Jinwoo Kim

Quorum sensing and the LysR-type transcriptional activator ToxR regulate toxoflavin biosynthesis and transport in Burkholderia glumae.

Burkholderia glumae BGR1 produces a broad‐host range phytotoxin, called toxoflavin, which is a key pathogenicity factor in rice grain rot and wilt in many field crops. Our molecular and genetic analyses of toxoflavin‐deficient mutants demonstrated that gene clusters for toxoflavin production consist of four transcriptional units. The toxoflavin biosynthesis genes were composed of five genes, toxA to toxE, as Suzuki etu2003al. (2004) reported previously. Genes toxF to toxI, which are responsible for toxoflavin transport, were polycistronic and similar to the genes for resistance‐nodulation‐division (RND) efflux systems. Using Tn3‐gusA reporter fusions, we found that ToxR, a LysR‐type regulator, regulates both the toxABCDE and toxFGHI operons in the presence of toxoflavin as a coinducer. In addition, the expression of both operons required a transcriptional activator, ToxJ, whose expression is regulated by quorum sensing. TofI, a LuxI homologue, was responsible for the biosynthesis of both N‐hexanoyl homoserine lactone and N‐octanoyl homoserine lactone (C8‐HSL). C8‐HSL and its cognate receptor TofR, a LuxR homologue, activated toxJ expression. This is the first report that quorum sensing is involved in pathogenicity by the regulation of phytotoxin biosynthesis and its transport in plant pathogenic bacteria.

Pyrroloquinoline Quinone Is a Plant Growth Promotion Factor Produced by Pseudomonas fluorescens B16

Pseudomonas fluorescens B16 is a plant growth-promoting rhizobacterium. To determine the factors involved in plant growth promotion by this organism, we mutagenized wild-type strain B16 using ΩKm elements and isolated one mutant, K818, which is defective in plant growth promotion, in a rockwool culture system. A cosmid clone, pOK40, which complements the mutant K818, was isolated from a genomic library of the parent strain. Tn3-gusA mutagenesis of pOK40 revealed that the genes responsible for plant growth promotion reside in a 13.3-kb BamHI fragment. Analysis of the DNA sequence of the fragment identified 11 putative open reading frames, consisting of seven known and four previously unidentified pyrroloquinoline quinone (PQQ) biosynthetic genes. All of the pqq genes showed expression only in nutrient-limiting conditions in a PqqH-dependent manner. Electrospray ionization-mass spectrometry analysis of culture filtrates confirmed that wild-type B16 produces PQQ, whereas mutants defective in plant growth promotion do not. Application of wild-type B16 on tomato (Solanum lycopersicum) plants cultivated in a hydroponic culture system significantly increased the height, flower number, fruit number, and total fruit weight, whereas none of the strains that did not produce PQQ promoted tomato growth. Furthermore, 5 to 1,000 nm of synthetic PQQ conferred a significant increase in the fresh weight of cucumber (Cucumis sativus) seedlings, confirming that PQQ is a plant growth promotion factor. Treatment of cucumber leaf discs with PQQ and wild-type B16 resulted in the scavenging of reactive oxygen species and hydrogen peroxide, suggesting that PQQ acts as an antioxidant in plants.

Toxoflavin Produced by Burkholderia glumae Causing Rice Grain Rot Is Responsible for Inducing Bacterial Wilt in Many Field Crops

Severe wilt symptoms similar to bacterial wilt caused by Ralstonia solanacearum were observed in tomato, hot pepper, eggplant, potato, perilla, sesame, and sunflower in 2000 and 2001 in Korea. From diseased crops at 65 different locations, we obtained 106 isolates that produced green pigment on CPG medium; 36 were isolated from discolored rice panicles. The causal pathogen was identified as Burkholderia glumae based on its biochemical characteristics, fatty acid methyl ester analysis, and 16S rRNA gene sequence. Nine representative isolates produced toxoflavin, as determined by electrospray ionization mass spectrometry using a direct inlet system and TLC analyses, and caused bacterial wilt on tomato, sesame, perilla, eggplant, and hot pepper. However, BGR12, a wild-type isolate lacking toxoflavin production and toxoflavin-deficient mutants generated by Tn5lacZ failed to cause bacterial wilt on those five field crops. Cells of B. glumae and synthetic toxoflavin caused wilt symptoms on field crops, demonstrating a lack of host specificity. Synthetic toxoflavin caused wilt symptoms on tomato, sesame, perilla, eggplant, and hot pepper at 10 μg/ml concentration 1 day after treatment. This is the first report of bacterial wilt on various crops caused by B. glumae, and our results clearly demonstrate that toxoflavin is a key factor in wilt symptom development.

Regulation of polar flagellum genes is mediated by quorum sensing and FlhDC in Burkholderia glumae.

The bacterium Burkholderia glumae causes rice grain rot by producing toxoflavin, whose expression is regulated by quorum sensing (QS). We report a major deviation from the current paradigm for the regulation of bacterial polar flagellum genes. The N‐octanoyl homoserine lactone (C8‐HSL)‐deficient mutant of B.u2003glumae is aflagellate and has lost the ability to swim and swarm at 37°C. Mutagenesis of the bacterium with the mini‐Tn5rescue identified an IclR‐type transcriptional regulator, called QsmR, which is important for flagellum formation. TofR, which is a cognate C8‐HSL receptor, activated qsmR expression by binding directly to the qsmR promoter region. From the flagellum gene cluster, we identified flhDC homologues that are directly activated by QsmR. FlhDC in turn activates the expression of genes involved in flagellum biosynthesis, motor functions and chemotaxis in B.u2003glumae. Non‐motile qsmR, fliA and flhDC mutants produced toxoflavin, but lost pathogenicity for rice. The unexpected discovery of FlhDC in a polarly flagellate bacterium suggests that exceptions to the typical regulatory mechanisms of flagellum genes exist in Gram‐negative bacteria. The finding that functional flagella play critical roles in the pathogenicity of B.u2003glumae suggests that either QS or flagellum formation constitutes a good target for the control of rice grain rot.

Genetic Diversity and Distribution of Korean Isolates of Ralstonia solanacearum

Genetic diversity among 478 isolates of Ralstonia solanacearum collected from various plants in Korea between 1997 and 2005 was determined based on biovar, pathogenicity, amplified fragment length polymorphism (AFLP), 16S rRNA, endoglucanase, hrpB, and mutS gene sequence analyses. Of the isolates, 440 belonged to biovars 1, 3, or 4, and 38 belonged to biovar 2. Biovar N2 isolates were not found. The biovar 1 and 2 isolates were found mainly in southern Korea, whereas the biovar 3 and 4 isolates were widely distributed throughout all nine provinces. AFLP analysis divided the 109 representative Korean isolates into six clusters that were distinct from most of the foreign isolates. Grouping of 8 representative isolates based on their 16S rRNA gene sequences indicated that biovars 1, 3, and 4 belonged to division 1, while biovar 2 belonged to subdivision 2b. Sequence analysis of the endoglucanase, hrpB, and mutS genes from the same isolates indicated that the biovar 1, 3, and 4 isolates belonged to phylotype I, while the biovar 2 isolate belonged to phylotype IV. This study is the first comprehensive analysis of genetic diversity among Korean isolates of R. solanacearum.

Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase

Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In Gram-negative bacteria, QS is often mediated by N-acyl-l-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the Gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-l-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein. Moreover, the reaction byproduct, 5′-methylthioadenosine, independently binds to the binding site for a second substrate, S-adenosyl-l-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents.

Complete Genome Sequence of Burkholderia gladioli BSR3

We report the complete genome sequence of Burkholderia gladioli BSR3, isolated from a diseased rice sheath in South Korea.

The Quorum Sensing-Dependent Gene katG of Burkholderia glumae Is Important for Protection from Visible Light

Quorum sensing (QS) plays important roles in the pathogenicity of Burkholderia glumae, the causative agent of bacterial rice grain rot. We determined how QS is involved in catalase expression in B. glumae. The QS-defective mutant of B. glumae exhibited less catalase activity than wild-type B. glumae. A beta-glucuronidase assay of a katG::Tn3-gusA78 reporter fusion protein revealed that katG expression is under the control of QS. Furthermore, katG expression was upregulated by QsmR, a transcriptional activator for flagellar-gene expression that is regulated by QS. A gel mobility shift assay confirmed that QsmR directly activates katG expression. The katG mutant produced toxoflavin but exhibited less severe disease than BGR1 on rice panicles. Under visible light conditions and a photon flux density of 61.6 micromol(-1) m(-2), the survival rate of the katG mutant was 10(5)-fold lower than that of BGR1. This suggests that KatG is a major catalase that protects bacterial cells from visible light, which probably results in less severe disease caused by the katG mutant.

Biochemical Evidence for ToxR and ToxJ Binding to the tox Operons of Burkholderia glumae and Mutational Analysis of ToxR

Burkholderia glumae produces toxoflavin, a phytotoxin with a broad host range, which is a key virulence factor in bacterial rice grain rot. Based on genetic analysis, we previously reported that ToxR, a LysR-type regulator, activates both the toxABCDE (toxoflavin biosynthesis genes) and toxFGHI (toxoflavin transporter genes) operons in the presence of toxoflavin as a coinducer. Quorum sensing regulates the expression of the transcriptional activator ToxJ that is required for tox gene expression. Here, we used gel mobility shift and DNase I protection analyses to demonstrate that both ToxR and ToxJ bind simultaneously to the regulatory regions of both tox operons. ToxR and ToxJ both bound to the toxA and toxF regulatory regions, and the sequences for the binding of ToxR to the regulatory regions of both tox operons possessed T-N(11)-A motifs. Following random mutagenesis of toxR, 10 ToxR mutants were isolated. We constructed a reporter strain, S6K34 (toxRA::Omega toxF::Tn3-gusA34) to evaluate which amino acid residues are important for ToxR activity. Several single amino acid substitutions identified residues that might be important for ToxR binding to DNA and toxoflavin binding. When various toxoflavin derivatives were tested to determine whether toxoflavin is a specific coinducer of ToxR in the S6K34 strain, ToxR, together with toxoflavin, conferred toxF expression, whereas 4,8-dihydrotoxoflavin did so only slightly. With these results, we have demonstrated biochemically that B. glumae cells control toxoflavin production tightly by the requirement of both ToxJ and toxoflavin as coinducers of ToxR.

Quorum Sensing Controls Flagellar Morphogenesis in Burkholderia glumae

Burkholderia glumae is a motile plant pathogenic bacterium that has multiple polar flagella and one LuxR/LuxI-type quorum sensing (QS) system, TofR/TofI. A QS-dependent transcriptional regulator, QsmR, activates flagellar master regulator flhDC genes. FlhDC subsequently activates flagellar gene expression in B. glumae at 37°C. Here, we confirm that the interplay between QS and temperature is critical for normal polar flagellar morphogenesis in B. glumae. In the wild-type bacterium, flagellar gene expression and flagellar number were greater at 28°C compared to 37°C. The QS-dependent flhC gene was significantly expressed at 28°C in two QS-defective (tofI::Ω and qsmR::Ω) mutants. Thus, flagella were present in both tofI::Ω and qsmR::Ω mutants at 28°C, but were absent at 37°C. Most tofI::Ω and qsmR::Ω mutant cells possessed polar or nonpolar flagella at 28°C. Nonpolarly flagellated cells processing flagella around cell surface of both tofI::Ω and qsmR::Ω mutants exhibited tumbling and spinning movements. The flhF gene encoding GTPase involved in regulating the correct placement of flagella in other bacteria was expressed in QS mutants in a FlhDC-dependent manner at 28°C. However, FlhF was mislocalized in QS mutants, and was associated with nonpolar flagellar formation in QS mutants at 28°C. These results indicate that QS-independent expression of flagellar genes at 28°C allows flagellar biogenesis, but is not sufficient for normal polar flagellar morphogenesis in B. glumae. Our findings demonstrate that QS functions together with temperature to control flagellar morphogenesis in B. glumae.