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Dissection of quorum-sensing genes in Burkholderia glumae reveals non-canonical regulation and the new regulatory gene tofM for toxoflavin production.

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, ?tofI and ?tofR 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 (? 10(11) CFU/ml) on solid media, including LB agar and King's B (KB) agar media. The ?tofI/?tofR 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 ?tofM 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 ?tofI strain, LSUPB145, was restored by 1 µM N-octanoyl homoserine lactone (C8-HSL).

SUBMITTER: Chen R

PROVIDER: S-EPMC3527420 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Dissection of quorum-sensing genes in Burkholderia glumae reveals non-canonical regulation and the new regulatory gene tofM for toxoflavin production.

Chen Ruoxi R Barphagha Inderjit K IK Karki Hari S HS Ham Jong Hyun JH

PloS one 20121220 12

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 resea ...[more]

PMID: 23284909

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Project description:BACKGROUND: Bacterial panicle blight caused by the bacterium Burkholderia glumae is an emerging disease of rice in the United States. Not much is known about this disease, the disease cycle or any source of disease resistance. To understand the interaction between rice and Burkholderia glumae, we used transcriptomics via next-generation sequencing (RNA-Seq) and bioinformatics to identify differentially expressed transcripts between resistant and susceptible interactions and formulate a model for rice resistance to the disease. RESULTS: Using inoculated young seedlings as sample tissues, we identified unique transcripts involved with resistance to bacterial panicle blight, including a PIF-like ORF1 and verified differential expression of some selected genes using qRT-PCR. These transcripts, which include resistance genes of the NBS-LRR type, kinases, transcription factors, transporters and expressed proteins with functions that are not known, have not been reported in other pathosystems including rice blast or bacterial blight. Further, functional annotation analysis reveals enrichment of defense response and programmed cell death (biological processes); ATP and protein binding (molecular functions); and mitochondrion-related (cell component) transcripts in the resistant interaction. CONCLUSION: Taken together, we formulated a model for rice resistance to bacterial panicle blight that involves an activation of previously unknown resistance genes and their activation partners upon challenge with B. glumae. Other interesting findings are that 1) though these resistance transcripts were up-regulated upon inoculation in the resistant interaction, some of them were already expressed in the water-inoculated control from the resistant genotype, but not in the water- and bacterium-inoculated samples from the susceptible genotype; 2) rice may have co-opted an ORF that was previously a part of a transposable element to aid in the resistance mechanism; and 3) resistance may have existed immediately prior to rice domestication.

Project description:Rhamnolipids are surface-active agents of microbial origin used as alternatives to synthetic surfactants. Burkholderia thailandensis is a non-pathogenic rhamnolipid-producing bacterium that could represent an interesting candidate for use in commercial processes. However, current bioprocesses for rhamnolipid production by this bacterium are not efficient enough, mainly due to low yields. Since regulation of rhamnolipid biosynthesis in B. thailandensis remains poorly understood, identifying new regulatory factors could help increase the production of these valuable metabolites. We performed a random transposon mutagenesis screening to identify genes directing rhamnolipid production in B. thailandensis E264. The most efficient rhamnolipid producer we identified harbored an inactivating transposon insertion in the scmR gene, which was recently described to encode as a secondary metabolite regulator in B. thailandensis. We investigated the impact of scmR loss on rhamnolipid biosynthesis and cell growth. Because biosynthesis of rhamnolipids and polyhydroxyalkanoates (PHAs) could share the same pool of lipid precursors, we also investigate the effect of ScmR on PHA production. We found that production of both rhamnolipids and PHAs are modulated by ScmR during the logarithmic growth phase and demonstrate that ScmR downregulates the production of rhamnolipids by affecting the expression of both rhl biosynthetic operons. Furthermore, our results indicate that PHA biosynthesis is reduced in the scmR- mutant, as ScmR promotes the transcription of the phaC and phaZ genes. By studying the relationship between ScmR and quorum sensing (QS) regulation we reveal that QS acts as an activator of scmR transcription. Finally, we pinpoint the QS-3 system as being involved in the regulation of rhamnolipid and PHA biosynthesis. We conclude that ScmR negatively affects rhamnolipid production, whereas it positively impacts PHAs biosynthesis. This could provide an interesting approach for future strain engineering, leading to improved yields of these valuable metabolites.

Dataset Information

Dissection of quorum-sensing genes in Burkholderia glumae reveals non-canonical regulation and the new regulatory gene tofM for toxoflavin production.

Publications

Dissection of quorum-sensing genes in Burkholderia glumae reveals non-canonical regulation and the new regulatory gene tofM for toxoflavin production.

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