Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers.

The non-domesticated Bacillus subtilis strain 3610 displays, over a wide range of humidity, hyper-branched, dendritic, swarming-like migration on a minimal agar medium. At high (70 %) humidity, the laboratory strain 168 sfp+ (producing surfactin) behaves very similarly, although this strain carries a frameshift mutation in swrA, which another group has shown under their conditions (which include low humidity) is essential for swarming. We reconcile these different results by demonstrating that, while swrA is essential for dendritic migration at low humidity (30-40 %), it is dispensable at high humidity. Dendritic migration (flagella- and surfactin-dependent) of strains 168 sfp+ swrA and 3610 involves elongation of dendrites for several hours as a monolayer of cells in a thin fluid film. This enabled us to determine in situ the spatiotemporal pattern of expression of some key players in migration as dendrites develop, using gfp transcriptional fusions for hag (encoding flagellin), comA (regulation of surfactin synthesis) as well as eps (exopolysaccharide synthesis). Quantitative (single-cell) analysis of hag expression in situ revealed three spatially separated subpopulations or cell types: (i) networks of chains arising early in the mother colony (MC), expressing eps but not hag; (ii) largely immobile cells in dendrite stems expressing intermediate levels of hag; and (iii) a subpopulation of cells with several distinctive features, including very low comA expression but hyper-expression of hag (and flagella). These specialized cells emerge from the MC to spearhead the terminal 1 mm of dendrite tips as swirling and streaming packs, a major characteristic of swarming migration. We discuss a model for this swarming process, emphasizing the importance of population density and of the complementary roles of packs of swarmers driving dendrite extension, while non-mobile cells in the stems extend dendrites by multiplication.

[1]  R. Losick,et al.  Cell population heterogeneity during growth of Bacillus subtilis. , 2005, Genes & development.

[2]  J. O'Brien,et al.  Genetic Organization of the Region Encoding Regulation, Biosynthesis, and Transport of Rhizobactin 1021, a Siderophore Produced by Sinorhizobium meliloti , 2001, Journal of bacteriology.

[3]  L. Limat,et al.  Spreading of liquid drops on Agar gels , 2009 .

[4]  I. Aranson,et al.  Model for dynamical coherence in thin films of self-propelled microorganisms. , 2007, Physical review. E, Statistical, nonlinear, and soft matter physics.

[5]  S. Séror,et al.  In situ localisation and quantification of surfactins in a Bacillus subtilis swarming community by imaging mass spectrometry , 2008, Proteomics.

[6]  Kazuo Kobayashi.  SlrR/SlrA controls the initiation of biofilm formation in Bacillus subtilis , 2008, Molecular microbiology.

[7]  K. Bisset.  The zonation phenomenon and structure of the swarm colony in Proteus mirabilis. , 1973, Journal of medical microbiology.

[8]  R. Losick,et al.  Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility , 2004, Molecular microbiology.

[9]  H. Berg,et al.  The upper surface of an Escherichia coli swarm is stationary , 2009, Proceedings of the National Academy of Sciences.

[10]  R. Losick,et al.  A master regulator for biofilm formation by Bacillus subtilis , 2004, Molecular microbiology.

[11]  Leo Eberl,et al.  Surface Motility of Serratia liquefaciens MG1 , 1999, Journal of bacteriology.

[12]  Howard C. Berg,et al.  Visualization of Flagella during Bacterial Swarming , 2010, Journal of bacteriology.

[13]  R. Harshey,et al.  Bacterial motility on a surface: many ways to a common goal. , 2003, Annual review of microbiology.

[14]  A. Kornberg,et al.  Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D. Weibel,et al.  Studying the Dynamics of Flagella in Multicellular Communities of Escherichia coli by Using Biarsenical Dyes , 2009, Applied and Environmental Microbiology.

[16]  H. Vlamakis,et al.  Biofilm development with an emphasis on Bacillus subtilis. , 2008, Current topics in microbiology and immunology.

[17]  R. Losick,et al.  Fruiting body formation by Bacillus subtilis , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[18]  L. McCarter.  Bacterial Acrobatics on a Surface: Swirling Packs, Collisions, and Reversals during Swarming , 2010, Journal of bacteriology.

[19]  H. Berg,et al.  Dynamics of bacterial swarming. , 2010, Biophysical journal.

[20]  Michael D. Abràmoff,et al.  Image processing with ImageJ , 2004 .

[21]  R. Losick,et al.  Paralogous antirepressors acting on the master regulator for biofilm formation in Bacillus subtilis , 2009, Molecular microbiology.

[22]  Kazuo Kobayashi.  Bacillus subtilis Pellicle Formation Proceeds through Genetically Defined Morphological Changes , 2007, Journal of bacteriology.

[23]  Roberto Kolter,et al.  Control of cell fate by the formation of an architecturally complex bacterial community. , 2008, Genes & development.

[24]  J. Henrichsen,et al.  Bacterial surface translocation: a survey and a classification. , 1972, Bacteriological reviews.

[25]  H. Berg,et al.  A Molecular Clutch Disables Flagella in the Bacillus subtilis Biofilm , 2008, Science.

[26]  R. Chen,et al.  Role of the σD-Dependent Autolysins in Bacillus subtilis Population Heterogeneity , 2008, Journal of bacteriology.

[27]  S. Séror,et al.  Comparative Analysis of the Development of Swarming Communities of Bacillus subtilis 168 and a Natural Wild Type: Critical Effects of Surfactin and the Composition of the Medium , 2005, Journal of bacteriology.

[28]  R. Losick,et al.  Targets of the master regulator of biofilm formation in Bacillus subtilis , 2006, Molecular microbiology.

[29]  P. Stragier,et al.  Plasmids for ectopic integration in Bacillus subtilis. , 1996, Gene.

[30]  R. Losick,et al.  A novel regulatory protein governing biofilm formation in Bacillus subtilis , 2008, Molecular microbiology.

[31]  I. Aranson,et al.  Concentration dependence of the collective dynamics of swimming bacteria. , 2007, Physical review letters.

[32]  S. Engelmann,et al.  Expression of a stress- and starvation-induced dps/pexB-homologous gene is controlled by the alternative sigma factor sigmaB in Bacillus subtilis , 1997, Journal of bacteriology.

[33]  Roberto Kolter,et al.  Extracellular signals that define distinct and coexisting cell fates in Bacillus subtilis. , 2010, FEMS microbiology reviews.

[34]  R. Losick,et al.  Swarming motility in undomesticated Bacillus subtilis , 2003, Molecular microbiology.

[35]  Loralyn M. Cozy,et al.  Gene position in a long operon governs motility development in Bacillus subtilis , 2010, Molecular microbiology.

[36]  R. Marti,et al.  The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties , 2006, Archives of Microbiology.

[37]  Benoît Perthame,et al.  Models of Self-Organizing Bacterial Communities and Comparisons with Experimental Observations , 2010 .

[38]  G. O’Toole,et al.  Rhamnolipids Modulate Swarming Motility Patterns of Pseudomonas aeruginosa , 2005, Journal of bacteriology.

[39]  Joyce E. Patrick,et al.  Laboratory Strains of Bacillus subtilis Do Not Exhibit Swarming Motility , 2009, Journal of bacteriology.

[40]  G. Fraser,et al.  Swarming motility. , 1999, Current opinion in microbiology.

[41]  M. Nakano,et al.  Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis , 1992, Molecular and General Genetics MGG.

[42]  C J Weijer,et al.  Periodic phenomena in Proteus mirabilis swarm colony development , 1996, Journal of bacteriology.

[43]  S. Séror,et al.  Branched swarming patterns on a synthetic medium formed by wild-type Bacillus subtilis strain 3610: detection of different cellular morphologies and constellations of cells as the complex architecture develops. , 2004, Microbiology.

[44]  A. Sękowska,et al.  Identification of genes required for different stages of dendritic swarming in Bacillus subtilis, with a novel role for phrC. , 2009, Microbiology.

[45]  I. Tuval,et al.  Bacterial swimming and oxygen transport near contact lines. , 2005, Proceedings of the National Academy of Sciences of the United States of America.