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Bacteriology Group, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy,1 Plant Bacteriology Group, International Centre for Genetic Engineering and Biotechnology, Biosafety Outstation, Via Piovega 23, 31050 Ca'Tron di Roncade, Treviso, Italy,2 Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Korea,3 Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan 481094
Received 16 January 2007/ Accepted 30 May 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Very few genetic or molecular studies related to the virulence of this bacterium on rice have been reported. B. glumae is known to produce the phytotoxin toxoflavin {1,6-dimethylpyrimido[5,4-E]-1,2,4-triazine-5,7(1H,6H)-dione}, a yellow pigment essential for pathogenicity (23, 37). Toxoflavin production by B. glumae reduces the growth of both leaves and roots of rice seedlings, induces chlorotic symptoms on rice panicles, and is responsible for causing bacterial wilt in many field crops (22, 37). The biosynthesis of toxoflavin has been elucidated involving the toxABCDE operon and is believed to be synthesized in a pathway common to the synthesis of riboflavin (37). Regulation of the toxABCDE operon has been studied recently, and it was demonstrated that the operon is regulated by the LysR family regulator ToxR and that toxoflavin acts as a coinducer (23). Regulation of this operon also requires another regulator called ToxJ whose expression is regulated by the TofI or TofR quorum-sensing (QS) system (23). B. glumae is very closely related to the nine Burkholderia species belonging to the Burkholderia cepacia complex, which are known for their abilities to colonize several environmental niches and to cause chronic infections in humans (10, 27). Members of the B. cepacia complex are not considered significant pathogens for the population of healthy humans but are considered a serious threat for specific groups of patients, such as cystic fibrosis patients (10) and patients with chronic granulomatous disease (CGD) (46). One Burkholderia species that is also very closely related phylogenetically to the B. cepacia complex is Burkholderia gladioli; this species is also pathogenic to rice and has also been associated with chronic infections of cystic fibrosis (40).
In this study we report that a strain of B. glumae, isolated from an infant with CGD, retained the ability to cause severe disease in rice. We also investigated the roles of two global regulation systems in panicle blight of this B. glumae strain. The two systems, QS and the stationary-phase alternative sigma factor RpoS, are known to play critical roles in plant-bacterium interactions (45). QS is a cell-density-dependent regulation system that relies on signal molecules that accumulate in the medium to which bacteria respond at quorum concentration via transcriptional regulation of target genes. N-acyl homoserine lactone (AHL) signal molecules are most commonly used in gram-negative bacteria. They are produced by an AHL synthase enzyme which most commonly belongs to the LuxI protein family. At quorum concentrations, AHLs then form a complex with a transcriptional regulator belonging to the LuxR family which then affects the transcriptional status of target genes (15). QS-dependent regulation is most beneficial to a community of bacteria, for example for bacteria producing extracellular enzymes and virulence factors (15, 45, 47). AHL QS in several phytopathogenic bacteria regulates virulence factors, for example in extracellular enzyme production in Erwinia carotovora, conjugation in Agrobacterium tumefaciens, and as previously mentioned, toxin production in an environmental isolate of B. glumae. AHL QS also plays a very important role in the human chronic lung infections caused by Pseudomonas aeruginosa and members of the B. cepacia complex in patients with cystic fibrosis (12, 34, 43). The stationary-phase RpoS sigma factor regulates many genes in stationary phase and is considered a master stress response (17, 21, 42). RpoS has also been reported to play important roles in plant-bacterium interactions in both plant pathogenic and plant beneficial bacteria (4, 8, 9, 29). The RpoS and QS regulons can overlap, meaning that the two global regulatory systems can cross-regulate each other and/or regulate a similar set of genes (33, 42). In the clinical B. glumae isolate reported here, AHL QS played a crucial role in rice pathogenesis, whereas the stationary-phase RpoS sigma factor did not. In addition, it was established that a lipase, which was regulated by QS, was involved in rice pathogenicity. We also determined that a nonpathogenic strain of B. glumae had lost its capacity to cause disease in rice, since it contained a nonfunctional AHL QS system. The rice-Burkholderia model could be a valuable system to study Burkholderia pathogenesis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(16) and E. coli HB101 (31) were used. Burkholderia strains were routinely grown in KB medium (24) or minimal M9 medium (31) supplemented with citric acid (0.3%) as the carbon source. Antibiotics for Burkholderia growth were added to media at the following concentrations: kanamycin, 100 µg/ml; gentamicin, 100 µg/ml; and tetracycline, 40 µg/ml. Antibiotics for E. coli growth were added to media at the following concentrations: ampicillin, 100 µg/ml; kanamycin, 100 µg/ml; gentamicin, 20 µg/ml; and tetracycline, 10 µg/ml.
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(pRK2013) (13). A B. glumae ATCC 33617T cosmid library was constructed by cloning partially digested EcoRI genomic DNA in pLAFR3 (36), which was then packaged into lambda phages using the Gigapack III XL kit as described by the supplier (Stratagene, La Jolla, CA) and subsequently used to infect E. coli HB101 cells.
Purification, detection, and visualization of signal (AHL) molecules.
The purification, detection, and visualization of AHL signal molecules from culture supernatants were performed essentially as described previously (1). Synthetic AHLs were purchased from Fluka Chemie AG (Buchs, CH).
Cloning of the tofI and tofR QS genes of B. glumae and inactivation of tofI and tofR.
About 4 x 109 cells each of E. coli HB101 harboring the B. glumae ATCC 33617T cosmid library, E. coli(pRK2013), and 2 x 108 cells of C. violaceum CVO26 were mixed. The suspension was applied to a 0.45-µm membrane filter (Millipore Corp.) on an LB plate. After overnight incubation at 30°C, the cells were resuspended and spread on LB plates containing ampicillin (100 µg/ml), kanamycin (100 µg/ml), streptomycin (100 µg/ml), and tetracycline (20 µg/ml). Strain CVO26 is naturally resistant to ampicillin and streptomycin and is resistant to kanamycin due to the mini-Tn5 present in the chromosome. Tetracycline will result in selection of transconjugants having received the pLAFR3-based cosmid clone. These plates were incubated for 48 h at 30°C, and transconjugants that turned purple were further assayed. One cosmid (pCOSGLU) from the cosmid library could restore purple pigmentation in strain CVO26. Further subcloning experiments confirmed that a 2.4-kb XhoI fragment cloned in pLAFR3 (creating pLGLU24) could restore pigmentation in strain CVO26. The 2.4-kb XhoI fragment was cloned in the corresponding site in pBluescript KS (generating pBGLU), sequenced, and contained the AHL QS locus of B. glumae 33617T composed of a luxI family member designated tofI and a luxR family member designated tofR (Fig. 1A). It was established that the 2.4-kb XhoI fragment did not contain the stop codon of tofR; hence, a XhoI-BglII fragment was cloned in pBluescript KS (generating pBGLU2) in order to completely sequence tofR (Fig. 1A). In order to create a tofI knockout genomic mutant of strain ATCC 33617T, a Km resistance gene cassette from pUC4K was cloned in the PvuII site of the tofI gene at position 102 in plasmid clone pLGLU24, creating pLGL24::Km (Fig. 1A). This latter plasmid was homogenized with the corresponding target regions in pCOSGLU, creating pCOSGLU::Km, which was subsequently used in marker exchange experiment using pPH1JI as the incompatible incoming plasmid. This generated a tofI genomic mutant designated B. glumae 33617TOFI. The fidelity of the marker exchange event was confirmed by Southern analysis (data not shown).
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The B. glumae AU6208 tofI and tofR genes were in part amplified by PCR in order to generate independent genomic mutations in B. glumae AU6208. The central part of the tofI gene was amplified by PCR as a 461-bp fragment using primers 5'-CCAGCGTCTGCGCGTCGATG-3' and 5'-GGCAGCTGCCGTCCGAAGAC-3' and cloned as an EcoRI fragment in the corresponding sites in pKNOCK-Km (2), generating pKmtofI. The central part of the tofR gene was amplified by PCR as a 601-bp fragment using primers 5'-GCAGGATGCCTACCACCAG-3' and 5'-TGCGCTCGGAGATGTTGAG-3' and cloned as a KpnI-XbaI fragment in the corresponding sites in pKNOCK-Km (2), generating pKmtofR. The pKmtofI and pKmtofR plasmids were then used as a suicide delivery system in order to create tofI and tofR single knockout mutants in strain AU6208 as described previously (2), generating AU6208TOFI2 and AU6208TOFR, respectively. The fidelity of the marker exchange events were confirmed by Southern analysis (data not shown).
Cloning and inactivation of the rpoS gene of B. glumae.
The rpoS gene of B. glumae ATCC 33617T was identified by screening the cosmid gene bank using the rpoS gene of closely related B. cepacia as a probe (the probe consisted of a 1-kb PstI fragment of pRBS-3 [2]). A cosmid designated pCOSBR-A was identified, and the rpoS gene was further localized in a 6-kb SmaI fragment (Fig. 1C), which was subsequently cloned in the corresponding site in pBluescript KS, yielding pBPR-A, and sequenced. The cloned rpoS gene from B. glumae 33617T was inactivated by cloning in its EcoRV site at position 698 a Km gene resistance cassette from pUC4K (Fig. 1C) in pCOSBR-A, yielding pCOSBR::Km. This latter plasmid was used in a marker exchange technique (see above) in order to introduce site-specific insertions in the rpoS gene of B. glumae ATCC 33617T, creating a rpoS knockout mutant designated 33617RPOS. The fidelity of the marker exchange event was confirmed by Southern analysis (data not shown). The plasmid construct pCOSBR::Km was also used in the same marker exchange technique in order to create a rpoS knockout mutant in B. glumae AU6208, which was designated B. glumae AU6208RPOS.
Analysis of B. glumae secreted proteins and inactivation of the lipA gene of B. glumae AU6208.
Total secreted proteins were isolated and characterized as follows. Cells from 10-ml overnight cultures were pelleted by centrifugation for 10 min at 8,000 x g. Cells remaining in the supernatant were removed by an additional centrifugation step for 3 min at 15,000 x g. Proteins in the cell-free supernatant were then precipitated with 10% (wt/vol) trichloroacetic acid, dried, and resuspended in sample buffer. After the suspension was boiled for 10 min, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on gels containing 12% (wt/vol) polyacrylamide.
The selected protein band was identified as follows: the band was cut out from the Coomassie brilliant blue-stained gel (see Fig. 3A) and placed in a siliconized microcentrifuge tube that had been rinsed with water and ethanol. The band was digested with trypsin, and the resulting peptides were extracted with water and 60% acetonitrile-1% trifluoroacetic acid. The fragments were then analyzed by mass spectroscopy (an internal sequence analysis of the protein spots was performed by using an electron spray ionization mass spectrometer [LCQ DECA XP; ThermoFinnigan]), and the lipase protein was identified by analysis of the peptides and by using protein data banks.
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Rice plant pathogenicity and toxoflavin assays.
For panicle blight assays, rice plants (Oryza sativa cv. Milyang 23) were grown in a greenhouse, inoculated at the flowering stage with a bacterial suspension (108 to 109 CFU/ml–1), and kept in a greenhouse. Disease in the rice plants was evaluated daily for 10 days, using the following scale: a score of 0 for a healthy panicle, 1 for a panicle 0 to 20% discolored, 2 for a panicle 20 to 40% discolored, 3 for a panicle 40 to 60% discolored, 4 for a panicle 60 to 80% discolored, and 5 for a panicle 80 to 100% discolored. Disease severity was determined using the following: disease degree = 
(number of samples with each rating x rating value)/total number of panicles.
For the seed germination assay, B. glumae strains were grown in KB liquid medium at 37°C and seeds of rice (Oryza sativa cv. Baldo) were pregerminated at 37°C in a bacterial suspension of 5 x 106 CFU/ml–1. Two days later, germinating seeds were transferred to a growth chamber under 30°C ± 6°C and high relative humidity (close to 100%) in petri plates, under 400-V lamps with a 16-h light-8-h darkness photoperiod. The disease evaluation was performed 7 days after inoculation, using a severity scale from 1 to 6. The severity scores were as follows: 6 for coleoptile and plumule macerated, not standing upright; 5 for aerial plant part developed but completely discolored or growth limited to less than 1 cm high; 4 for aerial plant part developed but partially discolored on more than 50% of surface; 3 for aerial plant part developed but partially discolored on less than 50% of surface; 2 for completely green seedlings but roots and aerial part less vigorous compared to noninfected control; and 1 for completely green seedlings and as vigorous as the noninfected control. The experiments were repeated three times. Score ratings were analyzed statistically in the SPSS program using the Kruskal-Wallis multiple-comparison test and the Mann-Whitney comparison test. Score ratings were grouped into three classes for visual presentation as follows: class 1; scores 1 and 2, class 2; scores 3 and 4; and class 3; scores 5 and 6.
The phytotoxins and toxoflavin assay have been previously described (21).
Nucleotide sequence accession numbers.
The nucleotide sequences have been deposited in GenBank/EMBL/DDBJ under the following accession numbers: B. glumae ATCC 33617 tofI and tofR genes, AM422469; B. glumae AU6208 tofI and tofR genes, AM22470; and B. glumae ATCC 33617 rpoS gene, AM422471.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The tofI AHL synthase of B. glumae AU6208 was inactivated, generating AU6208TOFI, and this mutant no longer produced the C6- and C8-AHL (Fig. 1D), indicating that it was responsible for producing the two AHLs and that TofR, due to its genetic location and identity to CepR (see above), was therefore most probably sensing and responding to C8-AHL. In order to determine whether AHL QS of strain AU6208 played a role in rice pathogenicity, panicle blight and seed germination assays were performed with the AU6208TOFI knockout mutant. The AHL QS deficient mutants displayed significant decreases in the ability to cause panicle blight (disease index of 0.5; Fig. 2A) and also significant decreases in disease symptoms in the seed germination assay (Fig. 2B). It was established that as in B. glumae strain BGR1 (21) and also in strain AU6208, AHL QS regulates toxoflavin production (Fig. 3C).
The B. glumae rpoS gene consisted of 1,094 nucleotides (Fig. 1C) encoding a protein of 364 amino acids displaying over 90% identity with other RpoS proteins of Burkholderia spp. (data not shown), 77% with RpoS of Ralstonia solanacearum and approximately 50% with RpoS sigma factors from Gammaproteobacteria. The B. glumae AU6208RPOS mutant was as pathogenic to rice as the wild-type parent strain in both panicle and seed germination assays (Fig. 2A and C).
The TofI-TofR system of B. glumae AU6208 regulates a lipase which is involved in rice pathogenicity.
Once established that the TofI-TofR system was a major player in rice pathogenicity of strain AU6208, we analyzed the profile of secreted proteins of the wild-type strain versus the profile of the AHL QS mutant AU6208TOFI in order to determine whether any secreted proteins were regulated by AHL QS. In this experiment we also included the type strain ATCC 33617 and tofI knockout mutant derivative 33617TOFI. As depicted in Fig. 3A, in ATCC 33617T and tofI mutant derivative 33617TOFI, the secreted protein profiles were identical, whereas in AU6208TOFI, a secreted protein of approximately 30 kDa was no longer present compared to the parent strain, indicating that AHL QS was important for its production. This protein was digested with trypsin and analyzed by mass spectroscopy resulting in the determination of the following peptides: VYVANLSGFQSDDGPNGR, GEQLLAYVK, VNLIGHSQGGLTSR, GSEFADFVQDV, and ASGQNDGLVSR. By performing a BLAST analysis, the peptides were 100% identical to parts of the identified and characterized lipase LipA of B. glumae (14) (accession number CAA01279). Apparently this protein was not present in both spent supernatants of B. glumae ATCC 33617T and tofI knockout derivative 33617TOFI, indicating that under the conditions tested, this lipase was not produced by this strain (Fig. 3A). In addition, we performed lipase enzyme assays and determined that the tofI mutant of strain AU6208 displayed considerably less lipase enzyme activity than the parent wild-type strain (Fig. 3B). The stationary-phase RpoS sigma factor was found not to be involved in lipA or LipA regulation (Fig. 3B).
A lipA mutant, designated AU6208LIPA, was constructed and tested in a rice panicle blight and seed germination assay. As can be seen in Fig. 2, the lipA mutant has dramatically lost its capacity to cause disease in rice in both pathogenicity assays, making it an important virulence factor.
TofR of B. glumae ATCC 33617T is nonfunctional.
It was observed that when we tried to complement the B. glumae clinical isolate tofI mutant AU6208TOFI for lipase activity by adding 1 µM C8-AHL to the growth media, we found that this did not result in the restoration of lipase enzyme activity. We did however observe complementation for lipase activity if the tofR gene of strain AU6208 was provided in trans in strain AU6208TOFI via plasmid pMPTOFR together with exogenously provided 1 µM C8-AHL (Fig. 3B). The reason for this is that AU6208TOFI was constructed via a marker exchange technique using a plasmid incompatibility technique using the tofI or tofR locus having tofI::Km and surrounding genetic loci of strain ATCC 33617T (as described in Materials and Methods). This marker exchange resulted in the transfer to the AU6208 chromosome of the tofI::Km locus as well as the wild-type tofR locus of B. glumae strain ATCC 33617T. The AU6208TOFI strain was also complemented for lipase activity when both the tofI and tofR genes of strain AU6208 were introduced in the mutant with plasmid pMPTOFIR (data not shown). As mentioned above, the TofR protein of strain ATCC 33617T is 100 amino acids longer and could be nonfunctional. Importantly, it was also determined that if the tofR gene of strain AU6208 was provided in trans in strain ATCC 33617T via plasmid pMPTOFR, the strain was then also able to regain its ability to produce toxoflavin as well as lipase activity (Fig. 3B and C). It was therefore concluded that TofR of strain ATCC 33617T was nonfunctional and most probably the major reason why the strain did not produce toxoflavin and lipase enzyme.
Having established that the AU6208TOFI mutant constructed above was a tofIR double mutant (tofI::Tn5 and nonfunctional tofR from strain ATCC 33617T), we constructed tofI and tofR single mutants in order to clarify the role of each component of the AHL QS system. Consequently, tofI and tofR mutants, designated AU6208TOFI2 and AU6208TOFR, respectively, were constructed as described in Materials and Methods. Interestingly, it was observed that the single tofI mutant displayed considerably slow and very poor growth in rich medium, while the tofR mutant displayed comparable growth compared to the parent strain. AU6208TOFR was less pathogenic to rice and could be complemented for virulence by introducing in trans pMPTOFR (data not shown); this mutant however was more pathogenic than AU6208TOFI, indicating that both TofR and AHL are necessary for maximum virulence. Strain AU6208TOFI2 (single tofI knockout mutant) could not be tested due to the very poor growth ability and low survival rates of this strain.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It was determined that in clinical isolate B. glumae AU6208 and in B. glumae ATCC 33617T, AHL QS is present and very well conserved with the TofI-TofR system of B. glumae BGR1 (23), the PlaI-PlaR system of B. plantarii (35), and the CepI-CepR system of members of the B. cepacia complex (12, 43). AHL QS in members of the B. cepacia complex regulate several important processes including those involved in virulence (12, 43). The system in strain AU6208 is playing a pivotal role in rice pathogenesis, since in two different assays, the tofI synthase knockout mutant dramatically lost its pathogenicity towards rice. It was previously also observed that in B. glumae BGR1, AHL QS is involved in regulating the expression of toxoflavin genes indirectly (23); thus, it is very likely that the TofI-TofR system could also be involved in a similar regulatory cascade of toxoflavin regulation in strain AU6208. In strain AU6208, the secreted LipA lipase was determined to be regulated by AHL QS, since the protein was produced in much lower quantities in the AU6208TOFI strain, and lipase activity was significantly reduced. In Burkholderia thailandensis, a nonpathogenic species very closely related to the human pathogen Burkholderia pseudomallei, lipase production was found to be both positively and negatively regulated by AHL QS (39). In B. cepacia complex member Burkholderia cenocepacia, lipase production is dependent on the growth phase, with maximal activity produced in stationary phase. AHL QS mutants of B. cenocepacia produced 40 to 50% less lipase activity than the parent strain at stationary phase (25). For both B. thailandensis and B. cenocepacia however, the genetic determinants encoding lipase enzymes that are regulated by AHL QS have not been determined. Importantly, the B. glumae AU6208 lipA mutant dramatically lost its capability to cause rice pathogenicity in two different assays. The reason most probably being that the LipA lipase plays a crucial role as a cell wall-degrading enzyme; this enzyme in cooperation with other cell wall-degrading enzymes breaks down components of cell walls. Plant cell walls are composed of cellulose and xylan components which provide strength and rigidity as well as acting as a barrier for pathogens. Few lipases have been reported to be virulence factors in microbial phytopathogens; a secreted lipase of fungal pathogen Fusarium graminearum is important in causing virulence to wheat and maize (44). Similarly, in rice BLAST pathogen Xanthomonas oryzae pv. oryzae, a lipase has been reported to be involved in pathogenicity (30). Lipases may be involved in hydrolyzing ester bonds in xylan, thus promoting xylan degradation; alternatively, they could also be involved in the degradation of epicuticular waxes which cover host epidermal cells, providing an efficient barrier. Lipases are ubiquitous, having been found in animals, plants, fungi, and bacteria, and are also being studied for their many potential industrial applications (7). Importantly, we determined that the expression of lipA in B. glumae is AHL QS dependent, and at present, it is not known whether it is direct or indirect via another regulator. The LipA protein of B. glumae is secreted by the type II secretion pathway; thus, the possibility that AHL QS regulates the gene(s) encoding the protein(s) involved in secretion and not the lipase structural gene cannot be excluded. However, the observation that the secretion profile of the wild type versus the AHL QS mutant differs only in this protein is an indication that QS most probably regulates the lipA structural gene. Therefore, in B. glumae, two pivotal virulence targets for AHL QS have now been identified (toxoflavin and lipase production), and the degree of cross talk and/or synergy merits attention.
Interestingly, it was established that the type strain B. glumae ATCC 33617, originally isolated from diseased rice, has lost its ability to be pathogenic to rice. Under laboratory growth conditions, the type strain is not yellow pigmented like strain AU6208, and it has lost the capability to produce the yellow phytotoxic pigment toxoflavin. It is possible that the phytopathogenic B. glumae strain can undergo phenotypic variation due to spontaneous mutations which result in morphological variants that do not express secondary metabolites. These mutations are most likely to occur in global regulatory genes. This variation or phenotypic conversion is known to occur in many bacterial species in order to generate population diversity thought to be important for niche adaptation (32). Interestingly, the TofR AHL receptor of strain ATCC 33617T was found to be 110 amino acids longer and was determined to be not functional. A spontaneous mutation most probably occurred, resulting in a mutated TofR; thus, AHL QS is not operational. However, this strain produced comparable quantities of AHLs (C8-AHL and C6-AHL) to the other B. glumae strains which are pathogenic to rice. In fact, introducing into strain ATCC 33617T the tofR gene of strain AU6208 harbored on a plasmid allowed the strain to regain its ability to produce the virulence factors toxoflavin and a secreted lipase. Growing B. glumae in laboratory media could have resulted in phenotype conversion through a mutation in tofR, making the strain no longer fit for colonization and infection of rice. More studies of B. glumae isolates derived from rice are necessary in order to establish whether this is a common phenomenon in this species. Defective AHL QS systems have also been reported in isolates of other bacterial species; thus, the possibility that active AHL QS systems under certain environmental growth conditions could have a negative impact on the organisms' fitness cannot be excluded (18, 19). In addition, we also observed that a single tofI mutant of pathogenic B. glumae AU6208 is rather sick, as it grows very slowly and has a high rate of death. The reason for this is currently unknown; interestingly, however, if the tofR gene is also inactivated in this mutant, the resulting double mutant is vital comparable to the wild type.
The stationary-phase RpoS sigma factor does not play a major role in B. glumae rice pathogenesis as observed here in two different and distinct pathogenicity assays. This is in contrast to what observed in B. plantarii where rpoS mutants are impaired in seedling blight (35). RpoS in bacteria is known to be important for adaptation and survival under several stress conditions (17). For some bacterial plant pathogens thus far, it has been reported to play marginal roles (1, 3, 4), while in others, it plays an important role (4, 8, 9, 27). RpoS is not involved in regulation of AHL QS in B. glumae, since rpoS mutants produced levels of AHLs comparable to those observed for the parent strain (data not shown).
In summary, in this report we describe a B. glumae clinical isolate (i) which has retained the capability of causing severe virulence to rice (ii) wherein AHL QS is important for its pathogenicity (iii) in which the lipA lipase is regulated by AHL QS and is very important for rice pathogenicity and (iv) that RpoS does not play a role in pathogenesis and regulation of AHL QS. In addition, B. glumae could undergo phenotypic conversion through a mutation in tofR, rendering its pathogenic potential unstable when this species is grown under laboratory conditions. This study highlights that B. glumae could be an important bacterium to address for studying Burkholderia pathogenesis having the convenient and reliable rice-pathogen model. Finally, following the increasing importance of this plant pathogen, understanding the major virulence factors is an important step towards designing possible control mechanisms of this important disease of a very important cereal.
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ACKNOWLEDGMENTS |
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We thank coresearchers I. Bertani, L. Steindler, and S. Ferluga for stimulating discussions.
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FOOTNOTES |
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Published ahead of print on 8 June 2007.
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S (RpoS) subunit of RNA polymerase. Microbiol. Mol. Biol. Rev. 66:373-395.This article has been cited by other articles:
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