Dissection of Quorum-Sensing Genes in Burkholderia glumae Reveals Non-Canonical Regulation and the New Regulatory Gene tofM for Toxoflavin Production

Burkholderia glumae causes bacterial panicle blight of rice and produces major virulence factors, including toxoflavin, under the control of the quorum-sensing (QS) system mediated by the luxI homolog, tofI, and the luxR homolog, tofR. In this study, a series of markerless deletion mutants of B. glumae for tofI and tofR were generated using the suicide vector system, pKKSacB, for comprehensive characterization of the QS system of this pathogen. Consistent with the previous studies by other research groups, ΔtofI and ΔtofR strains of B. glumae did not produce toxoflavin in Luria-Bertani (LB) broth. However, these mutants produced high levels of toxoflavin when grown in a highly dense bacterial inoculum (∼ 1011 CFU/ml) on solid media, including LB agar and King’s B (KB) agar media. The ΔtofI/ΔtofR strain of B. glumae, LSUPB201, also produced toxoflavin on LB agar medium. These results indicate the presence of previously unknown regulatory pathways for the production of toxoflavin that are independent of tofI and/or tofR. Notably, the conserved open reading frame (locus tag: bglu_2g14480) located in the intergenic region between tofI and tofR was found to be essential for the production of toxoflavin by tofI and tofR mutants on solid media. This novel regulatory factor of B. glumae was named tofM after its homolog, rsaM, which was recently identified as a novel negative regulatory gene for the QS system of another rice pathogenic bacterium, Pseudomonas fuscovaginae. The ΔtofM strain of B. glumae, LSUPB286, produced a less amount of toxoflavin and showed attenuated virulence when compared with its wild type parental strain, 336gr-1, suggesting that tofM plays a positive role in toxoflavin production and virulence. In addition, the observed growth defect of the ΔtofI strain, LSUPB145, was restored by 1 µM N-octanoyl homoserine lactone (C8-HSL).

[1]  Vittorio Venturi,et al.  The virtue of temperance: built‐in negative regulators of quorum sensing in Pseudomonas , 2011, Molecular microbiology.

[2]  E. Greenberg,et al.  Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. , 2001, Annual review of genetics.

[3]  N. Schaad,et al.  Laboratory guide for identification of plant pathogenic bacteria , 1988 .

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

[5]  Hiroaki Suga,et al.  Quorum sensing and the LysR‐type transcriptional activator ToxR regulate toxoflavin biosynthesis and transport in Burkholderia glumae , 2004, Molecular microbiology.

[6]  A. Eberhard.  Inhibition and Activation of Bacterial Luciferase Synthesis , 1972, Journal of bacteriology.

[7]  P. Dunlap,et al.  Quorum regulation of luminescence in Vibrio fischeri. , 1999, Journal of molecular microbiology and biotechnology.

[8]  E. Greenberg,et al.  Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators , 1994, Journal of bacteriology.

[9]  Fumihiko Suzuki,et al.  Molecular characterization of the tox operon involved in toxoflavin biosynthesis of Burkholderia glumae , 2004, Journal of General Plant Pathology.

[10]  I. Hwang,et al.  Structural and Functional Analysis of Phytotoxin Toxoflavin-Degrading Enzyme , 2011, PloS one.

[11]  G. Bertani,et al.  Lysogeny at Mid-Twentieth Century: P1, P2, and Other Experimental Systems , 2004, Journal of bacteriology.

[12]  D. Roop,et al.  Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. , 1995, Gene.

[13]  D. Hanahan,et al.  Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[14]  G. Ditta,et al.  Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[15]  J M Pemberton,et al.  An improved suicide vector for construction of chromosomal insertion mutations in bacteria. , 1992, Gene.

[16]  J. Ham,et al.  Burkholderia glumae: next major pathogen of rice? , 2011, Molecular plant pathology.

[17]  M. Alexeyev.  The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria. , 1999, BioTechniques.

[18]  D. Helinski,et al.  Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Vittorio Venturi,et al.  The plant pathogen Pseudomonas fuscovaginae contains two conserved quorum sensing systems involved in virulence and negatively regulated by RsaL and the novel regulator RsaM. , 2011, Environmental microbiology.

[20]  A conserved two-component regulatory system, PidS/PidR, globally regulates pigmentation and virulence-related phenotypes of Burkholderia glumae. , 2012, Molecular plant pathology.

[21]  James J. Smith,et al.  Identification and Onion Pathogenicity of Burkholderia cepacia Complex Isolates from the Onion Rhizosphere and Onion Field Soil , 2008, Applied and Environmental Microbiology.

[22]  M. Nei,et al.  MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. , 2011, Molecular biology and evolution.

[23]  Vittorio Venturi,et al.  Involvement of a Quorum-Sensing-Regulated Lipase Secreted by a Clinical Isolate of Burkholderia glumae in Severe Disease Symptoms in Rice , 2007, Applied and Environmental Microbiology.

[24]  D. Groth,et al.  Burkholderia glumae and B. gladioli Cause Bacterial Panicle Blight in Rice in the Southern United States. , 2009, Plant disease.

[25]  A. Pühler,et al.  A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative Bacteria , 1983, Bio/Technology.

[26]  B. Bassler,et al.  Quorum sensing in bacteria. , 2001, Annual review of microbiology.

[27]  K. Iiyama,et al.  A role of phytotoxin in virulence of Pseudomonas glumae Kurita et Tabei , 1995 .

[28]  Jae Woo Han,et al.  Diversities in Virulence, Antifungal Activity, Pigmentation and DNA Fingerprint among Strains of Burkholderia glumae , 2012, PloS one.

[29]  B. Wanner,et al.  Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria. , 1996, Plasmid.

[30]  Minkyun Kim,et al.  Regulation of polar flagellum genes is mediated by quorum sensing and FlhDC in Burkholderia glumae , 2007, Molecular microbiology.

[31]  J H Lamb,et al.  Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. , 1997, Microbiology.