Structural and Functional Analysis of Phytotoxin Toxoflavin-Degrading Enzyme

Pathogenic bacteria synthesize and secrete toxic low molecular weight compounds as virulence factors. These microbial toxins play essential roles in the pathogenicity of bacteria in various hosts, and are emerging as targets for antivirulence strategies. Toxoflavin, a phytotoxin produced by Burkholderia glumae BGR1, has been known to be the key factor in rice grain rot and wilt in many field crops. Recently, toxoflavin-degrading enzyme (TxDE) was identified from Paenibacillus polymyxa JH2, thereby providing a possible antivirulence strategy for toxoflavin-mediated plant diseases. Here, we report the crystal structure of TxDE in the substrate-free form and in complex with toxoflavin, along with the results of a functional analysis. The overall structure of TxDE is similar to those of the vicinal oxygen chelate superfamily of metalloenzymes, despite the lack of apparent sequence identity. The active site is located at the end of the hydrophobic channel, 9 Å in length, and contains a Mn(II) ion interacting with one histidine residue, two glutamate residues, and three water molecules in an octahedral coordination. In the complex, toxoflavin binds in the hydrophobic active site, specifically the Mn(II)-coordination shell by replacing a ligating water molecule. A functional analysis indicated that TxDE catalyzes the degradation of toxoflavin in a manner dependent on oxygen, Mn(II), and the reducing agent dithiothreitol. These results provide the structural features of TxDE and the early events in catalysis

Hierarchical regulation of Burkholderia glumae type III secretion system by GluR response regulator and Lon protease

Expression of type III secretion system (T3SS) genes, which are important for the virulence of phytopathogenic bacteria, is induced in the plant apoplastic environment or artificially amended growth conditions. Wild-type Burkholderia glumae BGR1, which causes rice panicle blight, induced a hypersensitive response (HR) in tobacco plants, whereas the T3SS genes were not significantly expressed in the commonly used hrp induction medium. T3SS gene expression in B. glumae was dependent on HrpB, a well known T3SS gene transcriptional regulator. Here, we report a stepwise mechanism of T3SS gene regulation by the GluR response regulator and Lon protease in addition to HrpB-mediated control of T3SS genes in B. glumae. The gluR mutant showed no HR in tobacco plants and exhibited attenuated virulence in rice plants. GluR directly activated hrpB expression, indicating that hrpB belongs to the GluR regulon. The lon mutation allowed high expression of the T3SS genes in nutrient-rich media. Lon directly activated gluR expression but repressed hrpB expression, indicating that Lon acts as a regulator rather than a protease. However, the lon mutant failed to induce an HR and virulence, suggesting that Lon not only acts as a negative regulator, but also has an essential, yet to be determined role for T3SS. Our results demonstrate the involvement of the two-component system response regulator GluR and Lon in T3SS gene regulation, providing new insight into the complex interplay mechanisms of regulators involved in T3SS gene expression in bacteria-plant interactions.N

Adverse effects of adaptive mutation to survive static culture conditions on successful fitness of the rice pathogen Burkholderia glumae in a host.

Bacteria often possess relatively flexible genome structures and adaptive genetic variants that allow survival in unfavorable growth conditions. Bacterial survival tactics in disadvantageous microenvironments include mutations that are beneficial against threats in their niche. Here, we report that the aerobic rice bacterial pathogen Burkholderia glumae BGR1 changes a specific gene for improved survival in static culture conditions. Static culture triggered formation of colony variants with deletions or point mutations in the gene bspP (BGLU_RS28885), which putatively encodes a protein that contains PDC2, PAS-9, SpoIIE, and HATPase domains. The null mutant of bspP survived longer in static culture conditions and produced a higher level of bis-(3'-5')-cyclic dimeric guanosine monophosphate than the wild type. Expression of the bacterial cellulose synthase regulator (bcsB) gene was upregulated in the mutant, consistent with the observation that the mutant formed pellicles faster than the wild type. Mature pellicle formation was observed in the bspP mutant before pellicle formation in wild-type BGR1. However, the population density of the bspP null mutant decreased substantially when grown in Luria-Bertani medium with vigorous agitation due to failure of oxalate-mediated detoxification of the alkaline environment. The bspP null mutant was less virulent and exhibited less effective colonization of rice plants than the wild type. All phenotypes caused by mutations in bspP were recovered to those of the wild type by genetic complementation. Thus, although wild-type B. glumae BGR1 prolonged viability by spontaneous mutation under static culture conditions, such genetic changes negatively affected colonization in rice plants. These results suggest that adaptive gene sacrifice of B. glumae to survive unfavorable growth conditions is not always desirable as it can adversely affect adaptability in the host

Influence of genomic structural variations and nutritional conditions on the emergence of quorum sensing-dependent gene regulation defects in Burkholderia glumae

Bacteria often change their genetic and physiological traits to survive in harsh environments. To determine whether, in various strains of Burkholderia glumae, genomic diversity is associated with the ability to adapt to ever-changing environments, whole genomes of 44 isolates from different hosts and regions were analyzed. Whole-genome phylogenetic analysis of the 44 isolates revealed six clusters and two divisions. While all isolates possessed chromosomes 1 and 2, strains BGR80S and BGR81S had one chromosome resulting from the merging of the two chromosomes. Upon comparison of genomic structures to the prototype BGR1, inversions, deletions, and rearrangements were found within or between chromosomes 1 and/or 2 in the other isolates. When three isolates-BGR80S, BGR15S, and BGR21S, representing clusters III, IV, and VI, respectively-were grown in Luria-Bertani medium, spontaneous null mutations were identified in qsmR encoding a quorum-sensing master regulator. Six days after subculture, qsmR mutants were found at detectable frequencies in BGR15S and BGR21S, and reached approximately 40% at 8 days after subculture. However, the qsmR mutants appeared 2 days after subculture in BGR80S and dominated the population, reaching almost 80%. No qsmR mutant was detected at detectable frequency in BGR1 or BGR13S. The spontaneous qsmR mutants outcompeted their parental strains in the co-culture. Daily addition of glucose or casamino acids to the batch cultures of BGR80S delayed emergence of qsmR mutants and significantly reduced their incidence. These results indicate that spontaneous qsmR mutations are correlated with genomic structures and nutritional conditions.N

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

Lethal Consequences of Overcoming Metabolic Restrictions Imposed on a Cooperative Bacterial Population

Quorum sensing (QS) controls cooperative activities in many Proteobacteria. In some species, QS-dependent specific metabolism contributes to the stability of the cooperation. However, the mechanism by which QS and metabolic networks have coevolved to support stable public good cooperation and maintenance of the cooperative group remains unknown. Here we explored the underlying mechanisms of QS-controlled central metabolism in the evolutionary aspects of cooperation. In Burkholderia glumae, the QS-dependent glyoxylate cycle plays an important role in cooperativity. A bifunctional QS-dependent transcriptional regulator, QsmR, rewired central metabolism to utilize the glyoxylate cycle rather than the tricarboxylic acid cycle. Defects in the glyoxylate cycle caused metabolic imbalance and triggered high expression of the stress-responsive chaperonin GroEL. High-level expression of GroEL in glyoxylate cycle mutants interfered with the biosynthesis of a public resource, oxalate, by physically interrupting the oxalate biosynthetic enzyme ObcA. Under such destabilized cooperativity conditions, spontaneous mutations in the qsmR gene in glyoxylate cycle mutants occurred to relieve metabolic stresses, but these mutants lost QsmR-mediated pleiotropy. Overcoming the metabolic restrictions imposed on the population of cooperators among glyoxylate cycle mutants resulted in the occurrence and selection of spontaneous qsmR mutants despite the loss of other important functions. These results provide insight into how QS bacteria have evolved to maintain stable cooperation via QS-mediated metabolic coordination

Flagellar number of wild-type strain BGR1 at 28°C and 37°C.

<p><b>A</b>. Schematic diagram showing the three divisions of the motility ring on 0.3% soft agar plates. Dotted and solid lines indicate the swimming regions, while the arrows indicate the direction of movement, of motile cells. Outside (O: edge of swimming ring), inside (I: center of swimming ring), and middle (M: middle of O and I) indicate the regions of the swim assay plates from which bacterial samples were taken. <b>B</b>. Distribution of BGR1 wild-type cells that had different numbers of flagella that were sampled from the O, M, I regions of the swim assay plates at 28°C and 37°C. Cells were counted based on TEM images (n = 100).</p

Genetic organization of <i>flhBAFG</i> and <i>fliA</i> genes, and <i>flhF</i> gene expression at 28°C.

<p><b>A</b>. Genetic organization and transcriptional units of <i>flhBAFG</i> and <i>fliA</i>, with arrows showing the direction of transcription. Recognition sites for <i>Eco</i>RI and <i>Stu</i>I are indicated by E and S, respectively. Vertical bars on the map denote the positions and orientation of the Tn<i>3</i>-<i>gusA</i> insertion. The two lines with arrowheads beneath the restriction enzyme map indicate the direction and extent of transcription. cDNA synthesized by reverse transcriptase with primers RT1 and RT2 was analyzed by PCR. Thick bars indicate the six expected PCR products, which were verified by electrophoresis on 1.5% agarose gel. <b>B</b>. Photographs of swim assay plates showing the swimming motility of the BGR1 mutant <i>fliA</i>::Tn<i>3</i>-<i>gusA45</i> and complementation with pBGFA carrying the <i>fliA</i> gene at 28°C and 37°C. <b>C</b>. Expression levels of the <i>flhF</i> gene in the wild-type BGR1, the <i>tofI</i> mutant BGS2, and the <i>qsmR</i> mutant BGS9, and the <i>flhC</i> mutant BGF41 at 28°C, based on qRT-PCR analysis. Vertical lines indicate the standard deviations of three independent experiments.</p

Swimming motility, movement, and flagellar morphology of the <i>flhF</i> mutant.

<p>After culturing on swim assay plates for 20-in of the ImageJ program. Each colored line shows the path of a different individual cell over a 2-s period. Flagellar morphologies of the wild-type BGR1 and the <i>flhF</i> mutant were determined by analyzing TEM images. Bars = 1 µm.</p

Expression of <i>flhC</i> and other flagellar genes in the QS mutants at 28°C and 37°C.

<p><b>A</b>. Expression levels of <i>flhC</i> in the wild-type BGR1, the <i>tofI</i> mutant BGS2, and the <i>qsmR</i> mutant BGS9 at 28°C, based on qRT-PCR analysis. Vertical lines indicate the standard deviations of three independent experiments. <b>B</b>. Expression levels of <i>fliC</i> and <i>flgK</i> genes were assessed in the wild-type BGR1, the <i>tofI</i> mutant BGS2, and the <i>qsmR</i> mutant BGS9 at 28°C and 37°C. BGF49, BGR1 <i>fliC</i>::Tn<i>3</i>-<i>gusA49</i>; S2F49, BGS2 <i>fliC</i>::Tn<i>3</i>-<i>gusA49</i>; S9F49, BGS9 <i>fliC</i>::Tn<i>3</i>-<i>gusA49</i>; BGF28, BGR1 <i>flgK</i>::Tn<i>3</i>-<i>gusA28</i>; S2F28, BGS2 <i>flgK</i>::Tn<i>3</i>-<i>gusA28</i>; S9F28, BGS9 <i>flgK</i>::Tn<i>3</i>- <i>gusA28</i>.</p