Mass Spectrometry Uncovers the Role of Surfactin as an Interspecies

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Mass spectrometry uncovers the role of surfactin as an interspecies recruitment factor Tal Luzzatto-Knaan, Alexey V. Melnik, and Pieter C. Dorrestein ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b01120 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 15, 2019

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Mass spectrometry uncovers the role of surfactin as an interspecies

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recruitment factor

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Tal Luzzatto-Knaan1*, Alexey V. Melnik1 and Pieter C. Dorrestein1*

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1Collaborative

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Pharmaceutical Sciences, University of California San Diego, CA 92093, USA.

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*Address correspondence to Tal Luzzatto-Knaan, [email protected] or Pieter

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Dorrestein, [email protected]

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Keywords: Mass spectrometry, Bacillus subtilis, Surfactin, Paenibacillus dendritiformis,

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Microbial ecology

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Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and

ABSTRACT Microbes use metabolic exchange to sense and respond to their changing environment.

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Surfactins, produced by Bacillus subtilis are extensively studied for its attributed role in biofilm

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formation, biosurfactant properties and antimicrobial activity, affecting the surrounding

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microbial consortia. Using mass spectrometry, we reveal that Paenibacillus dendritiformis,

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originally isolated with B. subtilis, is not antagonized by the presence of surfactins, and is

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actually attracted to it. We demonstrate here for the first time, that P. dendritiformis is also

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actively degrading surfactins produced by B. subtilis, and accumulating the degradation products

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that serve as territorial marker. This new attribute as an attractant of selected microbes and the

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conversion it into a deterrent, highlights the diverse role natural products have in shaping the

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environment and establishing mixed communities.

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INTRODUCTION Microbes often must respond and react to neighboring organisms. For this reason,

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microbes have evolved to produce niche specific chemistries to interact with their ecological

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environment such as soil, ocean water, or hosts such as plants, animal or even human cells 1.

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Humans have taken advantage of these molecules produced by microbes. They form our

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treatments for excessive iron accumulation (e.g. desferrioxamine) 2, prevent organs from being

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rejected during transplants (e.g. rapamycin) 3, we use them as antibiotics (e.g. erythromycin) 4, to

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treat insect pests of crops (e.g. bialaphos) 5, and as industrial biosurfactant (e.g. surfactin) 6 Such

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potent bioactivities have revolutionized medicine, biotechnology and agriculture 7, 8. Therefore,

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exploring the chemistry behind microbial interactions are of great interest. However, it is clear

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that these molecules, in the context of their ecological niche, must serve different and diverse

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functions. Such molecules are produced as a way to interact and influence the behavior and

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phenotype of self and neighboring cells or create an environmental niche favorable for

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supporting their life cycle 9. This metabolic exchange, the release of chemical cues and active

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natural products, may vary based on specific interactions 1.

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In this study, we focused on the chemical interplay between two Firmicutes by advanced

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mass spectrometry techniques, in order to unravel the diverse functionality of molecules in a

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defined ecological niche (Supplemental table S1). Bacillus subtilis, a well-studied and wide

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spared microbe, is also the eminent producer of surfactins 10. Surfactins are cyclic lipopeptides

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attributed with the natural functions in antagonism towards other organisms, motility on

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semisolid surfaces, attachment to surfaces and triggering biofilm formation 11-15. The other

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organism is the pattern forming Peanibacillus dendritiformis that considered to be highly

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responsive to small changes in the environment, which are expressed by visible changes in its 2 ACS Paragon Plus Environment


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social motility, known as swarming patterns 16. As a single colony, P. dendritiformis forms radial

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dendritic swarming patterns on solid surfaces 17-20. This extraordinary swarming capability

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makes P. dendritiformis an ideal model for characterizing interactions with its surrounding.

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Though some of these patterns have been widely described 16, 18, only little is known about the

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chemistry affecting its motility 17.

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Both species are ubiquitous and are commonly found in soil, plants and rhizosphere,

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insects, and many other environments 21. In fact, P. dendritiformis was initially isolated with B.

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subtilis and classified as such - meaning they are naturally interacting 22. Here we demonstrate a

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new interpretation for the chemical “give and take” between these two co-occurring microbes.

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RESULTS AND DISCUSSION

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P. dendritiformis is chemo-attracted to B. subtillis

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We observed that when P. dendritiformis is co-inoculated with Bacillus subtilis NCIB

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3610 at various distances (three colonies of each microbe at 1, 2, 3 cm apart), a distinct change in

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the swarming pattern of P. dendritiformis occurs. The first P. dendritiformis colony that senses

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the presence of B. subtilis, changed its swarming phenotype towards and around the B. subtilis

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colony forming tight dendrites (Figure 1A). In this case, the unusual phenotype was only

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detected for the closest P. dendritiformis colonies (1 cm apart) while the more distant colonies,

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that were 2 and 3 cm apart were halted, and did not exhibit this phenotype. Not only attracted

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and accelerated movement of P. dendritiformis around the adjacent colony of B. subtilis, the

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same P. dendritiformis colony enclosed the more distant B. subtilis colonies, preventing

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additional P. dendritiformis sibling colonies to engage in interaction (Fig 1A). Interactions

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among kin-species were recently reported, relating the various phenotypes to phylogeny 23. 3 ACS Paragon Plus Environment

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While B. subtilis sibling colony swarms merge, other non-kin strains form distinct boundaries

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between swarms often affected by the release of antimicrobial compounds 23. Conversely, kin-

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recognition has been investigated for P. dendritiformis in great detail and this kin-inhibition, as

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we see here by halting the more distant sibling colonies (Fig 1A), is attributed to the production

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of a sibling lethal factor that prevents swarms from merging and restricting colonial territory 24,

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25.

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response to non-kin-neighboring microbial interactions 23-25. Previous work with Paenibacillus

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vortex suggested airborne substances as inducers of motility, benefiting the non-motile microbe

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Xanthomonas perforans 26. However, the increased motility phenotype of P. dendritiformis was

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not a result of volatile molecules, as co-inoculation on partitioned plates cultured under identical

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conditions presented no such swarming (Fig 1B). This indicated that the attractive chemical

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signal is more likely transferring through the media.

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Figure 1: Paenibacillus dendritiformis (Pd) and Bacillus subtilis NCIB 3610 (Bs3610) grown in

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co-culture. (A) Colonies inoculated at 1,2,3 cm apart on peptone media and (B) on a partitioned

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plate.

Yet, the attraction of P. dendritiformis as shown here, has not been reported before in

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Targeting chemo-attractive molecules by mass spectrometry To understand the nature of the attraction of P. dendritiformis towards B. subtilis at the

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chemical level, we employed orthogonal mass spectrometry-based methods, imaging mass

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spectrometry (IMS) and molecular networking (Figure 2) 8. To visualize the molecules that are

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present in the agar, the co-culture was transferred onto a matrix assisted laser

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desorption/ionization (MALDI) target plate, covered with an organic matrix to assist in the

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desorption and ionization of the molecules and subjected to MALDI-IMS. Imaging mass

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spectrometry enabled the visualization of the spatial distribution of molecules associated with

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this interaction. In IMS, the selected parent mass ions of the molecules are displayed by pseudo-

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color, based on their intensity in each pixel creating a spatial image 27, 28. Optical images are then

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overlaid by such ion image distributions to understand the relationships between molecules with

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respect to the observed phenotypes. The IMS experiments also included the following controls;

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growth medium alone, culture of P. dendritiformis, two adjacent sibling cultures of P.

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dendritiformis, and B. subtilis alone (Figure 2A). The interaction and controls were all placed on

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the same target plate so that the relative intensities can be directly compared. Representative

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signals not found in the background agar are shown in Figure 2B. Some signals were observed in

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only one of the mono-cultures, while others were found only when interacting. Of these m/z

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248.5 and 659.5 were only detected on the P. dendritiformis colonies themselves and decreased

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or even disappeared to below detectable levels when P. dendritiformis was co-cultured adjacent

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to B. subtilis. The signal at m/z 427.6, although present in the background media, was also

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observed to increase around the colony of P. dendritiformis, while m/z 615.8 was B. subtilis

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colony associated and m/z 714.2 was observed most intensely at the outer edges of the B. subtilis

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colony. The m/z 1058.9 was associated with the B. subtilis colony as well as secreted into the 5 ACS Paragon Plus Environment

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agar but depleted as this signal gets closer to P. dendritiformis. At the same time there were

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several signals observed only in the interaction and not in any of the controls. Examples include

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m/z 622.4, and 658.4. The ions at m/z 615.8, 714.2 and 1058.9 are consistent with polyglutamates

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and the surfactin, previously detected in B. subtilis by imaging mass spectrometry 29. None of the

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other ions had been previously investigated.

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Figure 2: Bacillus subtilis NCIB 3610 (Bs3610) and Paenibacillus dendritiformis (Pd)

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interactions investigated by mass spectrometry-based approach combining IMS and tandem MS. 6 ACS Paragon Plus Environment


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(A) Experimental setup of interactions includes: media blank, Pd single colony and Pd vs. Pd

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sibling interaction, Bs3610 single colony and Bs3610 vs. Pd co-culture interaction. (B) Matrix

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assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) demonstrating the

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spatial distribution of selected ions of Pd and Bs3610 in mono and co-culture. (C) Tandem mass

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spectrometry of corresponding samples analyzed by GNPS molecular networking. Node fill

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color represents the sample origin: Red= Bs3610, Blue/Cyan= Pd, Pd vs. Pd, Purple= Bs3610 vs.

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Pd, Gray> 2 groups.

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Surfactin and associated products are involved in P. dendritiformis attraction to B.

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subtilis

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Because parent masses provide little structural insight, microbial IMS was complemented

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with liquid chromatography based untargeted tandem mass spectrometry (LC-MS/MS). For this

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experiment, the corresponding interaction and controls (growth medium alone, P. dendritiformis

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and B. subtilis) were extracted with EtOAc/MeOH (1:1) and subjected to LC-MS/MS analysis.

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During the elution, the ions were subjected to fragmentation in a data dependent fashion. The

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untargeted mass spectrometry was followed by molecular networking in the crowd-sourced

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analysis infrastructure called Global Natural Products Social (GNPS) molecular networking

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(Figure 2C) 30. Molecular networking clusters the spectral relationships of the fragmented ions

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that were detected by mass spectrometry from which one can infer the structural relationships of

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the molecules and enables direct dereplication against public reference spectra 8, 30-32. We set out

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to inspect the molecular families -a cluster of related spectra- in this molecular network in more

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detail (Figure 3). For most ions, no spatial information was available due to the different


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ionization methods of MALDI and electron spray ionization (ESI). However, one of the families

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was linked to two different surfactins (m/z 1022.6767, 1036.6944, corresponding to C14, C15,

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[M+H]+ surfactins) (Figure 3A-C), based on manual inspection of all the neighboring nodes, for

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which no spectral references were matched. These nodes were found to be related by CH2

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additions or deletions, resulting from substitutions seen by promiscuous adenylation domain

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specificities of the surfactin synthetases, and were annotated as C13-C17 surfactins and

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derivatives (Supplemental figure S1) 9-11, 29. Spatial mapping of the surfactins observed in the B.

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subtilis control sample by IMS revealed they have the same distributions (Figure 3B). This

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molecular family also had four purple nodes (representing mainly [M+H2O+2H]+2), indicating

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that these derivatives were observed at interaction (Figure 3). The possibility is that by

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interaction, surfactin is altered in some fashion such as hydrolysis, as similarly reported before

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when B. subtilis was cultured with a Streptomyces sp. 29.

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An additional molecular family was found associated with the interaction between P.

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dendritiformis and B. subtilis. The corresponding IMS spatial distribution of m/z 608.4, 622.4,

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636.4, 650.4 ions suggest that they are related to P. dendritiformis (Figure 3D-F). Notably,

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neither one of these masses are detected in monoculture swarming and sibling colony interaction

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controls of P. dendritiformis, nor detected in the monoculture of B. subtilis. Further examination

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of the fragmentation pattern generated by tandem MS showed two putative Leu/Ile mass shifts

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and an additional fragment differing by 14Da between the four compounds, suggesting a

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lipopeptidic nature. A deeper analysis uncovered a perfect match of these structures to the

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lipopeptidic tails of surfactins produced by B. subtilis (C13, C14, C15, C16) differing by the

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variable length of the acyl chain (Figure 3F,) 11. 8 ACS Paragon Plus Environment


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Figure 3: Annotation and spatial pattern of surfactin and surfactin-like lipopeptidic tail

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molecular families in Bacillus subtilis NCIB 3610 and Paenibacillus dendritiformis interactions.

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Upper panel: (A) Molecular network of detected molecules and annotation of surfactins by

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match to the GNPS spectral library. (B) Spatial distribution of surfactin ions [M+Na]+ by

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MALDI IMS. (C) Tandem MS spectra and structure of surfactins C14, C15. Selected nodes

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highlighted in colored circle corresponding to the spatial pattern pseudo color. Lower panel: (D)

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A molecular family of molecules detected in co-culture from LC-MS/MS molecular network (E)

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Spatial distribution of selected ions (m/z 608.4,622.4, 636.4 and 650.4 [M+Na]+) by MALDI

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IMS. (F) Detected tandem MS spectra and structure of surfactins-like lipopeptidic tail. Selected

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nodes highlighted in colored circle corresponding to the spatial pattern and annotated lipid tail.

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Node fill color represents the sample origin: Red= Bs3610, Blue/Cyan= Pd, Pd vs. Pd, Purple=

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Bs3610 vs. Pd, Gray> 2 groups.

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The IMS image overlay of surfactin C15 produced by B. subtilis and the corresponding

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lipopeptide tail (m/z 636.4 [M+Na]+ ) provides a complementary image (Figure 4A) suggesting

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that these molecules are anticorrelated. The residual peptidic fragment of the breakdown m/z

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459.28 [M+H]+ that is common among the four surfactins was annotated by b and y ions of the

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MS spectrum (Figure 4B/C). The IMS spatial distribution of the peptidic product m/z 481.3

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[M+Na]+ correlated with degradation of the lipopeptidic tails (Supplemental figure S2). The

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overlap of the surfactin degradation products with the region of increased motility led us to ask

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the following two questions: First, is surfactin involved in the increased motility and the

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attraction of P. dendritiformis when it is adjacent to B. subtilis? and secondly, can P.

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dendritiformis directly degrade surfactin? 10 ACS Paragon Plus Environment


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Figure 4: Complementary analysis of surfactin C15 degradation products. (A) IMS

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complementary image of surfactin C15 as produced by Bacillus subtilis and its lipopeptidic m/z

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636.42 [M+Na]+ and peptidic m/z 459.28 [M+H]+ degradation products. (B) Extracted ion

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chromatogram of the four lipopeptidic tails and the common peptidic product. (C) Annotation of

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the peptidic product by b and y ions.

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Surfactin act as an attractant and is modified by P.dendritiformis To test the hypothesis that surfactins are involved in the increased motility phenotype, we

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examined the swarming response of P. dendritiformis towards the wild type strain B. subtilis

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NCIB 3610 versus the mutated B. subtilis NCIB 3610 strain that lacks surfactin synthetase

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capabilities (∆srfAA). ∆srfAA is deficient in the first loading domain of the biosynthetic pathway

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of surfactins disabling the production of all structurally related surfactins 28, 33. Our results reveal

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an attraction of P. dendritiformis to the wild type Bs3610 and no attraction to the non-producing

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mutant (Figure 5A, Supplemental figure S3A, B). These results are consistent with the

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interpretation that surfactin is a key molecule required for this phenotype. However, mutation

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may result in additional effects on other molecules that are present. Therefore, we set out to

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determine if surfactin on its own is able to induce the motility behavior in P. dendritiformis. A

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disc diffusion assay with commercially available B. subtilis surfactin C15 (Sigma-Aldrich CAS#

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24730-31-2, 0.01mg used based on previous reports for effective dose 17, 34, 35) and a dose-

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response assay (Supplemental figure S3C-E), recapitulated the P. dendritiformis’s motility

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phenotype (Figure 5B,C). To verify that P. dendritiformis was responsible for the degradation of

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surfactin, we extracted disc diffusion assay plates and found the corresponding degradation

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products as in Figure 4 (by parent mass and MS/MS) to be present only when P. dendritiformis

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was present (Supplemental figure S4). These results support our hypothesis, confirming the

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necessity of surfactin for enhanced motility and its active degradation by P. dendritiformis.

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Figure 5: Paenibacillus dendritiformis (Pd)

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attraction to surfactin produced by Bacillus

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subtilis NCIB 3610 (Bs3610). (A) Mono and

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co-inoculation of Pd with the wild type

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Bs3610 and the surfactin deficient mutant

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(∆srfAA). (B) Disc diffusion assay of Pd with

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Bacillus subtilis commercial surfactin (Sigma-

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Aldrich CAS# 24730-31-2). (C) Co-

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inoculation of Pd with Bs3610 vs. commercial

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surfactin. Scale bar represent 5mm.

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The role of surfactin and its degradation products IMS experiments, show that the degradation products of surfactins are incorporated into

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the P. dendritiformis colony (Figure 3E) and not accumulated at the interaction zone 29 nor

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directly consumed as a carbon source 36. Therefore, we wondered how these molecules affect P.

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dendritiformis, and if they are associated with the enhanced motility as seen with the intact

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surfactin. By MS guided purification we have isolated both the lipopeptidic product (m/z

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614.4364 [M+H]+ , 636.4199 [M+Na]+ ) and the peptidic residue m/z 459.2830 [M+H]+ and

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tested the effect of these products on P. dendritiformis colonies response. The two degradation

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products had different affects on the swarming pattern of P. dendritiformis. While the peptidic

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tail showed no change in pattern similar to the negative control, the lipopeptidic tail restricted the

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growth around the applied area (Growth inhibition p value of p=0.000303 measured based on

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inhibition diameter by pairwise t-test) (Figure 6). Because we see no evidence of bactericidal or

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bacterial growth effects and see these exact molecules incorporated into the colonies, it is not

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expected to be toxic to P. dendritiformis. Initially, we suspected that these lipopeptide products

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might be assisting with swarming of P. dendritiformis, therefore, introducing these products

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would be attractive as well. However, the “pre-cut” product (i.e. the lipopeptidic tail) seem to

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lose its attractive properties following degradation and is not an attractive agent on its own and is

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not promoting swarming. Thus, it appears that modification of the attractant (surfactin) becomes

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a restricting territorial marker (lipopeptidic degradation product) for P. dendritiformis colonies.

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Figure 6: The effect of surfactin degradation products on the swarming of Paenibacillus

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dendritiformis (Pd). (A) Diffusion assay with purified degradation products of surfactin and (B)

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solvent and commercial surfactin C15 (Sigma-Aldrich CAS# 24730-31-2) as reference control.

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Assay was performed in triplicates. Swarm inhibition measured based on inhibition diameter, p

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value of p=0.000303 by pairwise t-test.

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Bacillus subtilis, though studied for decades, is still considered a treasure trove for the

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potential of its antimicrobial chemical arsenal 37. Surfactins are a great example of these vastly

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explored molecules, due to their biosurfactant and antimicrobial properties attributed to their

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ability to interact with membranes 10, 34, 38. These attributes are essential for the plant growth

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promoting rhizobacteria (PGPR) properties of B. subtilis that shape the microbial communities in

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the rhizosphere 10, 11, 39. Modification of antimicrobial agents have been described before by

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enzymatic inactivation of specific functional groups such as in β-lactamases resistance 40, 41. 15 ACS Paragon Plus Environment

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Modification of B. subtilis surfactins was also proposed as part of a defense mechanism of

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Streptomyces sp. Mg1 to detoxify the antimicrobial effect of surfactin, by secreting hydrolyzing

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enzymes, while the inactivated product was detected at the inhibition zone by IMS 29. In contrast,

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our findings show that surfactins produced by B. subtilis are the source of attraction for P.

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dendritiformis and not an antimicrobial threat, while the degradation products of surfactin are

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incorporated into the P. dendritiformis colony and not restricted to the interaction zone, as

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uncovered by IMS. Hence, one of the questions that should be asked is, what is the effect of the

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degradation of surfactins on the PGPR ability of B. subtilis? On the other hand, it was proposed

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that members of the Paenibacillus family also possess PGPR characteristics 42. Therefore, if P.

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dendritiformis is attracted to B. subtilis surfactins, that highlight their potential role in recruiting

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additional beneficial bacteria to the rhizosphere. Indeed, P. dendritiformis initially isolated with

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B. subtilis, highlighting their natural co-occurrence 22.

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The unique motility of P. dendritiformis colonies was carefully studied, concluding that

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the main factor affecting colonial growth is the presence or production of surface active

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(surfactants) molecules 17. One possible hypothesis is the exploitation of public goods, in this

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case, secreted surfactins produced by B. subtilis utilized by the non-producing P. dendritiformis

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community 23, 43. That can explain the rapid colonial growth of P. dendritiformis that does not

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pay the metabolic costs of surfactin production. In evolutionary terms, P. dendritiformis could

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create dependency on surfactin production by B. subtilis compatible with the emerging Black

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Queen Hypothesis 44. Meaning that an organism is capable of losing traits that are energetically

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expensive, yet still thrive in the compatible neighboring consortia. In that sense, it could be that

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while diverging phylogenetically from B. subtilis, P. dendritiformis had already lost the ability to

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produce surfactins (while able to produce other biosurfactants) but kept the ability to degrade and 16 ACS Paragon Plus Environment


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utilize them if provided as public goods, hence the attraction 23. It should be noted here, that

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while P. dendritiformis is enclosing B. subtilis and restricting its growth, B. subtilis is kept alive

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and vital, still producing surfactins.

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CONCLUSIONS

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Recent literature show that the plant microbiome assembly varies depending of the plant

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species and the soil conditions in what is termed “plant-soil feedbacks” (PSF) affected by the

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plants’ ability to recruit soil microbes to its rhizosphere 45. It was also suggested that addition of

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B. subtilis to tomato plant’s rhizosphere results in transient impact on the microbiome

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composition 46. Therefore, as we suggested here, some of these bacteria are able to recruit

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additional microbial species to form compatible assemblages. This behavior has vast potential

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applications in agriculture, biotechnology, and human health and are the inspiration of the

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emerging field of synthetic ecology of microbes, that predicts potential chemical interactions

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among microbes based on their genomic information 47. Predictions can be made by

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mathematical modeling to generate hypotheses that may be further studied with the various

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genomic and transcriptomic methods that are currently available. However, the application of

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spatial metabolomics, as used in this study, highlights microbial interactions that could not be

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explored by molecular approaches alone. The active degradation of surfactins as shown here, is

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an important example of metabolic exchange and the various utilities of different microbes. The

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chemical interplay shown by these two laboratory model systems is a clever strategy of one

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microbe sends out a signal to recruit other organisms to its ecological niche to establish favorable

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mixed communities. Such give and take of chemical signals among complex microbial

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communities are critical in understanding the interactions of microbial ecology and encourages

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multidisciplinary synergistic collaborations. 17 ACS Paragon Plus Environment

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METHODS

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Bacterial Strains and Media

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The strains used for this study were Paenibacillus dendritiformis, Bacillus subtilis NCIB 3610,

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∆srfAA (see Supplemental table S1). Peptone media reagents were used unless otherwise

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indicated. All chemical reagents were purchased from Sigma-Aldrich unless stated otherwise.

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All solvents used were LCMS grade. Bacteria suspension (5µl) inoculated from overnight

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cultures in LB liquid media for microbial interactions on 0.5% peptone/1% agar, incubated at

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30oC. Plates were incubated at 30oC for 24 hours before recording.

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MALDI IMS

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Samples were transferred onto a MALDI target plate and mounted with universal matrix as

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described by Yang et al. 27. Samples were analyzed by Autoflex speed MALDI ToF (Bruker

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Daltonics) instrument in 300um raster for selected regions with 55% laser and 12.0 gain. Data

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visualized using the FlexImaging software 4.1 (Bruker Daltonics) normalized to RMS.

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LCMS analysis

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Samples extracted from solid media with EtOAc /MeOH (1:1) sonicated for 10 min. Samples

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were dried out and re-suspended in MeOH. Liquid Chromatography High Resolution tandem

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Mass Spectrometry (LC-HRMS/MS): Samples were analyzed using an LC system Dionex

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UltiMate 3000 (Thermo) using a Phenomenex Kinetex C-18 column (1.7 µm, 50 × 2.1 mm) A=

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98%H2O/2%ACN/0.1%FA, B=98%ACN/2%H2O/0.1%FA in the following method:

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Chromatographic separation performed by a linear gradient: 0–1 min 5% B, 1–2 min 5–50% B,

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2–4 min 50-55% B, 4–10 min 50-100% B, 10–11.5 min 100% B, 11.5-12 min 100-5%B and 12-



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12.5 min 5%B. MS spectra were acquired in a positive ion mode on a Maxis QTOF mass

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spectrometer (Bruker Daltonics), equipped with ESI source as described by Bouslimani et al. 48.

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Molecular networking

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A molecular network was created using the online workflow at GNPS. The data was filtered by

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removing all MS/MS peaks within +/- 17 Da of the precursor m/z. MS/MS spectra were window

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filtered by choosing only the top 6 peaks in the +/- 50Da window throughout the spectrum. The

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data was then clustered with MS-Cluster with a parent mass tolerance of 1.0 Da and a MS/MS

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fragment ion tolerance of 0.5 Da to create consensus spectra. Further, consensus spectra that

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contained less than 2 spectra were discarded. A network was then created where edges were

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filtered to have a cosine score above 0.6 and more than 4 matched peaks. Further edges between

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two nodes were kept in the network if and only if each of the nodes appeared in each other's

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respective top 10 most similar nodes. The spectra in the network were then searched against

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GNPS' spectral libraries. The library spectra were filtered in the same manner as the input data.

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All matches kept between network spectra and library spectra were required to have a score

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above 0.6 and at least 4 matched peaks 30.

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Mutant assay/ Disc diffusion assay

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Five microliters of an overnight bacterial culture were inoculated as co-cultures on a

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peptone/agar media 1cm apart. For surfactin disc assay, gradual concentration of commercial

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surfactin C15 (Sigma-Aldrich CAS# 24730-31-2) was tested (in MeOH, supplemental figure

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S3C). Selected concentration of 0.01mg was plated on a Whatman paper disc. Plates were

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incubated at 30oC for 24 hours before recording.

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MS guided purification 19 ACS Paragon Plus Environment

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The isolation of degradation products (lipopeptidic tails combined / peptidic product) carried out

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on a semi-preparative column Luna C18, 5µM, 100Å, 250 x 4.6 mm (Phenomenex) with guard

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column of the same stationary phase attached with chromatographic method as mentioned above

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at a flow rate of 1 mL min-1. 30 fractions were collected. All fractions were combined and dried

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to roughly 1mL volume in a rotary evaporator then transferred to pre-weighed and dried

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microcentrifuge tubes and lyophilized to dryness. The resultant powder of purified sample was

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solubilized in MeOH at 1mg mL-1 for further experiments.

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Acknowledgments

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Authors would like to thank the Ben-Jacob Lab for kindly providing Paenibacillus

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dendritiformis strain. This work was supported by BARD, the United States - Israel Binational

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Agricultural Research and Development Fund, Vaadia-BARD Postdoctoral Fellowship Award

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no. FI-494–13.

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Competing interests

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The authors declare no competing interests.

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Supporting Information Available: This material is available free of charge via the internet

378

at http://pubs.acs.org. Files include:

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Table of strains, Annotation of surfactins, IMS of peptidic product, Mutant and disc diffusion

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assay, Degradation of commercial surfactin, Link to data on GNPS.

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Author Contributions

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TLK and PCD designed the project



383

TLK and AVM performed the research

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TLK and AVM analyzed data

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TLK, AVM and PCD wrote the paper

386

REFERENCES

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1. Phelan, V. V., Liu, W. T., Pogliano, K., and Dorrestein, P. C. (2012) Microbial metabolic exchange--the chemotype-to-phenotype link, Nat Chem Biol 8, 26-35. 2. Hider, R. C., and Kong, X. (2010) Chemistry and biology of siderophores, Nat Prod Rep 27, 637-657. 3. Schwecke, T., Aparicio, J. F., Molnar, I., Konig, A., Khaw, L. E., Haydock, S. F., Oliynyk, M., Caffrey, P., Cortes, J., and Lester, J. B. (1995) The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin, Proc Natl Acad Sci U S A 92, 7839-7843. 4. Preston, D. A. (1986) Microbiological aspects of erythromycin, Pediatr Infect Dis 5, 120-123. 5. Dayan, F. E., Cantrell, C. L., and Duke, S. O. (2009) Natural products in crop protection, Bioorg Med Chem 17, 4022-4034. 6. Peypoux, F., Bonmatin, J. M., and Wallach, J. (1999) Recent trends in the biochemistry of surfactin, Appl Microbiol Biotechnol 51, 553-563. 7. Garg, N., Luzzatto-Knaan, T., Melnik, A. V., Caraballo-Rodriguez, A. M., Floros, D. J., Petras, D., Gregor, R., Dorrestein, P. C., and Phelan, V. V. (2017) Natural products as mediators of disease, Nat Prod Rep 34, 194-219. 8. Luzzatto-Knaan, T., Melnik, A. V., and Dorrestein, P. C. (2015) Mass spectrometry tools and workflows for revealing microbial chemistry, Analyst 140, 4949-4966. 9. Tyc, O., Song, C., Dickschat, J. S., Vos, M., and Garbeva, P. (2017) The Ecological Role of Volatile and Soluble Secondary Metabolites Produced by Soil Bacteria, Trends Microbiol 25, 280-292. 10. Stein, T. (2005) Bacillus subtilis antibiotics: structures, syntheses and specific functions, Mol Microbiol 56, 845-857. 11. Raaijmakers, J. M., De Bruijn, I., Nybroe, O., and Ongena, M. (2010) Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics, FEMS Microbiol Rev 34, 1037-1062. 12. Ron, E. Z., and Rosenberg, E. (2001) Natural roles of biosurfactants, Environ Microbiol 3, 229-236. 13. Chen, Y., Cao, S., Chai, Y., Clardy, J., Kolter, R., Guo, J. H., and Losick, R. (2012) A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants, Mol Microbiol 85, 418-430. 14. Gonzalez, D. J., Haste, N. M., Hollands, A., Fleming, T. C., Hamby, M., Pogliano, K., Nizet, V., and Dorrestein, P. C. (2011) Microbial competition between Bacillus subtilis and Staphylococcus aureus monitored by imaging mass spectrometry, Microbiology 157, 2485-2492. 15. Rosenberg, G., Steinberg, N., Oppenheimer-Shaanan, Y., Olender, T., Doron, S., Ben-Ari, J., SirotaMadi, A., Bloom-Ackermann, Z., and Kolodkin-Gal, I. (2016) Not so simple, not so subtle: the interspecies competition between Bacillus simplex and Bacillus subtilis and its impact on the evolution of biofilms, NPJ Biofilms Microbiomes 2, 15027. 16. Ben-Jacob, E., Cohen, I., and Gutnick, D. L. (1998) Cooperative organization of bacterial colonies: from genotype to morphotype, Annu Rev Microbiol 52, 779-806. 21 ACS Paragon Plus Environment

Page 22 of 25

Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470


17. Be'er, A., Smith, R. S., Zhang, H. P., Florin, E. L., Payne, S. M., and Swinney, H. L. (2009) Paenibacillus dendritiformis bacterial colony growth depends on surfactant but not on bacterial motion, J Bacteriol 191, 5758-5764. 18. Be'er, A., Strain, S. K., Hernandez, R. A., Ben-Jacob, E., and Florin, E. L. (2013) Periodic reversals in Paenibacillus dendritiformis swarming, J Bacteriol 195, 2709-2717. 19. Tcherpakov, M., Ben-Jacob, E., and Gutnick, D. L. (1999) Paenibacillus dendritiformis sp. nov., proposal for a new pattern-forming species and its localization within a phylogenetic cluster, Int J Syst Bacteriol 49 Pt 1, 239-246. 20. Ben-Jacob, E., Schochet, O., Tenenbaum, A., Cohen, I., Czirok, A., and Vicsek, T. (1994) Generic modelling of cooperative growth patterns in bacterial colonies, Nature 368, 46-49. 21. McSpadden Gardener, B. B. (2004) Ecology of Bacillus and Paenibacillus spp. in Agricultural Systems, Phytopathology 94, 1252-1258. 22. Ben-Jacob, E., Shmueli, H., Shochet, O., and Tenenbaum, A. (1992) Adaptive self-organization during growth of bacterial colonies, Physica A 187, 378-424. 23. Lyons, N. A., and Kolter, R. (2017) Bacillus subtilis Protects Public Goods by Extending Kin Discrimination to Closely Related Species, MBio 8. 24. Be'er, A., Ariel, G., Kalisman, O., Helman, Y., Sirota-Madi, A., Zhang, H. P., Florin, E. L., Payne, S. M., Ben-Jacob, E., and Swinney, H. L. (2010) Lethal protein produced in response to competition between sibling bacterial colonies, Proc Natl Acad Sci U S A 107, 6258-6263. 25. Be'er, A., Florin, E. L., Fisher, C. R., Swinney, H. L., and Payne, S. M. (2011) Surviving bacterial sibling rivalry: inducible and reversible phenotypic switching in Paenibacillus dendritiformis, MBio 2, e00069-00011. 26. Hagai, E., Dvora, R., Havkin-Blank, T., Zelinger, E., Porat, Z., Schulz, S., and Helman, Y. (2014) Surfacemotility induction, attraction and hitchhiking between bacterial species promote dispersal on solid surfaces, Isme J 8, 1147-1151. 27. Yang, J. Y., Phelan, V. V., Simkovsky, R., Watrous, J. D., Trial, R. M., Fleming, T. C., Wenter, R., Moore, B. S., Golden, S. S., Pogliano, K., and Dorrestein, P. C. (2012) Primer on agar-based microbial imaging mass spectrometry, J Bacteriol 194, 6023-6028. 28. Yang, Y. L., Xu, Y., Straight, P., and Dorrestein, P. C. (2009) Translating metabolic exchange with imaging mass spectrometry, Nat Chem Biol 5, 885-887. 29. Hoefler, B. C., Gorzelnik, K. V., Yang, J. Y., Hendricks, N., Dorrestein, P. C., and Straight, P. D. (2012) Enzymatic resistance to the lipopeptide surfactin as identified through imaging mass spectrometry of bacterial competition, Proc Natl Acad Sci U S A 109, 13082-13087. 30. Wang, M., and Carver, J. J., and Phelan, V. V., and Sanchez, L. M., and Garg, N., and Peng, Y., and Nguyen, D. D., and Watrous, J., and Kapono, C. A., and Luzzatto-Knaan, T., and Porto, C., and Bouslimani, A., and Melnik, A. V., and Meehan, M. J., and Liu, W. T., and Crusemann, M., and Boudreau, P. D., and Esquenazi, E., and Sandoval-Calderon, M., and Kersten, R. D., and Pace, L. A., and Quinn, R. A., and Duncan, K. R., and Hsu, C. C., and Floros, D. J., and Gavilan, R. G., and Kleigrewe, K., and Northen, T., and Dutton, R. J., and Parrot, D., and Carlson, E. E., and Aigle, B., and Michelsen, C. F., and Jelsbak, L., and Sohlenkamp, C., and Pevzner, P., and Edlund, A., and McLean, J., and Piel, J., and Murphy, B. T., and Gerwick, L., and Liaw, C. C., and Yang, Y. L., and Humpf, H. U., and Maansson, M., and Keyzers, R. A., and Sims, A. C., and Johnson, A. R., and Sidebottom, A. M., and Sedio, B. E., and Klitgaard, A., and Larson, C. B., and Boya, P. C., and Torres-Mendoza, D., and Gonzalez, D. J., and Silva, D. B., and Marques, L. M., and Demarque, D. P., and Pociute, E., and O'Neill, E. C., and Briand, E., and Helfrich, E. J., and Granatosky, E. A., and Glukhov, E., and Ryffel, F., and Houson, H., and Mohimani, H., and Kharbush, J. J., and Zeng, Y., and Vorholt, J. A., and Kurita, K. L., and Charusanti, P., and McPhail, K. L., and Nielsen, K. F., and Vuong, L., and Elfeki, M., and Traxler, M. F., and Engene, N., and Koyama, N., and Vining, O. B., 22 ACS Paragon Plus Environment


471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516

and Baric, R., and Silva, R. R., and Mascuch, S. J., and Tomasi, S., and Jenkins, S., and Macherla, V., and Hoffman, T., and Agarwal, V., and Williams, P. G., and Dai, J., and Neupane, R., and Gurr, J., and Rodriguez, A. M., and Lamsa, A., and Zhang, C., and Dorrestein, K., and Duggan, B. M., and Almaliti, J., and Allard, P. M., and Phapale, P., and Nothias, L. F., and Alexandrov, T., and Litaudon, M., and Wolfender, J. L., and Kyle, J. E., and Metz, T. O., and Peryea, T., and Nguyen, D. T., and VanLeer, D., and Shinn, P., and Jadhav, A., and Muller, R., and Waters, K. M., and Shi, W., and Liu, X., and Zhang, L., and Knight, R., and Jensen, P. R., and Palsson, B. O., and Pogliano, K., and Linington, R. G., and Gutierrez, M., and Lopes, N. P., and Gerwick, W. H., and Moore, B. S., and Dorrestein, P. C., and Bandeira, N. (2016) Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking, Nat Biotechnol 34, 828-837. 31. Garg, N., Zeng, Y., Edlund, A., Melnik, A. V., Sanchez, L. M., Mohimani, H., Gurevich, A., Miao, V., Schiffler, S., Lim, Y. W., Luzzatto-Knaan, T., Cai, S., Rohwer, F., Pevzner, P. A., Cichewicz, R. H., Alexandrov, T., and Dorrestein, P. C. (2016) Spatial Molecular Architecture of the Microbial Community of a Peltigera Lichen, mSystems 1. 32. Luzzatto Knaan, T., Garg, N., Wang, M., Glukhov, E., Peng, Y., Ackermann, G., Amir, A., Duggan, B. M., Ryazanov, S., Gerwick, L., Knight, R., Alexandrov, T., Bandeira, N., Gerwick, W. H., and Dorrestein, P. C. (2017) Digitizing mass spectrometry data to explore the chemical diversity and distribution of marine cyanobacteria and algae, Elife 6. 33. Straight, P. D., Willey, J. M., and Kolter, R. (2006) Interactions between Streptomyces coelicolor and Bacillus subtilis: Role of surfactants in raising aerial structures, J Bacteriol 188, 4918-4925. 34. Cochrane, S. A., and Vederas, J. C. (2014) Lipopeptides from Bacillus and Paenibacillus spp.: A Gold Mine of Antibiotic Candidates, Med Res Rev. 35. Xu, W., Liu, H., Wang, X., and Yang, Q. (2016) Surfactin Induces Maturation of Dendritic Cells in vitro, Biosci Rep. 36. Lima, T. M., Procopio, L. C., Brandao, F. D., Carvalho, A. M., Totola, M. R., and Borges, A. C. (2011) Biodegradability of bacterial surfactants, Biodegradation 22, 585-592. 37. Nonejuie, P., Trial, R. M., Newton, G. L., Lamsa, A., Ranmali Perera, V., Aguilar, J., Liu, W. T., Dorrestein, P. C., Pogliano, J., and Pogliano, K. (2016) Application of bacterial cytological profiling to crude natural product extracts reveals the antibacterial arsenal of Bacillus subtilis, J Antibiot (Tokyo) 69, 353-361. 38. Traxler, M. F., and Kolter, R. (2015) Natural products in soil microbe interactions and evolution, Nat Prod Rep. 39. Aleti, G., Lehner, S., Bacher, M., Compant, S., Nikolic, B., Plesko, M., Schuhmacher, R., Sessitsch, A., and Brader, G. (2016) Surfactin variants mediate species-specific biofilm formation and root colonization in Bacillus, Environ Microbiol 18, 2634-2645. 40. De Pascale, G., and Wright, G. D. (2010) Antibiotic resistance by enzyme inactivation: from mechanisms to solutions, Chembiochem 11, 1325-1334. 41. Liu, Y., Kyle, S., and Straight, P. D. (2018) Antibiotic Stimulation of a Bacillus subtilis Migratory Response, mSphere 3. 42. Grady, E. N., MacDonald, J., Liu, L., Richman, A., and Yuan, Z. C. (2016) Current knowledge and perspectives of Paenibacillus: a review, Microb Cell Fact 15, 203. 43. Drescher, K., Nadell, C. D., Stone, H. A., Wingreen, N. S., and Bassler, B. L. (2014) Solutions to the public goods dilemma in bacterial biofilms, Curr Biol 24, 50-55. 44. Morris, J. J., Lenski, R. E., and Zinser, E. R. (2012) The Black Queen Hypothesis: evolution of dependencies through adaptive gene loss, MBio 3.


Page 24 of 25

Page 25 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

517 518 519 520 521 522 523 524 525 526


45. Fitzpatrick, C. R., Copeland, J., Wang, P. W., Guttman, D. S., Kotanen, P. M., and Johnson, M. T. J. (2018) Assembly and ecological function of the root microbiome across angiosperm plant species, Proc Natl Acad Sci U S A 115, E1157-E1165. 46. Qiao, J., Yu, X., Liang, X., Liu, Y., and Borriss, R. (2017) Addition of plant-growth-promoting Bacillus subtilis PTS-394 on tomato rhizosphere has no durable impact on composition of root microbiome, BMC Microbiol 17, 131. 47. Zomorrodi, A. R., and Segre, D. (2016) Synthetic Ecology of Microbes: Mathematical Models and Applications, J Mol Biol 428, 837-861. 48. Bouslimani, A., Sanchez, L. M., Garg, N., and Dorrestein, P. C. (2014) Mass spectrometry of natural products: current, emerging and future technologies, Nat Prod Rep.

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Mass Spectrometry Uncovers the Role of Surfactin as an Interspecies

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