High Spatial Resolution Imaging Mass Spectrometry Reveals Chemical Heterogeneity Across Bacterial Microcolonies

Abstract

Microbes interact with the world around them at the chemical level. However, directly examining the chemical exchange between microbes and microbes and their environment, at ecological scales, i.e., the scale of a single bacterial cell or small groups of cells, remains a key challenge. Here we address this obstacle by presenting a methodology that enables matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) of bacterial microcolonies. By combining optimized sample preparation with subatmospheric pressure MALDI, we demonstrate that chemical output from groups of as few as ∼50 cells can be visualized with MALDI-IMS. Application of this methodology to Bacillus subtilis and Streptomyces coelicolor revealed heterogeneity in chemical output across microcolonies and asymmetrical metabolite production when cells grew within physiological gradients produced by Medicago sativa roots. Taken together, these results indicate that MALDI-IMS can readily visualize metabolites made by very small assemblages of bacterial cells and that even these small groups of cells can differentially produce metabolites in response to local chemical gradients.

Multiple lineages of Streptomyces produce antimicrobials within passalid beetle galleries across eastern North America

“…A key obstacle in the study of the ecology of specialized metabolites is the detection of these compounds in situ . Historically, this detection has been challenging for multiple reasons: i) specialized metabolites exist at relatively low concentrations within complex chemical environments (Grenni et al, 2018; Kellner & Dettner, 1995; Schoenian et al, 2011); ii) their production probably occurs in a dynamic spatio-temporal manner (Debois et al, 2014; Pessotti et al, 2019); iii) additional factors like light, temperature, pH might alter their chemical structures and stabilities (Boreen et al, 2004; Cycoń et al, 2019; Edhlund et al, 2006; Gothwal & Shashidhar, 2015; Mitchell et al, 2014; Thiele-Bruhn & Peters, 2007); and iv) some compounds are likely degraded by surrounding microbes (Barra Caracciolo et al, 2015; Gothwal & Shashidhar, 2015; Grenni et al, 2018). For these reasons, knowledge of when and where microbes produce specialized metabolites in natural settings is extremely limited.…”

Cooperation, Competition, and Specialized Metabolism in a Simplified Root Nodule Microbiome

“…Samples were thawed at room temperature inside a desiccator under vacuum (0.6 MPa) for 30 to 45 min. Micrographs of the root nodule slices were acquired using a Zeiss microscope (Zeiss Axio Zoom v.16 equipped with AxioCam 506 color camera), and a Super-DHB MALDI matrix (Sigma-Aldrich) was deposited on the top of the samples using a sublimation method described previously by Pessotti et al (38) to achieve a spatial resolution of 10 m. MALDI-IMS was performed in positive mode using a SubAP/MALDI (nanogram) source (MassTech, Columbia, MD) coupled to a Thermo Q-Exactive HRMS. Full MS1 scans were acquired in positive mode at a resolution of 35,000 FWHM (full width at half-maximum), a mass range of m/z 100 to 2,000, an AGC target of 1 ϫ 10 6 ions, and a maximum IT of 400 ms.…”

“…Members of the genus Pseudomonas and the order Rhizobiales are notable exceptions, as genetic approaches have been used in these organisms to demonstrate the effect of specialized metabolites which inhibited fungal pathogens ( 33 , 34 ) and mediated microbe/host plant communication ( 35 , 36 ), respectively. From a chemical perspective, our knowledge of specialized metabolism in planta is much more limited, with only a few reports of demonstrations of detection of antimicrobials in planta ( 37 – 40 ). Thus, while specialized metabolisms appear to be widespread in plant microbiomes, many questions remain regarding when and where these molecules are produced in planta and what their impact may be within these microbial communities.…”

Direct Imaging of Plant Metabolites in the Rhizosphere Using Laser Desorption Ionization Ultra-High Resolution Mass Spectrometry

“…Molecular MSI via (MA)LDI-MS has been demonstrated for isolated roots ( Jun et al, 2010 ; Peukert et al, 2012 ; Hölscher et al, 2014 ; Rudolph-Mohr et al, 2015 ; Sarabia et al, 2018 ; Korenblum et al, 2020 ; Veličković et al, 2020a ; Döll et al, 2021 ), root nodules ( Ye et al, 2013 ), the ginger rhizome ( Harada et al, 2009 ), and bacterial micro-colonies on the root surface ( Debois et al, 2014 ; Pessotti et al, 2019 ). Derivatization reactions can be used to increase the sensitivity for the detection of metabolites in maize roots during the MSI ( Dueñas et al, 2019 ; O’Neill and Lee, 2020 ).…”

“…Compared with secondary ion mass spectrometry (SIMS), MALDI and laser desorption ionization (LDI) are considered soft ionization techniques and allow for a high spatial resolution ranging from 10 μm ( Harada et al, 2009 ; Hölscher et al, 2014 ; Pessotti et al, 2019 ) to 50 μm ( Rudolph-Mohr et al, 2015 ; Korenblum et al, 2020 ). This spatial resolution would allow the detection of metabolite gradients in the rhizosphere covering a few hundred micrometers ( Holz et al, 2019 ; Bilyera et al, 2021 ) up to several mm ( Schenck zu Schweinsberg-Mickan et al, 2010 ; Holz et al, 2018b ).…”