Multimodal Molecular Imaging Reveals a Novel Membrane Component in Sporangia of the Rare Actinomycete <i>Actinoplanes missouriensis</i>

The bacterium <i>Actinoplanes missouriensis</i> belongs to the genus <i>Actinoplanes</i>, a prolific source of useful natural products. This microbe forms globular structures called sporangia, which contain many dormant spores. Recent studies using transmission electron microscopy have shown that the <i>A. missouriensis</i> sporangium membrane has an unprecedented three-layer structure, but its molecular components remain unclear. Here, we present multimodal (spontaneous Raman scattering, coherent anti-Stokes Raman scattering, second harmonic generation, sum frequency generation, and third-order sum frequency generation) label-free molecular imaging of intact <i>A. missouriensis</i> sporangia. Spontaneous Raman imaging assisted with multivariate curve resolution-alternating least-squares analysis revealed a novel component in the sporangium membrane that exhibits unique Raman bands at 1550 and 1615 cm^-1 in addition to those characteristic of lipids. A plausible candidate for this component is an unsaturated carbonyl compound with an aliphatic moiety derived from fatty acid. Furthermore, second harmonic generation imaging revealed that a layer(s) of the sporangium membrane containing this unknown component has an ordered, noncentrosymmetric structure like fibrillar proteins and amylopectin. Our results suggest that the sporangium membrane is a new type of biological membrane, not only in terms of architecture but also in terms of components. We demonstrate that multimodal molecular imaging with Raman scattering as the core technology will provide a promising platform for interrogating the chemical components, whether known or unknown, of diverse biological structures produced by microbes.