Gamma-polyglutamic acid (gamma-PGA) produced by Bacillus amyloliquefaciens C06 promoting its colonization on fruit surface.

Bacillus amyloliquefaciens C06, an effective biological agent in controlling brown rot of stone fruit caused by Monilinia fructicola, was also found to produce extra-cellular mucilage and form mucoid colonies on semi-solid surfaces. This study aimed to characterize the extra-cellular mucilage produced by B. amyloliquefaciens C06 using transposon mutagenesis and biochemical and physical analyses. The mucilage production in B. amyloliquefaciens C06 was demonstrated to be associated with ywsC gene expression and characterized to be of high molecular weight, consisted of only glutamic acid and linked with non-peptide bonds, thus identified as gamma-polyglutamic acid (gamma-PGA). Compared with wild type B. amyloliquefaciens C06, its mutants deficient in producing gamma-PGA, e.g. M106 and C06DeltaywsC showed less efficiency in biofilm formation, surface adhesion and swarming ability. It was also demonstrated that gamma-PGA was not essential for C06 to form colony on semi-solid surfaces, but was able to improve its colony structure. In vivo evaluation showed that disruption of gamma-PGA production in C06DeltaywsC impaired its efficiency of colonizing apple surfaces.

[1]  T. Chin-A-Woeng,et al.  Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot. , 2000, Molecular plant-microbe interactions : MPMI.

[2]  J. Spizizen,et al.  TRANSFORMATION OF BIOCHEMICALLY DEFICIENT STRAINS OF BACILLUS SUBTILIS BY DEOXYRIBONUCLEATE. , 1958, Proceedings of the National Academy of Sciences of the United States of America.

[3]  C. B. Thorne,et al.  EFFECTS OF SOME METALLIC IONS ON GLUTAMYL POLYPEPTIDE SYNTHESIS BY BACILLUS SUBTILIS , 1958, Journal of bacteriology.

[4]  C. Kirschner,et al.  The role of intermolecular interactions: studies on model systems for bacterial biofilms. , 1999, International journal of biological macromolecules.

[5]  Å. Henriksson,et al.  Adhesion of bacillus spores in relation to hydrophobicity. , 1990, The Journal of applied bacteriology.

[6]  G. Bertani,et al.  STUDIES ON LYSOGENESIS I , 1951, Journal of bacteriology.

[7]  U. Rönner,et al.  Forces involved in adhesion of Bacillus cereus spores to solid surfaces under different environmental conditions. , 1990, The Journal of applied bacteriology.

[8]  R. Fall,et al.  Rapid Surface Motility in Bacillus subtilis Is Dependent on Extracellular Surfactin and Potassium Ion , 2003, Journal of bacteriology.

[9]  M. Jiang,et al.  Efficient production of poly(γ-glutamic acid) by newly isolated Bacillus subtilis NX-2 , 2005 .

[10]  I. Shih,et al.  The production of poly-(γ-glutamic acid) from microorganisms and its various applications , 2001 .

[11]  A. Fouet,et al.  Poly‐gamma‐glutamate in bacteria , 2006, Molecular microbiology.

[12]  B. Lazazzera,et al.  Defining the genetic differences between wild and domestic strains of Bacillus subtilis that affect poly‐γ‐dl‐glutamic acid production and biofilm formation , 2005, Molecular microbiology.

[13]  Mitsugu Matsushita,et al.  Morphological Changes in Growth Phenomena of Bacterial Colony Patterns , 1992 .

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

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

[16]  R. Gross,et al.  γ-Poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies , 1994 .

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

[18]  R. Harshey,et al.  Bees aren't the only ones: swarming in Gram‐negative bacteria , 1994, Molecular microbiology.

[19]  A. Galizzi,et al.  Surface-Associated Flagellum Formation and Swarming Differentiation in Bacillus subtilis Are Controlled by the ifm Locus , 2004, Journal of bacteriology.

[20]  T. Mascher,et al.  Regulatory Overlap and Functional Redundancy among Bacillus subtilis Extracytoplasmic Function σ Factors , 2007, Journal of bacteriology.

[21]  Monica Gupta,et al.  Epr plays a key role in DegU-mediated swarming motility of Bacillus subtilis. , 2009, FEMS microbiology letters.

[22]  W. Tsai,et al.  Surface characteristics of Bacillus cereus and its adhesion to stainless steel. , 2001, International journal of food microbiology.

[23]  S. G. Waley The structure of bacterial polyglutamic acid , 1955 .

[24]  S. Perotto,et al.  Mucoid mutants of the biocontrol strain pseudomonas fluorescens CHA0 show increased ability in biofilm formation on mycorrhizal and nonmycorrhizal carrot roots. , 2001, Molecular plant-microbe interactions : MPMI.

[25]  A. Sloma,et al.  Extracellular Proteolytic Activity Plays a Central Role in Swarming Motility in Bacillus subtilis , 2004, Journal of bacteriology.

[26]  Y. Le Breton,et al.  In Vivo Random Mutagenesis of Bacillus subtilis by Use of TnYLB-1, a mariner-Based Transposon , 2006, Applied and Environmental Microbiology.

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

[28]  G. Patra,et al.  Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization , 2004, BMC Genomics.

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

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

[31]  T. Zhou,et al.  Development of biocontrol agents from food microbial isolates for controlling post-harvest peach brown rot caused by Monilinia fructicola. , 2008, International journal of food microbiology.

[32]  S. Anand,et al.  Swarming: A coordinated bacterial activity , 2002 .

[33]  G. Amati,et al.  SwrAA activates poly-gamma-glutamate synthesis in addition to swarming in Bacillus subtilis. , 2009, Microbiology.

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

[35]  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.

[36]  S. Tokuyama,et al.  Characterization of the Bacillus subtilis ywsC Gene, Involved in γ-Polyglutamic Acid Production , 2002 .

[37]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[38]  Roberto Kolter,et al.  Biofilms: the matrix revisited. , 2005, Trends in microbiology.