assay lacking SAM. D) Control enzyme assay with boiled NcpB. E)–G) Other pyrone substrates were not accepted by the enzyme, using the same reaction conditions as shown in panel B.

Figure 5. Genes for nocapyrone synthesis are found in cone snail tissue

The N. alba CR167 16S gene, ncpB and ncpC were amplified from both C. rolani and C. tribblei metagenome DNA by nested PCR experiments. The PCR products were confirmed to be identical to the positive controls by Sanger sequencing the gel-extracted PCR products. A) Primers noc16s_Fwd and noc16s_Rev. B) Primers MT_in_Fwd and MT_in_Rev. C) Primers PKS_in_Fwd and PKS_in_Rev. Template DNA: 1) C. tribblei; 2) C. rolani; 3) N. alba CR167. 4) no DNA control; 0) DNA ladder.

Figure 6. Activity of compound 12 observed by calcium imaging of dissociated DRG neurons in culture

Each trace is the response of a single neuron. Responses from ~100 neurons were monitored individually and simultaneously in a given experimental trial. Selected traces are shown from a single experimental trial. The Y-axis is a measure of relative intracellular (cytoplasmic) calcium concentration, [Ca²⁺]_i, obtained by standard ratiometric calcium-imaging techniques (i.e. ratio of 340 nm/380 nm excitation while monitoring fluorescence emission at 510 nm). The X-axis is time in minutes. Increases in [Ca²⁺]_i were elicited by briefly depolarizing the neurons with 25 mM potassium (KCl pulse) at regular seven-minute intervals, as indicated by vertical arrows. Each KCl pulse elicited an increase in [Ca²⁺]_i (by activating voltage-gated calcium channels) that is observed as a peak in each trace. Capsaicin (300 nM) was applied to the neurons for one minute at the end of the experiment, as indicated by the black circle. After the fourth KCl pulse, the compound was applied to the cells for six minutes (horizontal bar). In many small-diameter, capsaicin-sensitive DRG neurons, the compound caused an amplification of the calcium-transient elicited by a KCl pulse (bottom three traces) or directly elicited a calcium-transient (fourth trace from the bottom). In contrast, in many large-diameter, capsaicin-resistant neurons, the compound partially blocked the calcium-transient elicited by a KCl pulse (top four traces). All effects of the compound were reversible, as shown.