Abstract
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).
Highlights
Burkholderia glumae, the primary causal agent of bacterial panicle blight (BPB) of rice, is one of the most important disease problems affecting rice production in the southern United States, including Louisiana, Arkansas and Texas [1]
In Gram-negative bacteria, QS systems mediated by LuxI and LuxR-family proteins are involved in a diverse range of bacterial behaviors and traits, including formation of biofilm, production of virulence factors, conjugation, and antibiosis [9,10]
Mutations in tofI and tofR were confirmed with the biosensor strain, C. violaceum CV026, which produces the purple pigment, violacein, in the presence of acyl homoserine lactone (AHL) compounds, including C6-HSL and C8-HSL [20]
Summary
Burkholderia glumae, the primary causal agent of bacterial panicle blight (BPB) of rice, is one of the most important disease problems affecting rice production in the southern United States, including Louisiana, Arkansas and Texas [1]. This rice disease has been reported from many rice-growing areas around the world, including east Asia, southeast Asia and South America [1]. In Gram-negative bacteria, QS systems mediated by LuxI and LuxR-family proteins are involved in a diverse range of bacterial behaviors and traits, including formation of biofilm, production of virulence factors, conjugation, and antibiosis [9,10]. LuxI-family proteins are synthases that produce N-acyl homoserine lactone (AHL)-type intercellular signal molecules; LuxR-family proteins are cognate receptors that bind to the AHL molecules [13]
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