Interaction between microorganisms and higher organisms: MALDI-Imaging reveals secondary metabolites

Over the recent years MALDI-Imaging analysis has gained high interest and the ever increasing number of publication demonstrates its wide applicability.   The aim of this study is to employ MALDI-FTMS Imaging to gain insight into interactions between microorganisms and higher organisms. The infection of potatoes with phytopathogenic bacteria of the Streptomyces genus is the cause for substantial loss in crop harvest. Thus the understanding of this interaction might lead to more efficient treatments to protect the crop. Using MALDI-FTMS imaging, any compound of interest can be assigned to an unique elemental composition by making use of the intrinsic mass accuracy of FTMS, its high resolution and the capability to use isotopic fine structures.   
 
Methods: Potato tuber sterilized in hot water, 70 % ethanol, 0.05 % citric acid. Inoculation in Petri dishes with perlite and 200 ?l mycelium. After 5 days the tuber was cut using a cryotome, slices were mounted on ITO slides (Bruker Daltonics). Matrix applicaton by vibrational nebulizing device (ImagePrep, Bruker Daltonics). MALDI-MS: HCCA coated slides were loaded into a 12T FTMS (solariX, Bruker Daltonics). MALDI images: Pixel size 50 ?m. For each pixel a single scan was recorded consisting of  200 laser shots (rep. rate 1 KHz). For each scan, an 8 MWord spectrum was acquired (m/z range 130 ? 1500), up to 18.000 pixels per given imaging experiment. External calibration: Arginine clusters (electrospray mode). Automatic lock mass calibration: Matrix peak ([2M+H]+).

Preliminary Data: Here we present results from an incubation experiment of a Streptomyces bottropensis on slices of potato tubers. During the analysis of the imaging data mass peaks were detected which were not seen in prior LC-MS/MS experiments conducted using extracts of similar inoculation experiments. These peaks gave rise to secondary metabolites emerging from S. bottropensis under these cultivation conditions. By using the high mass accuracy and mass resolving power of the FTMS we could unambiguously identify the elemental compositions of these compounds directly from the plant tissue. Key to this data assignment is the possibility to make use of the Isotopic Fine Structure (IFS), which limits the number of putative elemental compositions substantially. The series of compounds is detected as protonated species and potassium adducts which are both used for the  assignment of the elemental compositions. The derived compostions (C27H48N6On; 5<n<10) suggest that these metabolites are siderophores which enable the microorganism to sustain the physiological Fe-concentration. Comparing the distributions through a vertical slice of a potato the newly detected compounds are co-located with known Iromycins. The mass measurement accuracy was well below 1 ppm.