Identification and characterization of gut-associated lactic acid bacteria isolated from the bean bug, Riptortus pedestris (Hemiptera: Alydidae).

Lactic acid bacteria (LAB) are beneficial bacteria for humans and animals. However, the characteristics and functions of LAB in insects remain unclear. Here, we isolated LAB from the gut of Riptortus pedestris, a pest that is a significant problem in soybean cultivation in Korea, and identified two Lactococcus lactis and one Enterococcus faecalis using matrix-associated laser desorption/ionization-time of flight and 16S rRNA analyses. All three LAB strains survived at pH 8, and L. lactis B103 and E. faecalis B105 survived at pH 9 for 24 h. In addition, these strains survived well in simulated gastric juice of humans containing pepsin and exhibited high resistance to bile salts. Two strains of L. lactis and one of E. faecalis maintained constant density (> 104 colony-forming units [CFU]/mL) at pH 2.5, but viability at pH 2.2 was strain-dependent. The three LAB were reinoculated into second-instar nymphs of R. pedestris and colonized well, reaching a constant density (> 105 CFU/gut) in the adult insect gut. Interestingly, feeding of these LAB increased the survival rate of insects compared to the negative control, with the largest increase seen for L. lactis B103. However, the LAB did not increase the weight or length of adult insects. These results indicate that insect-derived LAB possess the traits required for survival under gastrointestinal conditions and have beneficial effects on insect hosts. The LAB infection frequency of the wild bean bug populations was 89% (n = 18) in Gyeongsangnam-do, South Korea. These LAB can be utilized as a novel probiotic in the cultivation of beneficial insects. This study provides fundamental information about the symbiosis between insects and LAB, and a novel concept for pest control.

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.

Membrane Depolarization and Apoptosis-Like Cell Death in an Alkaline Environment in the Rice Pathogen Burkholderia glumae.

The rice pathogen Burkholderia glumae uses amino acids as a principal carbon source and thus produces ammonia in amino acid-rich culture medium such as Luria-Bertani (LB) broth. To counteract ammonia-mediated environmental alkaline toxicity, the bacterium produces a public good, oxalate, in a quorum sensing (QS)-dependent manner. QS mutants of B. glumae experience alkaline toxicity and may undergo cell death at the stationary phase when grown in LB medium. Here, we show that the cell-death processes of QS mutants due to alkaline environmental conditions are similar to the apoptosis-like cell death reported in other bacteria. Staining QS mutants with bis-(1,3-dibutylbarbituric acid)-trimethine oxonol revealed membrane depolarization. CellROX™ staining showed excessive generation of reactive oxygen species (ROS) in QS mutants. The expression of genes encoding HNH endonuclease (BGLU_1G15690), oligoribonuclease (BGLU_1G09120), ribonuclease E (BGLU_1G09400), and Hu-beta (BGLU_1G13530) was significantly elevated in QS mutants compared to that in wild-type BGR1, consistent with the degradation of cellular materials as observed under transmission electron microscopy (TEM). A homeostatic neutral pH was not attainable by QS mutants grown in LB broth or by wild-type BGR1 grown in an artificially amended alkaline environment. At an artificially adjusted alkaline pH, wild-type BGR1 underwent apoptosis-like cell death similar to that observed in QS mutants. These results show that environmental alkaline stress interferes with homeostatic neutral cellular pH, induces membrane depolarization, and causes apoptosis-like cell death in B. glumae.

Mutations in the Two-Component GluS-GluR Regulatory System Confer Resistance to ß-Lactam Antibiotics in Burkholderia glumae.

Bacteria have specific signaling systems to overcome selective pressure, such as exposure to antibiotics. The two-component system (TCS) plays an important role in the development of antibiotic resistance. Using the rice pathogen Burkholderia glumae BGR1 as a model organism, we showed that the GluS (BGLU_1G13350) - GluR (BGLU_1G13360) TCS, consisting of a sensor kinase and response regulator, respectively, contributes to ß-lactam resistance through a distinct mechanism. Inactivation of gluS or gluR conferred resistance to ß-lactam antibiotics in B. glumae, whereas wild-type (WT) B. glumae was susceptible to these antibiotics. In gluS and gluR mutants, the expression of genes encoding metallo-ß-lactamases (MBLs) and penicillin-binding proteins (PBPs) was significantly higher than in the WT. GluR-His bound to the putative promoter regions of annotated genes encoding MBL (BGLU_1G21360) and PBPs (BGLU_1G13280 and BGLU_1G04560), functioning as a repressor. These results demonstrate that the potential to attain ß-lactam resistance may be genetically concealed in the TCS, in contrast to the widely accepted view of the role of TCS in antibiotic resistance. Our findings provide a new perspective on antibiotic resistance mechanisms, and suggest a different therapeutic approach for successful control of bacterial pathogens.

Identification of a Genetically Linked but Functionally Independent Two-Component System Important for Cell Division of the Rice Pathogen Burkholderia glumae.

Bacterial two-component regulatory systems control the expression of sets of genes to coordinate physiological functions in response to environmental cues. Here, we report a genetically linked but functionally unpaired two-component system (TCS) comprising the sensor kinase GluS (BGLU_1G13350) and the response regulator GluR (BGLU_1G13360), which is critical for cell division in the rice pathogen Burkholderia glumae BGR1. The gluR null mutant, unlike the gluS mutant, formed filamentous cells in Lysogeny Broth medium and was sensitive to exposure to 42°C. Expression of genes responsible for cell division and cell-wall (dcw) biosynthesis in the gluR mutant was elevated at transcription levels compared with the wild type. GluR-His bound to the putative promoter regions of ftsA and ftsZ is involved in septum formation, indicating that repression of genes in the dcw cluster by GluR is critical for cell division in B. glumae. The gluR mutant did not form filamentous cells in M9 minimal medium, whereas exogenous addition of glutamine or glutamate to the medium induced filamentous cell formation. These results indicate that glutamine and glutamate influence GluR-mediated cell division in B. glumae, suggesting that GluR controls cell division of B. glumae in a nutrition-dependent manner. These findings provide insight into how the recognition of external signals by TCS affects the sophisticated molecular mechanisms involved in controlling bacterial cell division.

Unraveling the role of quorum sensing-dependent metabolic homeostasis of the activated methyl cycle in a cooperative population of Burkholderia glumae.

The activated methyl cycle (AMC) is responsible for the generation of S-adenosylmethionine (SAM), which is a substrate of N-acylhomoserine lactone (AHL) synthases. However, it is unknown whether AHL-mediated quorum sensing (QS) plays a role in the metabolic flux of the AMC to ensure cell density-dependent biosynthesis of AHL in cooperative populations. Here we show that QS controls metabolic homeostasis of the AMC critical for AHL biosynthesis and cellular methylation in Burkholderia glumae, the causal agent of rice panicle blight. Activation of genes encoding SAM-dependent methyltransferases, S-adenosylhomocysteine (SAH) hydrolase, and methionine synthases involved in the AMC by QS is essential for maintaining the optimal concentrations of methionine, SAM, and SAH required for bacterial cooperativity as cell density increases. Thus, the absence of QS perturbed metabolic homeostasis of the AMC and caused pleiotropic phenotypes in B. glumae. A null mutation in the SAH hydrolase gene negatively affected AHL and ATP biosynthesis and the activity of SAM-dependent methyltransferases including ToxA, which is responsible for the biosynthesis of a key virulence factor toxoflavin in B. glumae. These results indicate that QS controls metabolic flux of the AMC to secure the biosynthesis of AHL and cellular methylation in a cooperative population.

Quorum Sensing-Independent Cellulase-Sensitive Pellicle Formation Is Critical for Colonization of Burkholderia glumae in Rice Plants.

Bacteria form biofilms as a means to adapt to environmental changes for survival. Pellicle is a floating biofilm formed at the air-liquid interface in static culture conditions; however, its functional roles have received relatively little attention compared to solid surface-associated biofilms in gram-negative bacteria. Here we show that the rice pathogen Burkholderia glumae BGR1 forms cellulase-sensitive pellicles in a bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)- and flagellum-dependent, but quorum sensing (QS)-independent, manner. Pellicle formation was more favorable at 28°C than at the optimum growth temperature (37°C), and was facilitated by constitutive expression of pelI, a diguanylate cyclase gene from B. glumae, or pleD, the GGDEF response regulator from Agrobacterium tumefaciens. Constitutive expression of pelI or pleD raised the levels of c-di-GMP, facilitated pellicle formation, and suppressed swarming motility in B. glumae. QS-defective mutants of B. glumae formed pellicles, while flagellum-defective mutants did not. Pellicles of B. glumae were sensitive to cellulase but not to proteinase K or DNase I. A gene cluster containing seven genes involved in bacterial cellulose biosynthesis, bcsD, bcsR, bcsQ, bcsA, bcsB, bcsZ, and bcsC, homologous to known genes involved in cellulose biosynthesis in other bacteria, was identified in B. glumae. Mutations in each gene abolished pellicle formation. These results revealed a positive correlation between cellulase-sensitive pellicles and putative cellulose biosynthetic genes. Pellicle-defective mutants did not colonize as successfully as the wild-type strain BGR1 in rice plants, which resulted in a significant reduction in virulence. Our findings show that cellulase-sensitive pellicles produced in a QS-independent manner play important roles in the interactions between rice plants and B. glumae.

Glutamate uptake is important for osmoregulation and survival in the rice pathogen Burkholderia glumae.

Bacteria exhibit an optimal growth rate in culture media with sufficient nutrients at an optimal temperature and pH. In addition, the concentration of solutes plays a critical role in bacterial growth and survival. Glutamate is known to be a major anionic solute involved in osmoregulation and the bacterial cell's response to changes in solute concentration. To determine how glutamate uptake is involved in osmoregulation in the rice bacterial pathogen Burkholderia glumae BGR1, we mutated the gltI gene encoding a periplasmic substrate binding protein of a glutamate transport system to abolish glutamate uptake, and monitored the growth of the gltI null mutant in Luria-Bertani medium. We found that the gltI null mutant showed a slower growth rate than the wild-type strain and experienced hyperosmotic stress resulting in water loss from the cytoplasm in stationary phase. When the incubation time was extended, the mutant population collapsed due to the hyperosmotic stress. The gltI null mutant exhibited loss of adaptability under both hypoosmotic and hyperosmotic stresses. The growth rate of the gltI null mutant was restored to the level of wild-type growth by exogenous addition of glycine betaine to the culture medium, indicating that glycine betaine is a compatible solute in B. glumae. These results indicate that glutamate uptake from the environment plays a key role in osmoregulation in B. glumae.

Critical role of quorum sensing-dependent glutamate metabolism in homeostatic osmolality and outer membrane vesiculation in Burkholderia glumae.

Metabolic homeostasis in cooperative bacteria is achieved by modulating primary metabolism in a quorum sensing (QS)-dependent manner. A perturbed metabolism in QS mutants causes physiological stress in the rice bacterial pathogen Burkholderia glumae. Here, we show that increased bacterial osmolality in B. glumae is caused by unusually high cellular concentrations of glutamate and betaine generated by QS deficiencies. QS negatively controls glutamate uptake and the expression of genes involved in the glutamine synthetase and glutamine oxoglutarate aminotransferase cycles. Thus, cellular glutamate levels were significantly higher in the QS mutants than in the wild type, and they caused hyperosmotic cellular conditions. Under the hypotonic conditions of the periplasm in the QS mutants, outer membrane bulging and vesiculation were observed, although these changes were rescued by knocking out the gltI gene, which encodes a glutamate transporter. Outer membrane modifications were not detected in the wild type. These results suggest that QS-dependent glutamate metabolism is critical for homeostatic osmolality. We suggest that outer membrane bulging and vesiculation might be the outcome of a physiological adaptation to relieve hypotonic osmotic stress in QS mutants. Our findings reveal how QS functions to maintain bacterial osmolality in a cooperative population.

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.IMPORTANCE We address how quorum sensing (QS) has coevolved with metabolic networks to maintain bacterial sociality. We found that QS-mediated metabolic rewiring is critical for sustainable bacterial cooperation in Burkholderia glumae The loss of the glyoxylate cycle triggered the expression of the stress-responsive molecular chaperonin GroEL. Excessive biosynthesis of GroEL physically hampered biosynthesis of a public good, oxalate. This is one good example of how molecular chaperones play critical roles in bacterial cooperation. In addition, we showed that metabolic restrictions in the glyoxylate cycle acted as a selection pressure on metabolic networks; there were spontaneous mutations in the qsmR gene to relieve such stresses. However, the presence of spontaneous qsmR mutants had tragic consequences for a cooperative population of B. glumae due to failure of qsmR-dependent activation of public good biosynthesis. These results provide a good example of a bacterial strategy for robust cooperation via QS-mediated metabolic rewiring.

Control of bacterial metabolism by quorum sensing.

Bacterial quorum sensing (QS)-dependent gene expression is a dynamic response to cell density. Bacteria produce costly public goods for the benefit of the population as a whole. As an example, QS rewires cellular metabolism to produce oxalate (a public good) to enable survival during the stationary phase in Burkholderia glumae, Burkholderia thailandensis, and Burkholderia pseudomallei. Recent reports showed that QS serves as a metabolic brake to maintain homeostatic primary metabolism in B. glumae and readjusts the central metabolism of Pseudomonas aeruginosa. In this review, we emphasize the dynamics and complexity of the control of gene expression by QS and discuss the metabolic costs and possible metabolic options to sustain cooperativity. We then focus on how QS influences bacterial central metabolism.

HopW1 from Pseudomonas syringae disrupts the actin cytoskeleton to promote virulence in Arabidopsis.

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.

Plant pathogenic bacteria target the actin microfilament network involved in the trafficking of disease defense components.

Cells of infected organisms transport disease defense-related molecules along actin filaments to deliver them to their sites of action to combat the pathogen. To accommodate higher demand for intracellular traffic, plant F-actin density increases transiently during infection or treatment of Arabidopsis with pathogen-associated molecules. Many animal and plant pathogens interfere with actin polymerization and depolymerization to avoid immune responses. Pseudomonas syringae, a plant extracellular pathogen, injects HopW1 effector into host cells to disrupt the actin cytoskeleton and reduce vesicle movement in order to elude defense responses. In some Arabidopsis accessions, however, HopW1 is recognized and causes resistance via an actin-independent mechanism. HopW1 targets isoform 7 of vegetative actin (ACT7) that is regulated by phytohormones and environmental factors. We hypothesize that dynamic changes of ACT7 filaments are involved in plant immunity.

Regulation of universal stress protein genes by quorum sensing and RpoS in Burkholderia glumae.

Burkholderia glumae possesses a quorum-sensing (QS) system mediated by N-octanoyl-homoserine lactone (C(8)-HSL) and its cognate receptor TofR. TofR/C(8)-HSL regulates the expression of a transcriptional regulator, qsmR. We identified one of the universal stress proteins (Usps), Usp2, from a genome-wide analysis of QS-dependent proteomes of B. glumae. In the whole genome of B. glumae BGR1, 11 usp genes (usp1 to usp11) were identified. Among the stress conditions tested, usp1 and usp2 mutants died 1 h after heat shock stress, whereas the other usp mutants and the wild-type strain survived for more than 3 h at 45°C. The expressions of all usp genes were positively regulated by QS, directly by QsmR. In addition, the expressions of usp1 and usp2 were dependent on RpoS in the stationary phase, as confirmed by the direct binding of RpoS-RNA holoenzyme to the promoter regions of the usp1 and usp2 genes. The expression of usp1 was upregulated upon a temperature shift from 37°C to either 28°C or 45°C, whereas the expression of usp2 was independent of temperature stress. This indicates that the regulation of usp1 and usp2 expression is different from what is known about Escherichia coli. Compared to the diverse roles of Usps in E. coli, Usps in B. glumae are dedicated to heat shock stress.

An HrpB-dependent but type III-independent extracellular aspartic protease is a virulence factor of Ralstonia solanacearum.

The host specificity of Ralstonia solanacearum, the causal organism of bacterial wilt on many solanaceous crops, is poorly understood. To identify a gene conferring host specificity of the bacterium, SL341 (virulent to hot pepper but avirulent to potato) and SL2029 (virulent to potato but avirulent to hot pepper) were chosen as representative strains. We identified a gene, rsa1, from SL2029 that confers avirulence to SL341 in hot pepper. The rsa1 gene encoding an 11.8-kDa protein possessed the perfect consensus hrp(II) box motif upstream of the gene. Although the expression of rsa1 was activated by HrpB, a transcriptional activator for hrp gene expression, Rsa1 protein was secreted in an Hrp type III secretion-independent manner. Rsa1 exhibited weak homology with an aspartic protease, cathepsin D, and possessed protease activity. Two specific aspartic protease inhibitors, pepstatin A and diazoacetyl-d,l-norleucine methyl ester, inhibited the protease activity of Rsa1. Substitution of two aspartic acid residues with alanine at positions 54 and 59 abolished protease activity. The SL2029 rsa1 mutant was much less virulent than the wild-type strain, but did not induce disease symptoms in hot pepper. These data indicate that Rsa1 is an extracellular aspartic protease and plays an important role for the virulence of SL2029 in potato.

Proteomic analysis of quorum sensing-dependent proteins in Burkholderia glumae.

Burkholderia glumae, the causal agent of bacterial rice grain rot, utilizes quorum sensing (QS) systems that rely on N-octanoyl homoserine lactone (synthesized by TofI) and its cognate receptor TofR to activate toxoflavin biosynthesis genes and an IclR-type transcriptional regulator gene, qsmR. Since QS is essential for B. glumae pathogenicity, we analyzed the QS-dependent proteome by 2-dimensional gel electrophoresis. A total of 79 proteins, including previously known QS-dependent proteins, were differentially expressed between the wild-type BGR1 and the tofI mutant BGS2 strains. Among this set, 59 proteins were found in the extracellular fraction, and 20 were cytoplasmic. Thirty-four proteins, including lipase and proteases, were secreted through the type II secretion system (T2SS). Real-time RT-PCR analysis showed that the corresponding genes of the 49 extracellular and 13 intracellular proteins are regulated by QS at the transcriptional level. The T2SS, encoded by 12 general secretion pathway (gsp) genes with 3 independent transcriptional units, was controlled by QS. beta-Glucuronidase activity analysis of gsp::Tn3-gusA gene fusions and electrophoretic mobility shift assays revealed that the expression of gsp genes is directly regulated by QsmR. T2SS-defective mutants exhibited reduced virulence, indicating that the T2SS-dependent extracellular proteins play important roles in B. glumae virulence.

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 (toxR'A'::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.

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.

Proteomic analysis of the proteins regulated by HrpB from the plant pathogenic bacterium Burkholderia glumae.

Plant pathogenic bacteria transfer effector proteins into plant cells via the hypersensitive response and pathogenicity (Hrp) type III protein secretion system (T3SS) during infection. The genes encoding the Hrp T3SS are expressed only under plant apoplast-mimicking conditions in an AraC-type transcriptional activator HrpB-dependent manner. To identify the proteins controlled by HrpB in Burkholderia glumae in vitro, we constitutively expressed hrpB and analyzed the proteins showing altered expression using 2-DE and ESI-MS/MS. Among 46 proteins exhibiting consistently altered expression, which were encoded by 34 different genes, 34 were secretory proteins and 12 were cytoplasmic. Twenty-eight of the secreted proteins showed increased accumulation, whereas the other six showed decreased accumulation. None of the HrpB-dependent proteins had significant homology to known T3SS-dependent proteins, except for HrpK from Pseudomonas syringae pv. syringae and two T3SS-associated cytoplasmic proteins from Ralstonia solanacearum. Twenty-one of the 34 genes had putative HrpB-binding sequences in their upstream regulatory regions. Secretion of all 34 extracellular proteins was independent of the Hrp T3SS, and 16 were secreted via a type II protein secretion system (T2SS). Mutants lacking the T2SS or Hrp T3SS produced toxoflavin but were less virulent to rice panicles, indicating the importance of these proteins in pathogenicity.

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. glumae is aflagellate and has lost the ability to swim and swarm at 37 degrees 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. glumae. 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. glumae suggests that either QS or flagellum formation constitutes a good target for the control of rice grain rot.