Arnaouteli, Sofia

Davidson, Fordyce A.

MacPhee, Cait E.

Matoz-Fernandez, Daniel

Porter, Michael

Stanley-Wall, Nicola R.

English

Discovery Research Portal

                                                                    University of DundeeComment on “Rivalry in Bacillus subtilis coloniesMatoz-Fernandez, Daniel; Arnaouteli, Sofia; Porter, Michael; MacPhee, Cait E.; Stanley-Wall,Nicola R.; Davidson, Fordyce A.Published in:Soft MatterDOI:10.1039/C9SM02141HPublication date:2020Document VersionPublisher's PDF, also known as Version of recordLink to publication in Discovery Research PortalCitation for published version (APA):Matoz-Fernandez, D., Arnaouteli, S., Porter, M., MacPhee, C. E., Stanley-Wall, N. R., & Davidson, F. A. (2020).Comment on “Rivalry in Bacillus subtilis colonies: enemy or family?”. Soft Matter, 16(13), 3344-3346.https://doi.org/10.1039/C9SM02141HGeneral rightsCopyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or othercopyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated withthese rights. • Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal.Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Download date: 28. Apr. 20203344 | Soft Matter, 2020, 16, 3344--3346 This journal is©The Royal Society of Chemistry 2020Cite this: SoftMatter, 2020,16, 3344Comment on ‘‘Rivalry in Bacillus subtilis colonies:enemy or family?’’Daniel Matoz-Fernandez, ab Sofia Arnaouteli, a Michael Porter, aCait E. MacPhee, c Nicola R. Stanley-Wall *a and Fordyce A. Davidson *bIt is well known that biofilms are one of themost widespread formsof life on Earth, capable of colonising almost any environmentfrom humans to metals.1 In general, biofilms manifest as self-organised multicellular communities embedded in a self-producedextracellular polymeric matrix that, among other functions, aidssignalling to the resident cells.2 For processes such as formationand dispersal, the importance of signalling at the cell-to-cell scalewithin the microbial community is widely recognised.3,4 However,in recent years it has been shown that bacteria in biofilms andother collectives of cells, can communicate effectively over largedistances both within and between collectives using a diverse rangeof mechanisms including quorum sensing,5 electrical signalling,6,7and mechanical transmissions.8In a recent paper, Paul et al.9 have shown proximal Bacillussubtilis subspecies spizizenii biofilms interact, leading to either‘‘demarcation’’ or ‘‘merging’’ of the initially spatially separatedcommunities. Explicit definitions of demarcation and mergingare not given, but it is inferred that the former implies that avisible gap is observed between two proximally located coloniesand that this gap is a (semi-)permanent feature once estab-lished. The centre of this approximately linear gap is defined asthe demarcation line (DL). Merging is the absence of this gapwhere proximal colonies grow until they visually appear tomeet and importantly, the authors make explicit reference to‘‘merging into a single colony’’. The outcome of the interactionwas found by Paul et al. to depend on the initial separationdistance at the point of inoculation and the substrate composi-tion. Moreover, by using an inert object (made from polydi-methylsiloxane (PDMS)), in place one of the living communities,the authors concluded that these outcomes are driven by bio-chemical signals, not mechanical cues. The generality of thesefindings was supported by the demonstration that Pseudomonasfluorescens biofilms respond in a similar way. Finally, the authorsexplored various hypotheses to explain their experimentalobservations via mathematical modelling. These data shedsignificant light on the complexities of biofilm growth andinteractions and provide impetus for future lines of research.In this commentary, we support the inference that the out-come of an interaction between two neighbouring colonies isgoverned by long-range (and therefore most likely) biochemicalsignalling of some form. Moreover, we agree that the simplerelationship presented between inoculum distance and diffusivitywithin the medium does capture the essence of the experimentalobservations. However, we argue that in general, the processesmediating the outcome of such interactions are far more complexthan the authors postulate. In particular, we highlight that (i) theirrelationship does not necessarily hold in broader, but closelyrelated contexts and (ii) the relationship does not elucidate theunderlying mechanism(s).With reference to the above, we found that if we establish twosibling Bacillus subtilis NCIB 3610 biofilms with an inoculationdistance of 10 mm, at 48 hours we observed an apparentdemarcation zone (Fig. 1a) similar to that presented in Paulet al. and reported by other authors cited in that paper (ofparticular relevance are ref. 10–12). However, we found that thisdemarcation zone resolved completely over time (Fig. 1b) andtherefore does not represent a quantitatively predictable end-point. Hence, we propose that evolution over realistic growthtimescales has to be factored into future investigations. We alsowish to highlight that confocal microscopy revealed that when twosibling biofilms appear to ‘‘merge’’ when imaged at the macroscale (i.e. there is no visible gap in between) they actually simplyabut, and remain as distinct spatially segregated communities(Fig. 1c). Therefore, we suggest that ‘‘abuttal’’ rather thana Division of Molecular Microbiology, School of Life Sciences, University of Dundee,Dundee DD1 5EH, UK. E-mail: n.r.stanleywall@dundee.ac.ukb Division of Mathematics, School of Science and Engineering, University of Dundee,Dundee DD1 4HN, UK. E-mail: f.a.davidson@dundee.ac.ukc School of Physics, University of Edinburgh, EH9 3FD, UKReceived 28th October 2019,Accepted 24th January 2020DOI: 10.1039/c9sm02141hrsc.li/soft-matter-journalSoft MatterCOMMENTOpen Access Article. Published on 24 March 2020. Downloaded on 4/1/2020 3:05:08 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View IssueThis journal is©The Royal Society of Chemistry 2020 Soft Matter, 2020, 16, 3344--3346 | 3345‘‘merging’’ may be a more appropriate term, and argue againstthe concept of ‘‘merging into a single colony’’, which impliesmixing. Indeed abuttal and true merging may be distinct out-comes – this requires deeper investigation in each case.Finally, in our opinion, the mathematical model presentedby Paul et al. does not support the conclusions of the paper. Thepaper proposes that biochemical signalling is responsible forcontrolling the interactions. This is in line with the work ofBe’er et al.12 The model of Be’er et al. includes explicit production,sensing and reaction to a diffusible toxin and demarcation results asan emergent feature of the interaction of these fields. On the otherhand, the model of Paul et al. includes no explicit reference to abiochemical signalling agent. Rather, the outcomes of their modelare governed by a nutrient-dependentmortality rate (see eqn (8)) thatis mediated by carefully selected time and space constraints. Thesetemporal and geometric conditions are necessary to locate the DL atthe interfacial gap between the colonies. These conditions thereforedrive the conclusions of the model and hence, with respect and inour opinion, represent a circular argument. The authors are clear intheir intention that the model should be general. It is our opinionthat this generality precludes further understanding of the under-lying mechanisms.It could well be that biochemical signalling is exactly themechanism that allows sibling colonies to determine both theirtemporal state and their relative spatial location. Investigatingthese hypotheses would be of great interest. We note however,that nutrient limitation in itself is sufficient to inhibit growth(see for example the models of ref. 10 and 11). In particular, inArnaouteli et al., 201913 we demonstrated that proximal siblingcolonies can have a marked effect on the growth dynamics of eachcolony where demarcation or abuttal was determined by nutrientlimitation (see e.g. Fig. 6c13). The experimental results in Arnaou-teli et al., 2019 were rationalised using a mathematical model thattook full account of the main components of the underlyinghypotheses (in that case the production of pulcherriminic acidto chelate free iron in the medium). Growth arrest was anemergent property of this model. Of particular interest is thatgrowth arrest of the expanding outer colony edge was observed insingle biofilms – no interactions were necessary (although inter-actions were considered). The mathematical model elucidatedthis phenomenon to be induced by a wave of iron depletion thatovertook the expanding colony margin. We therefore propose thatwhen studying colony interactions, self-limitation is a feature thatcannot be dismissed without further investigation.We fully agree with the authors that proximal growth can have adistinct effect on the physical appearance of biofilms at themacroscale. For example, when we inoculated four sibling biofilmsat sufficient distance to preclude abutting, a marked asymmetry ofradial growth and of material properties as indicated by changes inwrinkle structure was still observed (Fig. 1d, see also ref. 11). This issuggestive of long-range (biochemical) signal(s) being transmittedand received at the cellular level and manifest through macro-scaleremodelling of the biofilm architecture. However, our previouswork and the experiments detailed here, lead us to conclude thatin general the interaction between proximal biofilms is mediatedby a complex growth response. This response can be mediatedby separation and substrate stiffness, but is also dependent onmultiple other factors including inoculum density and size, genus,media composition and other experimental constraints.In conclusion, there is clear evidence that bacterial biofilmsinteract over short and long distances and that these interac-tions are in part governed by biochemical signalling. Moreover,truly understanding how microbes colonise and interact withtheir environment requires the scientific investigation of multi-biofilm assays. The physics/mathematics of soft matter com-munities are likely to have a significant input to future scien-tific studies in this area and contributions will best be made bycareful matching of experimental data and biological under-standing with mathematical models. This calibrated interdis-ciplinary approach is well-suited to shine further light on thefundamental role of microbes in the environment.MethodsBiofilm formationB. subtilisNCIB 3610 was grown onMSggmedium (5mMpotassiumphosphate and 100 mM MOPS at pH 7.0 supplemented withFig. 1 Interacting Bacillus subtilis biofilms. B. subtilis isolate NCIB 3610 wasinoculated on 1.5% (w/v) agar MSggmedium at 30 1C.14 The otherwise isogenicstrains were distinguished by expression of either mKate2 or gfp mut2. Thefounding cells of the biofilms were initially inoculated at a separation distanceof 10 mm and imaged at (a) 48 h, where a distinctive demarcation zone isvisible and indicated by a visible gap and resultant asymmetry of radial growthmanifest as flattened edges along the interaction zone [expressing gfp – leftand mKate2 – right]; and (b) at 96 hours, where the demarcation zone hasresolved and the visible gap has disappeared. The biofilms now seem to‘‘merge’’ as defined in Paul et al. (c) Merging of proximal biofilms using confocalmicroscopy maximum projection image for x–y plane (top panel) and z-plane(bottom panel) of interacting B. subtilis colonies [expressing gfp – left andmKate2 – right] at 48 hours, separated by an initial distance of 5 mm growingon 1.5% (w/v) agar MSgg medium at 30 1C. (d) B. subtilis sibling biofilms exhibitcomplex interactions. Four NCIB 3610 biofilm sibling colonies (two expressinggfp [top left and bottom right] and two expressing mKate2 [top right andbottom left]) were founded at 20 mm distance on 1.5% (w/v) agar MSggmedium at 30 1C and incubated for 96 h before imaging.Comment Soft MatterOpen Access Article. Published on 24 March 2020. Downloaded on 4/1/2020 3:05:08 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online3346 | Soft Matter, 2020, 16, 3344--3346 This journal is©The Royal Society of Chemistry 20202 mM MgCl2, 700 mM CaCl2, 50 mM MnCl2, 1 mM ZnCl2, 2 mMthiamine, 0.5% glycerol, 0.5% glutamate) solidified with 1.5%select agar (Invitrogen) at 30 1C for the indicated time points aspreviously described14 with the exception that 1 ml of culturewas used for biofilm initiation. Images of colony biofilms wererecorded using a Nikon D3200 digital camera mounted on a KaiserRS3XA copy stand or using a Leica MZ16FA stereomicroscope.Confocal microscopy4 ml of MSgg medium supplemented with 1.5% (w/v) agar wasplaced into a 35 mm diameter Petri dish and dried for 1 hour ina laminar flow hood. NCIB 3610 strain constitutively producingGFP-16 or mKate217 was spotted into the centre of the agar inthe Petri dish and incubated at 30 1C for the indicated timeperiod. A Leica SP8 upright confocal was used to image theedge of the biofilm using a 10 0.3 N.A. air objective and aheated chamber that was pre-warmed to 30 1C. A cling film tentwas draped from around the objective and tucked loosely underthe stage to eliminate airflow across the plate and minimisedehydration (and therefore shrinkage) of the agar. An additional35 mm diameter Petri dish was filled with water and placed nextto the biofilm plate to increase the humidity inside the tent. Anargon-ion laser was used to excite the GFP at 488 nm and2% power. Z-stacks capturing the full height of the biofilmborder were specified based on the presence of GFP-containingcells and planes of 1024  1024 pixels were acquired quicklyusing a resonant mirror, averaging 16 scans per line. Imageswere imported into an OMERO15 server and figures were pre-pared using OMERO figure (http://figure.openmicroscopy.org/).Conflicts of interestThere are no conflicts to declare.AcknowledgementsWork in the NSW, CEM, and FAD groups is supported bythe Biotechnology and Biological Sciences Research Council[BB/P001335/1; BB/R012415/1]. We would like to acknowledgethe Dundee Imaging Facility, Dundee, which is supported by the‘Wellcome Trust Technology Platform’ award [097945/B/11/Z]and the ‘MRC Next Generation Optical Microscopy’ award[MR/K015869/1]. Thank you to Akos Kovacs and colleagues forthe mKate labelled strain.References1 H. C. Flemming, J. Wingender, U. Szewzyk, P. Steinberg,S. A. Rice and S. Kjelleberg, Biofilms: an emergent form ofbacterial life, Nat. Rev. Microbiol., 2016, 14(9), 563–575.2 A. Dragos and A. T. Kovacs, The Peculiar Functions of theBacterial Extracellular Matrix, Trends Microbiol., 2017, 25(4),257–266.3 L. S. Cairns, L. Hobley and N. R. Stanley-Wall, Biofilmformation by Bacillus subtilis: new insights into regulatorystrategies and assembly mechanisms, Mol. Microbiol., 2014,93(4), 587–598.4 E. Nagar and R. Schwarz, To be or not to be planktonic? Self-inhibition of biofilm development, Environ. Microbiol.,2015, 17(5), 1477–1486.5 J. L. Connell, A. K. Wessel, M. R. Parsek, A. D. Ellington,M.Whiteley and J. B. Shear, Probing prokaryotic social behaviorswith bacterial ‘‘lobster traps’’, mBio, 2010, 1(4), e00202-10.6 A. Prindle, J. Liu, M. Asally, S. Ly, J. Garcia-Ojalvo andG. M. Suel, Ion channels enable electrical communicationin bacterial communities, Nature, 2015, 527(7576), 59–63.7 J. Liu, R. Martinez-Corral, A. Prindle, D. D. Lee, J. Larkin,M. Gabalda-Sagarra, J. Garcia-Ojalvo and G. M. Suel, Cou-pling between distant biofilms and emergence of nutrienttime-sharing, Science, 2017, 356(6338), 638–642.8 D. R. Espeso, E. Martinez-Garcia, V. de Lorenzo and A. Goni-Moreno, Physical Forces Shape Group Identity of SwimmingPseudomonas putida Cells, Front. Microbiol., 2016, 7, 1437.9 R. Paul, T. Ghosh, T. Tang and A. Kumar, Rivalry in Bacillussubtilis colonies: enemy or family?, Soft Matter, 2019, 15(27),5400–5411.10 P. Patra, C. N. Vassallo, D. Wall and O. A. Igoshin, Mecha-nism of Kin-Discriminatory Demarcation Line Formationbetween Colonies of Swarming Bacteria, Biophys. J., 2017,113(11), 2477–2486.11 A. Sekowska, J. B. Masson, A. Celani, A. Danchin andM. Vergassola, Repulsion and metabolic switches in thecollective behavior of bacterial colonies, Biophys. J., 2009,97(3), 688–698.12 A. Be’er, G. Ariel, O. Kalisman, Y. Helman, A. Sirota-Madi,H. P. Zhang, E. L. Florin, S. M. Payne, E. Ben-Jacob and H. L.Swinney, Lethal protein produced in response to competitionbetween sibling bacterial colonies, Proc. Natl. Acad. Sci. U. S. A.,2010, 107(14), 6258–6263.13 S. Arnaouteli, D. A. Matoz-Fernandez, M. Porter, M. Kalamara,J. Abbott, C. E. MacPhee, F. A. Davidson and N. R. Stanley-Wall,Pulcherrimin formation controls growth arrest of the Bacillussubtilis biofilm, Proc. Natl. Acad. Sci. U. S. A., 2019, 116(27),13553–13562.14 S. S. Branda, J. E. Gonzalez-Pastor, S. Ben-Yehuda, R. Losickand R. Kolter, Fruiting body formation by Bacillus subtilis,Proc. Natl. Acad. Sci. U. S. A., 2001, 98(20), 11621–11626.15 C. Allan, J. M. Burel, J. Moore, C. Blackburn, M. Linkert,S. Loynton, D. Macdonald, W. J. Moore, C. Neves, A. Patterson,M. Porter, A. Tarkowska, B. Loranger, J. Avondo, I. Lagerstedt,L. Lianas, S. Leo, K. Hands, R. T. Hay, A. Patwardhan, C. Best,G. J. Kleywegt, G. Zanetti and J. R. Swedlow, OMERO: flexible,model-driven data management for experimental biology, Nat.Methods, 2012, 9(3), 245–253.16 J. van Gestel, F. J. Weissing, O. P. Kuipers and A. T. Kova´cs,Density of founder cells affects spatial pattern formationand cooperation in Bacillus subtilis biofilms, ISME J., 2014,8(10), 2069–2079.17 D. T. Verhamme, T. B. Kiley and N. R. Stanley-Wall, DegUco-ordinates multicellular behaviour exhibited by Bacillussubtilis, Mol. Microbiol., 2007, 65, 554–568.Soft Matter CommentOpen Access Article. Published on 24 March 2020. Downloaded on 4/1/2020 3:05:08 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

Comment on “Rivalry in <i>Bacillus subtilis</i> colonies:enemy or family?”

                                                                    University of DundeeComment on “Rivalry in Bacillus subtilis coloniesMatoz-Fernandez, Daniel; Arnaouteli, Sofia; Porter, Michael; MacPhee, Cait E.; Stanley-Wall,Nicola R.; Davidson, Fordyce A.Published in:Soft MatterDOI:10.1039/C9SM02141HPublication date:2020Licence:CC BYDocument VersionPublisher's PDF, also known as Version of recordLink to publication in Discovery Research PortalCitation for published version (APA):Matoz-Fernandez, D., Arnaouteli, S., Porter, M., MacPhee, C. E., Stanley-Wall, N. R., & Davidson, F. A. (2020).Comment on “Rivalry in Bacillus subtilis colonies: enemy or family?”. Soft Matter, 16(13), 3344-3346.https://doi.org/10.1039/C9SM02141HGeneral rightsCopyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or othercopyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated withthese rights. • Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal.Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Download date: 19. Apr. 20233344 | Soft Matter, 2020, 16, 3344--3346 This journal is©The Royal Society of Chemistry 2020Cite this: SoftMatter, 2020,16, 3344Comment on ‘‘Rivalry in Bacillus subtilis colonies:enemy or family?’’Daniel Matoz-Fernandez, ab Sofia Arnaouteli, a Michael Porter, aCait E. MacPhee, c Nicola R. Stanley-Wall *a and Fordyce A. Davidson *bIt is well known that biofilms are one of the most widespread formsof life on Earth, capable of colonising almost any environmentfrom humans to metals.1 In general, biofilms manifest as self-organised multicellular communities embedded in a self-producedextracellular polymeric matrix that, among other functions, aidssignalling to the resident cells.2 For processes such as formationand dispersal, the importance of signalling at the cell-to-cell scalewithin the microbial community is widely recognised.3,4 However,in recent years it has been shown that bacteria in biofilms andother collectives of cells, can communicate effectively over largedistances both within and between collectives using a diverse rangeof mechanisms including quorum sensing,5 electrical signalling,6,7and mechanical transmissions.8In a recent paper, Paul et al.9 have shown proximal Bacillussubtilis subspecies spizizenii biofilms interact, leading to either‘‘demarcation’’ or ‘‘merging’’ of the initially spatially separatedcommunities. Explicit definitions of demarcation and mergingare not given, but it is inferred that the former implies that avisible gap is observed between two proximally located coloniesand that this gap is a (semi-)permanent feature once estab-lished. The centre of this approximately linear gap is defined asthe demarcation line (DL). Merging is the absence of this gapwhere proximal colonies grow until they visually appear tomeet and importantly, the authors make explicit reference to‘‘merging into a single colony’’. The outcome of the interactionwas found by Paul et al. to depend on the initial separationdistance at the point of inoculation and the substrate composi-tion. Moreover, by using an inert object (made from polydi-methylsiloxane (PDMS)), in place one of the living communities,the authors concluded that these outcomes are driven by bio-chemical signals, not mechanical cues. The generality of thesefindings was supported by the demonstration that Pseudomonasfluorescens biofilms respond in a similar way. Finally, the authorsexplored various hypotheses to explain their experimentalobservations via mathematical modelling. These data shedsignificant light on the complexities of biofilm growth andinteractions and provide impetus for future lines of research.In this commentary, we support the inference that the out-come of an interaction between two neighbouring colonies isgoverned by long-range (and therefore most likely) biochemicalsignalling of some form. Moreover, we agree that the simplerelationship presented between inoculum distance and diffusivitywithin the medium does capture the essence of the experimentalobservations. However, we argue that in general, the processesmediating the outcome of such interactions are far more complexthan the authors postulate. In particular, we highlight that (i) theirrelationship does not necessarily hold in broader, but closelyrelated contexts and (ii) the relationship does not elucidate theunderlying mechanism(s).With reference to the above, we found that if we establish twosibling Bacillus subtilis NCIB 3610 biofilms with an inoculationdistance of 10 mm, at 48 hours we observed an apparentdemarcation zone (Fig. 1a) similar to that presented in Paulet al. and reported by other authors cited in that paper (ofparticular relevance are ref. 10–12). However, we found that thisdemarcation zone resolved completely over time (Fig. 1b) andtherefore does not represent a quantitatively predictable end-point. Hence, we propose that evolution over realistic growthtimescales has to be factored into future investigations. We alsowish to highlight that confocal microscopy revealed that when twosibling biofilms appear to ‘‘merge’’ when imaged at the macroscale (i.e. there is no visible gap in between) they actually simplyabut, and remain as distinct spatially segregated communities(Fig. 1c). Therefore, we suggest that ‘‘abuttal’’ rather thana Division of Molecular Microbiology, School of Life Sciences, University of Dundee,Dundee DD1 5EH, UK. E-mail: n.r.stanleywall@dundee.ac.ukb Division of Mathematics, School of Science and Engineering, University of Dundee,Dundee DD1 4HN, UK. E-mail: f.a.davidson@dundee.ac.ukc School of Physics, University of Edinburgh, EH9 3FD, UKReceived 28th October 2019,Accepted 24th January 2020DOI: 10.1039/c9sm02141hrsc.li/soft-matter-journalSoft MatterCOMMENTOpen Access Article. Published on 24 March 2020. Downloaded on 4/1/2020 3:05:08 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View IssueThis journal is©The Royal Society of Chemistry 2020 Soft Matter, 2020, 16, 3344--3346 | 3345‘‘merging’’ may be a more appropriate term, and argue againstthe concept of ‘‘merging into a single colony’’, which impliesmixing. Indeed abuttal and true merging may be distinct out-comes – this requires deeper investigation in each case.Finally, in our opinion, the mathematical model presentedby Paul et al. does not support the conclusions of the paper. Thepaper proposes that biochemical signalling is responsible forcontrolling the interactions. This is in line with the work ofBe’er et al.12 The model of Be’er et al. includes explicit production,sensing and reaction to a diffusible toxin and demarcation results asan emergent feature of the interaction of these fields. On the otherhand, the model of Paul et al. includes no explicit reference to abiochemical signalling agent. Rather, the outcomes of their modelare governed by a nutrient-dependent mortality rate (see eqn (8)) thatis mediated by carefully selected time and space constraints. Thesetemporal and geometric conditions are necessary to locate the DL atthe interfacial gap between the colonies. These conditions thereforedrive the conclusions of the model and hence, with respect and inour opinion, represent a circular argument. The authors are clear intheir intention that the model should be general. It is our opinionthat this generality precludes further understanding of the under-lying mechanisms.It could well be that biochemical signalling is exactly themechanism that allows sibling colonies to determine both theirtemporal state and their relative spatial location. Investigatingthese hypotheses would be of great interest. We note however,that nutrient limitation in itself is sufficient to inhibit growth(see for example the models of ref. 10 and 11). In particular, inArnaouteli et al., 201913 we demonstrated that proximal siblingcolonies can have a marked effect on the growth dynamics of eachcolony where demarcation or abuttal was determined by nutrientlimitation (see e.g. Fig. 6c13). The experimental results in Arnaou-teli et al., 2019 were rationalised using a mathematical model thattook full account of the main components of the underlyinghypotheses (in that case the production of pulcherriminic acidto chelate free iron in the medium). Growth arrest was anemergent property of this model. Of particular interest is thatgrowth arrest of the expanding outer colony edge was observed insingle biofilms – no interactions were necessary (although inter-actions were considered). The mathematical model elucidatedthis phenomenon to be induced by a wave of iron depletion thatovertook the expanding colony margin. We therefore propose thatwhen studying colony interactions, self-limitation is a feature thatcannot be dismissed without further investigation.We fully agree with the authors that proximal growth can have adistinct effect on the physical appearance of biofilms at themacroscale. For example, when we inoculated four sibling biofilmsat sufficient distance to preclude abutting, a marked asymmetry ofradial growth and of material properties as indicated by changes inwrinkle structure was still observed (Fig. 1d, see also ref. 11). This issuggestive of long-range (biochemical) signal(s) being transmittedand received at the cellular level and manifest through macro-scaleremodelling of the biofilm architecture. However, our previouswork and the experiments detailed here, lead us to conclude thatin general the interaction between proximal biofilms is mediatedby a complex growth response. This response can be mediatedby separation and substrate stiffness, but is also dependent onmultiple other factors including inoculum density and size, genus,media composition and other experimental constraints.In conclusion, there is clear evidence that bacterial biofilmsinteract over short and long distances and that these interac-tions are in part governed by biochemical signalling. Moreover,truly understanding how microbes colonise and interact withtheir environment requires the scientific investigation of multi-biofilm assays. The physics/mathematics of soft matter com-munities are likely to have a significant input to future scien-tific studies in this area and contributions will best be made bycareful matching of experimental data and biological under-standing with mathematical models. This calibrated interdis-ciplinary approach is well-suited to shine further light on thefundamental role of microbes in the environment.MethodsBiofilm formationB. subtilis NCIB 3610 was grown on MSgg medium (5 mM potassiumphosphate and 100 mM MOPS at pH 7.0 supplemented withFig. 1 Interacting Bacillus subtilis biofilms. B. subtilis isolate NCIB 3610 wasinoculated on 1.5% (w/v) agar MSgg medium at 30 1C.14 The otherwise isogenicstrains were distinguished by expression of either mKate2 or gfp mut2. Thefounding cells of the biofilms were initially inoculated at a separation distanceof 10 mm and imaged at (a) 48 h, where a distinctive demarcation zone isvisible and indicated by a visible gap and resultant asymmetry of radial growthmanifest as flattened edges along the interaction zone [expressing gfp – leftand mKate2 – right]; and (b) at 96 hours, where the demarcation zone hasresolved and the visible gap has disappeared. The biofilms now seem to‘‘merge’’ as defined in Paul et al. (c) Merging of proximal biofilms using confocalmicroscopy maximum projection image for x–y plane (top panel) and z-plane(bottom panel) of interacting B. subtilis colonies [expressing gfp – left andmKate2 – right] at 48 hours, separated by an initial distance of 5 mm growingon 1.5% (w/v) agar MSgg medium at 30 1C. (d) B. subtilis sibling biofilms exhibitcomplex interactions. Four NCIB 3610 biofilm sibling colonies (two expressinggfp [top left and bottom right] and two expressing mKate2 [top right andbottom left]) were founded at 20 mm distance on 1.5% (w/v) agar MSggmedium at 30 1C and incubated for 96 h before imaging.Comment Soft MatterOpen Access Article. Published on 24 March 2020. Downloaded on 4/1/2020 3:05:08 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online3346 | Soft Matter, 2020, 16, 3344--3346 This journal is©The Royal Society of Chemistry 20202 mM MgCl2, 700 mM CaCl2, 50 mM MnCl2, 1 mM ZnCl2, 2 mMthiamine, 0.5% glycerol, 0.5% glutamate) solidified with 1.5%select agar (Invitrogen) at 30 1C for the indicated time points aspreviously described14 with the exception that 1 ml of culturewas used for biofilm initiation. Images of colony biofilms wererecorded using a Nikon D3200 digital camera mounted on a KaiserRS3XA copy stand or using a Leica MZ16FA stereomicroscope.Confocal microscopy4 ml of MSgg medium supplemented with 1.5% (w/v) agar wasplaced into a 35 mm diameter Petri dish and dried for 1 hour ina laminar flow hood. NCIB 3610 strain constitutively producingGFP-16 or mKate217 was spotted into the centre of the agar inthe Petri dish and incubated at 30 1C for the indicated timeperiod. A Leica SP8 upright confocal was used to image theedge of the biofilm using a 10 0.3 N.A. air objective and aheated chamber that was pre-warmed to 30 1C. A cling film tentwas draped from around the objective and tucked loosely underthe stage to eliminate airflow across the plate and minimisedehydration (and therefore shrinkage) of the agar. An additional35 mm diameter Petri dish was filled with water and placed nextto the biofilm plate to increase the humidity inside the tent. Anargon-ion laser was used to excite the GFP at 488 nm and2% power. Z-stacks capturing the full height of the biofilmborder were specified based on the presence of GFP-containingcells and planes of 1024  1024 pixels were acquired quicklyusing a resonant mirror, averaging 16 scans per line. Imageswere imported into an OMERO15 server and figures were pre-pared using OMERO figure (http://figure.openmicroscopy.org/).Conflicts of interestThere are no conflicts to declare.AcknowledgementsWork in the NSW, CEM, and FAD groups is supported bythe Biotechnology and Biological Sciences Research Council[BB/P001335/1; BB/R012415/1]. We would like to acknowledgethe Dundee Imaging Facility, Dundee, which is supported by the‘Wellcome Trust Technology Platform’ award [097945/B/11/Z]and the ‘MRC Next Generation Optical Microscopy’ award[MR/K015869/1]. Thank you to Akos Kovacs and colleagues forthe mKate labelled strain.References1 H. C. Flemming, J. Wingender, U. Szewzyk, P. Steinberg,S. A. Rice and S. Kjelleberg, Biofilms: an emergent form ofbacterial life, Nat. Rev. Microbiol., 2016, 14(9), 563–575.2 A. Dragos and A. T. Kovacs, The Peculiar Functions of theBacterial Extracellular Matrix, Trends Microbiol., 2017, 25(4),257–266.3 L. S. Cairns, L. Hobley and N. R. Stanley-Wall, Biofilmformation by Bacillus subtilis: new insights into regulatorystrategies and assembly mechanisms, Mol. Microbiol., 2014,93(4), 587–598.4 E. Nagar and R. Schwarz, To be or not to be planktonic? Self-inhibition of biofilm development, Environ. Microbiol.,2015, 17(5), 1477–1486.5 J. L. Connell, A. K. Wessel, M. R. Parsek, A. D. Ellington,M. Whiteley and J. B. Shear, Probing prokaryotic social behaviorswith bacterial ‘‘lobster traps’’, mBio, 2010, 1(4), e00202-10.6 A. Prindle, J. Liu, M. Asally, S. Ly, J. Garcia-Ojalvo andG. M. Suel, Ion channels enable electrical communicationin bacterial communities, Nature, 2015, 527(7576), 59–63.7 J. Liu, R. Martinez-Corral, A. Prindle, D. D. Lee, J. Larkin,M. Gabalda-Sagarra, J. Garcia-Ojalvo and G. M. Suel, Cou-pling between distant biofilms and emergence of nutrienttime-sharing, Science, 2017, 356(6338), 638–642.8 D. R. Espeso, E. Martinez-Garcia, V. de Lorenzo and A. Goni-Moreno, Physical Forces Shape Group Identity of SwimmingPseudomonas putida Cells, Front. Microbiol., 2016, 7, 1437.9 R. Paul, T. Ghosh, T. Tang and A. Kumar, Rivalry in Bacillussubtilis colonies: enemy or family?, Soft Matter, 2019, 15(27),5400–5411.10 P. Patra, C. N. Vassallo, D. Wall and O. A. Igoshin, Mecha-nism of Kin-Discriminatory Demarcation Line Formationbetween Colonies of Swarming Bacteria, Biophys. J., 2017,113(11), 2477–2486.11 A. Sekowska, J. B. Masson, A. Celani, A. Danchin andM. Vergassola, Repulsion and metabolic switches in thecollective behavior of bacterial colonies, Biophys. J., 2009,97(3), 688–698.12 A. Be’er, G. Ariel, O. Kalisman, Y. Helman, A. Sirota-Madi,H. P. Zhang, E. L. Florin, S. M. Payne, E. Ben-Jacob and H. L.Swinney, Lethal protein produced in response to competitionbetween sibling bacterial colonies, Proc. Natl. Acad. Sci. U. S. A.,2010, 107(14), 6258–6263.13 S. Arnaouteli, D. A. Matoz-Fernandez, M. Porter, M. Kalamara,J. Abbott, C. E. MacPhee, F. A. Davidson and N. R. Stanley-Wall,Pulcherrimin formation controls growth arrest of the Bacillussubtilis biofilm, Proc. Natl. Acad. Sci. U. S. A., 2019, 116(27),13553–13562.14 S. S. Branda, J. E. Gonzalez-Pastor, S. Ben-Yehuda, R. Losickand R. Kolter, Fruiting body formation by Bacillus subtilis,Proc. Natl. Acad. Sci. U. S. A., 2001, 98(20), 11621–11626.15 C. Allan, J. M. Burel, J. Moore, C. Blackburn, M. Linkert,S. Loynton, D. Macdonald, W. J. Moore, C. Neves, A. Patterson,M. Porter, A. Tarkowska, B. Loranger, J. Avondo, I. Lagerstedt,L. Lianas, S. Leo, K. Hands, R. T. Hay, A. Patwardhan, C. Best,G. J. Kleywegt, G. Zanetti and J. R. Swedlow, OMERO: flexible,model-driven data management for experimental biology, Nat.Methods, 2012, 9(3), 245–253.16 J. van Gestel, F. J. Weissing, O. P. Kuipers and A. T. Kovács,Density of founder cells affects spatial pattern formationand cooperation in Bacillus subtilis biofilms, ISME J., 2014,8(10), 2069–2079.17 D. T. Verhamme, T. B. Kiley and N. R. Stanley-Wall, DegUco-ordinates multicellular behaviour exhibited by Bacillussubtilis, Mol. Microbiol., 2007, 65, 554–568.Soft Matter CommentOpen Access Article. Published on 24 March 2020. Downloaded on 4/1/2020 3:05:08 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

                                                                    University of DundeeComment on “Rivalry in Bacillus subtilis coloniesMatoz-Fernandez, Daniel; Arnaouteli, Sofia; Porter, Michael; MacPhee, Cait E.; Stanley-Wall,Nicola R.; Davidson, Fordyce A.Published in:Soft MatterDOI:10.1039/C9SM02141HPublication date:2020Licence:CC BYDocument VersionPublisher's PDF, also known as Version of recordLink to publication in Discovery Research PortalCitation for published version (APA):Matoz-Fernandez, D., Arnaouteli, S., Porter, M., MacPhee, C. E., Stanley-Wall, N. R., & Davidson, F. A. (2020).Comment on “Rivalry in Bacillus subtilis colonies: enemy or family?”. Soft Matter, 16(13), 3344-3346.https://doi.org/10.1039/C9SM02141HGeneral rightsCopyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or othercopyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated withthese rights.Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Download date: 24. Sep. 20253344 | Soft Matter, 2020, 16, 3344--3346 This journal is©The Royal Society of Chemistry 2020Cite this: SoftMatter, 2020,16, 3344Comment on ‘‘Rivalry in Bacillus subtilis colonies:enemy or family?’’Daniel Matoz-Fernandez, ab Sofia Arnaouteli, a Michael Porter, aCait E. MacPhee, c Nicola R. Stanley-Wall *a and Fordyce A. Davidson *bIt is well known that biofilms are one of the most widespread formsof life on Earth, capable of colonising almost any environmentfrom humans to metals.1 In general, biofilms manifest as self-organised multicellular communities embedded in a self-producedextracellular polymeric matrix that, among other functions, aidssignalling to the resident cells.2 For processes such as formationand dispersal, the importance of signalling at the cell-to-cell scalewithin the microbial community is widely recognised.3,4 However,in recent years it has been shown that bacteria in biofilms andother collectives of cells, can communicate effectively over largedistances both within and between collectives using a diverse rangeof mechanisms including quorum sensing,5 electrical signalling,6,7and mechanical transmissions.8In a recent paper, Paul et al.9 have shown proximal Bacillussubtilis subspecies spizizenii biofilms interact, leading to either‘‘demarcation’’ or ‘‘merging’’ of the initially spatially separatedcommunities. Explicit definitions of demarcation and mergingare not given, but it is inferred that the former implies that avisible gap is observed between two proximally located coloniesand that this gap is a (semi-)permanent feature once estab-lished. The centre of this approximately linear gap is defined asthe demarcation line (DL). Merging is the absence of this gapwhere proximal colonies grow until they visually appear tomeet and importantly, the authors make explicit reference to‘‘merging into a single colony’’. The outcome of the interactionwas found by Paul et al. to depend on the initial separationdistance at the point of inoculation and the substrate composi-tion. Moreover, by using an inert object (made from polydi-methylsiloxane (PDMS)), in place one of the living communities,the authors concluded that these outcomes are driven by bio-chemical signals, not mechanical cues. The generality of thesefindings was supported by the demonstration that Pseudomonasfluorescens biofilms respond in a similar way. Finally, the authorsexplored various hypotheses to explain their experimentalobservations via mathematical modelling. These data shedsignificant light on the complexities of biofilm growth andinteractions and provide impetus for future lines of research.In this commentary, we support the inference that the out-come of an interaction between two neighbouring colonies isgoverned by long-range (and therefore most likely) biochemicalsignalling of some form. Moreover, we agree that the simplerelationship presented between inoculum distance and diffusivitywithin the medium does capture the essence of the experimentalobservations. However, we argue that in general, the processesmediating the outcome of such interactions are far more complexthan the authors postulate. In particular, we highlight that (i) theirrelationship does not necessarily hold in broader, but closelyrelated contexts and (ii) the relationship does not elucidate theunderlying mechanism(s).With reference to the above, we found that if we establish twosibling Bacillus subtilis NCIB 3610 biofilms with an inoculationdistance of 10 mm, at 48 hours we observed an apparentdemarcation zone (Fig. 1a) similar to that presented in Paulet al. and reported by other authors cited in that paper (ofparticular relevance are ref. 10–12). However, we found that thisdemarcation zone resolved completely over time (Fig. 1b) andtherefore does not represent a quantitatively predictable end-point. Hence, we propose that evolution over realistic growthtimescales has to be factored into future investigations. We alsowish to highlight that confocal microscopy revealed that when twosibling biofilms appear to ‘‘merge’’ when imaged at the macroscale (i.e. there is no visible gap in between) they actually simplyabut, and remain as distinct spatially segregated communities(Fig. 1c). Therefore, we suggest that ‘‘abuttal’’ rather thana Division of Molecular Microbiology, School of Life Sciences, University of Dundee,Dundee DD1 5EH, UK. E-mail: n.r.stanleywall@dundee.ac.ukb Division of Mathematics, School of Science and Engineering, University of Dundee,Dundee DD1 4HN, UK. E-mail: f.a.davidson@dundee.ac.ukc School of Physics, University of Edinburgh, EH9 3FD, UKReceived 28th October 2019,Accepted 24th January 2020DOI: 10.1039/c9sm02141hrsc.li/soft-matter-journalSoft MatterCOMMENTOpen Access Article. Published on 24 March 2020. Downloaded on 4/1/2020 3:05:08 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View IssueThis journal is©The Royal Society of Chemistry 2020 Soft Matter, 2020, 16, 3344--3346 | 3345‘‘merging’’ may be a more appropriate term, and argue againstthe concept of ‘‘merging into a single colony’’, which impliesmixing. Indeed abuttal and true merging may be distinct out-comes – this requires deeper investigation in each case.Finally, in our opinion, the mathematical model presentedby Paul et al. does not support the conclusions of the paper. Thepaper proposes that biochemical signalling is responsible forcontrolling the interactions. This is in line with the work ofBe’er et al.12 The model of Be’er et al. includes explicit production,sensing and reaction to a diffusible toxin and demarcation results asan emergent feature of the interaction of these fields. On the otherhand, the model of Paul et al. includes no explicit reference to abiochemical signalling agent. Rather, the outcomes of their modelare governed by a nutrient-dependent mortality rate (see eqn (8)) thatis mediated by carefully selected time and space constraints. Thesetemporal and geometric conditions are necessary to locate the DL atthe interfacial gap between the colonies. These conditions thereforedrive the conclusions of the model and hence, with respect and inour opinion, represent a circular argument. The authors are clear intheir intention that the model should be general. It is our opinionthat this generality precludes further understanding of the under-lying mechanisms.It could well be that biochemical signalling is exactly themechanism that allows sibling colonies to determine both theirtemporal state and their relative spatial location. Investigatingthese hypotheses would be of great interest. We note however,that nutrient limitation in itself is sufficient to inhibit growth(see for example the models of ref. 10 and 11). In particular, inArnaouteli et al., 201913 we demonstrated that proximal siblingcolonies can have a marked effect on the growth dynamics of eachcolony where demarcation or abuttal was determined by nutrientlimitation (see e.g. Fig. 6c13). The experimental results in Arnaou-teli et al., 2019 were rationalised using a mathematical model thattook full account of the main components of the underlyinghypotheses (in that case the production of pulcherriminic acidto chelate free iron in the medium). Growth arrest was anemergent property of this model. Of particular interest is thatgrowth arrest of the expanding outer colony edge was observed insingle biofilms – no interactions were necessary (although inter-actions were considered). The mathematical model elucidatedthis phenomenon to be induced by a wave of iron depletion thatovertook the expanding colony margin. We therefore propose thatwhen studying colony interactions, self-limitation is a feature thatcannot be dismissed without further investigation.We fully agree with the authors that proximal growth can have adistinct effect on the physical appearance of biofilms at themacroscale. For example, when we inoculated four sibling biofilmsat sufficient distance to preclude abutting, a marked asymmetry ofradial growth and of material properties as indicated by changes inwrinkle structure was still observed (Fig. 1d, see also ref. 11). This issuggestive of long-range (biochemical) signal(s) being transmittedand received at the cellular level and manifest through macro-scaleremodelling of the biofilm architecture. However, our previouswork and the experiments detailed here, lead us to conclude thatin general the interaction between proximal biofilms is mediatedby a complex growth response. This response can be mediatedby separation and substrate stiffness, but is also dependent onmultiple other factors including inoculum density and size, genus,media composition and other experimental constraints.In conclusion, there is clear evidence that bacterial biofilmsinteract over short and long distances and that these interac-tions are in part governed by biochemical signalling. Moreover,truly understanding how microbes colonise and interact withtheir environment requires the scientific investigation of multi-biofilm assays. The physics/mathematics of soft matter com-munities are likely to have a significant input to future scien-tific studies in this area and contributions will best be made bycareful matching of experimental data and biological under-standing with mathematical models. This calibrated interdis-ciplinary approach is well-suited to shine further light on thefundamental role of microbes in the environment.MethodsBiofilm formationB. subtilis NCIB 3610 was grown on MSgg medium (5 mM potassiumphosphate and 100 mM MOPS at pH 7.0 supplemented withFig. 1 Interacting Bacillus subtilis biofilms. B. subtilis isolate NCIB 3610 wasinoculated on 1.5% (w/v) agar MSgg medium at 30 1C.14 The otherwise isogenicstrains were distinguished by expression of either mKate2 or gfp mut2. Thefounding cells of the biofilms were initially inoculated at a separation distanceof 10 mm and imaged at (a) 48 h, where a distinctive demarcation zone isvisible and indicated by a visible gap and resultant asymmetry of radial growthmanifest as flattened edges along the interaction zone [expressing gfp – leftand mKate2 – right]; and (b) at 96 hours, where the demarcation zone hasresolved and the visible gap has disappeared. The biofilms now seem to‘‘merge’’ as defined in Paul et al. (c) Merging of proximal biofilms using confocalmicroscopy maximum projection image for x–y plane (top panel) and z-plane(bottom panel) of interacting B. subtilis colonies [expressing gfp – left andmKate2 – right] at 48 hours, separated by an initial distance of 5 mm growingon 1.5% (w/v) agar MSgg medium at 30 1C. (d) B. subtilis sibling biofilms exhibitcomplex interactions. Four NCIB 3610 biofilm sibling colonies (two expressinggfp [top left and bottom right] and two expressing mKate2 [top right andbottom left]) were founded at 20 mm distance on 1.5% (w/v) agar MSggmedium at 30 1C and incubated for 96 h before imaging.Comment Soft MatterOpen Access Article. Published on 24 March 2020. Downloaded on 4/1/2020 3:05:08 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online3346 | Soft Matter, 2020, 16, 3344--3346 This journal is©The Royal Society of Chemistry 20202 mM MgCl2, 700 mM CaCl2, 50 mM MnCl2, 1 mM ZnCl2, 2 mMthiamine, 0.5% glycerol, 0.5% glutamate) solidified with 1.5%select agar (Invitrogen) at 30 1C for the indicated time points aspreviously described14 with the exception that 1 ml of culturewas used for biofilm initiation. Images of colony biofilms wererecorded using a Nikon D3200 digital camera mounted on a KaiserRS3XA copy stand or using a Leica MZ16FA stereomicroscope.Confocal microscopy4 ml of MSgg medium supplemented with 1.5% (w/v) agar wasplaced into a 35 mm diameter Petri dish and dried for 1 hour ina laminar flow hood. NCIB 3610 strain constitutively producingGFP-16 or mKate217 was spotted into the centre of the agar inthe Petri dish and incubated at 30 1C for the indicated timeperiod. A Leica SP8 upright confocal was used to image theedge of the biofilm using a 10 0.3 N.A. air objective and aheated chamber that was pre-warmed to 30 1C. A cling film tentwas draped from around the objective and tucked loosely underthe stage to eliminate airflow across the plate and minimisedehydration (and therefore shrinkage) of the agar. An additional35 mm diameter Petri dish was filled with water and placed nextto the biofilm plate to increase the humidity inside the tent. Anargon-ion laser was used to excite the GFP at 488 nm and2% power. Z-stacks capturing the full height of the biofilmborder were specified based on the presence of GFP-containingcells and planes of 1024  1024 pixels were acquired quicklyusing a resonant mirror, averaging 16 scans per line. Imageswere imported into an OMERO15 server and figures were pre-pared using OMERO figure (http://figure.openmicroscopy.org/).Conflicts of interestThere are no conflicts to declare.AcknowledgementsWork in the NSW, CEM, and FAD groups is supported bythe Biotechnology and Biological Sciences Research Council[BB/P001335/1; BB/R012415/1]. We would like to acknowledgethe Dundee Imaging Facility, Dundee, which is supported by the‘Wellcome Trust Technology Platform’ award [097945/B/11/Z]and the ‘MRC Next Generation Optical Microscopy’ award[MR/K015869/1]. Thank you to Akos Kovacs and colleagues forthe mKate labelled strain.References1 H. C. Flemming, J. Wingender, U. Szewzyk, P. Steinberg,S. A. Rice and S. Kjelleberg, Biofilms: an emergent form ofbacterial life, Nat. Rev. Microbiol., 2016, 14(9), 563–575.2 A. Dragos and A. T. Kovacs, The Peculiar Functions of theBacterial Extracellular Matrix, Trends Microbiol., 2017, 25(4),257–266.3 L. S. Cairns, L. Hobley and N. R. Stanley-Wall, Biofilmformation by Bacillus subtilis: new insights into regulatorystrategies and assembly mechanisms, Mol. Microbiol., 2014,93(4), 587–598.4 E. Nagar and R. Schwarz, To be or not to be planktonic? Self-inhibition of biofilm development, Environ. Microbiol.,2015, 17(5), 1477–1486.5 J. L. Connell, A. K. Wessel, M. R. Parsek, A. D. Ellington,M. Whiteley and J. B. Shear, Probing prokaryotic social behaviorswith bacterial ‘‘lobster traps’’, mBio, 2010, 1(4), e00202-10.6 A. Prindle, J. Liu, M. Asally, S. Ly, J. Garcia-Ojalvo andG. M. Suel, Ion channels enable electrical communicationin bacterial communities, Nature, 2015, 527(7576), 59–63.7 J. Liu, R. Martinez-Corral, A. Prindle, D. D. Lee, J. Larkin,M. Gabalda-Sagarra, J. Garcia-Ojalvo and G. M. Suel, Cou-pling between distant biofilms and emergence of nutrienttime-sharing, Science, 2017, 356(6338), 638–642.8 D. R. Espeso, E. Martinez-Garcia, V. de Lorenzo and A. Goni-Moreno, Physical Forces Shape Group Identity of SwimmingPseudomonas putida Cells, Front. Microbiol., 2016, 7, 1437.9 R. Paul, T. Ghosh, T. Tang and A. Kumar, Rivalry in Bacillussubtilis colonies: enemy or family?, Soft Matter, 2019, 15(27),5400–5411.10 P. Patra, C. N. Vassallo, D. Wall and O. A. Igoshin, Mecha-nism of Kin-Discriminatory Demarcation Line Formationbetween Colonies of Swarming Bacteria, Biophys. J., 2017,113(11), 2477–2486.11 A. Sekowska, J. B. Masson, A. Celani, A. Danchin andM. Vergassola, Repulsion and metabolic switches in thecollective behavior of bacterial colonies, Biophys. J., 2009,97(3), 688–698.12 A. Be’er, G. Ariel, O. Kalisman, Y. Helman, A. Sirota-Madi,H. P. Zhang, E. L. Florin, S. M. Payne, E. Ben-Jacob and H. L.Swinney, Lethal protein produced in response to competitionbetween sibling bacterial colonies, Proc. Natl. Acad. Sci. U. S. A.,2010, 107(14), 6258–6263.13 S. Arnaouteli, D. A. Matoz-Fernandez, M. Porter, M. Kalamara,J. Abbott, C. E. MacPhee, F. A. Davidson and N. R. Stanley-Wall,Pulcherrimin formation controls growth arrest of the Bacillussubtilis biofilm, Proc. Natl. Acad. Sci. U. S. A., 2019, 116(27),13553–13562.14 S. S. Branda, J. E. Gonzalez-Pastor, S. Ben-Yehuda, R. Losickand R. Kolter, Fruiting body formation by Bacillus subtilis,Proc. Natl. Acad. Sci. U. S. A., 2001, 98(20), 11621–11626.15 C. Allan, J. M. Burel, J. Moore, C. Blackburn, M. Linkert,S. Loynton, D. Macdonald, W. J. Moore, C. Neves, A. Patterson,M. Porter, A. Tarkowska, B. Loranger, J. Avondo, I. Lagerstedt,L. Lianas, S. Leo, K. Hands, R. T. Hay, A. Patwardhan, C. Best,G. J. Kleywegt, G. Zanetti and J. R. Swedlow, OMERO: flexible,model-driven data management for experimental biology, Nat.Methods, 2012, 9(3), 245–253.16 J. van Gestel, F. J. Weissing, O. P. Kuipers and A. T. Kovács,Density of founder cells affects spatial pattern formationand cooperation in Bacillus subtilis biofilms, ISME J., 2014,8(10), 2069–2079.17 D. T. Verhamme, T. B. Kiley and N. R. Stanley-Wall, DegUco-ordinates multicellular behaviour exhibited by Bacillussubtilis, Mol. Microbiol., 2007, 65, 554–568.Soft Matter CommentOpen Access Article. Published on 24 March 2020. Downloaded on 4/1/2020 3:05:08 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

https://discovery.dundee.ac.uk/ws/files/48561558/c9sm02141h.pdf

Comment on “Rivalry in <i>Bacillus subtilis</i> colonies:enemy or family?”

Abstract

Similar works

Full text

Available Versions

Discovery Research Portal

Discovery Research Portal

Discovery Research Portal