Multimodal Molecular\nImaging Reveals a Novel Membrane\nComponent in Sporangia of the Rare Actinomycete Actinoplanes\nmissouriensis

The bacterium Actinoplanes missouriensis belongs to the genus Actinoplanes, a prolific source of useful natural\nproducts. This microbe forms\nglobular structures called sporangia, which contain many dormant spores.\nRecent studies using transmission electron microscopy have shown that\nthe A. missouriensis sporangium membrane\nhas an unprecedented three-layer structure, but its molecular components\nremain unclear. Here, we present multimodal (spontaneous Raman scattering,\ncoherent anti-Stokes Raman scattering, second harmonic generation,\nsum frequency generation, and third-order sum frequency generation)\nlabel-free molecular imaging of intact A. missouriensis sporangia. Spontaneous Raman imaging assisted with multivariate\ncurve resolution–alternating least-squares analysis revealed\na novel component in the sporangium membrane that exhibits unique\nRaman bands at 1550 and 1615 cm<sup>–1</sup> in addition to\nthose characteristic of lipids. A plausible candidate for this component\nis an unsaturated carbonyl compound with an aliphatic moiety derived\nfrom fatty acid. Furthermore, second harmonic generation imaging revealed\nthat a layer(s) of the sporangium membrane containing this unknown\ncomponent has an ordered, noncentrosymmetric structure like fibrillar\nproteins and amylopectin. Our results suggest that the sporangium\nmembrane is a new type of biological membrane, not only in terms of\narchitecture but also in terms of components. We demonstrate that\nmultimodal molecular imaging with Raman scattering as the core technology\nwill provide a promising platform for interrogating the chemical components,\nwhether known or unknown, of diverse biological structures produced\nby microbes.