CURRENT MICROBIOLOGY Vol. 47 (2003), pp. 174Ð179 DOI: 10.1007/s00284-002-3926-z Current Microbiology An International Journal © Springer-Verlag New York Inc. 2003

AHL-Deficient Mutants of ambifaria BC-F Have Decreased Antifungal Activity

Hongwei Zhou,1 Fude Yao,1 Daniel P. Roberts,2 Thomas G. Lessie1 1Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA 2Sustainable Agricultural Systems Laboratory, USDA-ARS, Bldg 001, Rm. 140, BARC-West, 10300 Baltimore Ave., Beltsville, MD 20705- 2350, USA

Received: 13 July 2002 / Accepted: 24 October 2002

Abstract. Burkholderia ambifaria BC-F, a biocontrol strain reported previously to exhibit broad- spectrum antifungal activity, was highly active in formation of N-acyl homoserine lactones (AHLs). We constructed AHL-deficient derivatives of strain BC-F in which the genes specifying AHL synthase (bafI) and AHL-binding transcriptional activator (bafR) were inactivated by allelic exchange. The resulting AHL-deficient mutants had decreased antifungal activity.

The newly designated species Burkholderia ambifaria plant pathogenic [27] and in plant disease sup- [4] belongs to the Burkholderia cepacia complex [5, 26], pression by certain plant beneficial bacteria [21]. a group of bacteria that first attracted attention because of In this report, we focus on the maize rhizosphere their unusual biochemical versatility [25]. Members of isolate B. ambifaria BC-F, a representative of genomo- the B. cepacia complex are widely distributed in the var VII of the B. cepacia complex [4, 14]. Strain BC-F environment and are common inhabitants of the rhizo- has been shown to suppress diseases of cucumber, pep- sphere of crop plants such as maize and rice [1, 8, 10, per, soybean, and tomato caused by the important soil- 20]. The B. cepacia complex includes plant beneficial borne fungal pathogens Fusarium oxysporum f.sp. lyco- microbes that fix atmospheric nitrogen, promote plant persici, Pythium ultimum, Rhizoctonia solani, and growth, produce antifungal agents, and suppress plant Sclerotium rolfsii [14, 16]. It also has been shown to disease [10, 14, 20]. Although bacteria belonging to this suppress populations of the plant parasitic nematode group represent a pool of naturally occurring bioreme- Meloidogyne incognita in vitro and on pepper roots [14, diation and biocontrol agents, concerns have been raised 18]. However, the means by which this versatile biocon- about their commercial use owing to their emergence as trol agent suppresses these diverse plant pathogens is a cause of respiratory tract infections in patients with unknown. We demonstrate here that the bafIR locus of cystic fibrosis [12]. strain BC-F is required for suppression of certain plant Most members of the B. cepacia complex produce pathogenic fungi. N-acyl homoserine lactones that are synthesized from S-adenosyl methionine and fatty acid-acyl carrier protein Materials and Methods by AHL synthases belonging to the LuxI family [11, 13, Bacterial strains and growth conditions. Burkholderia ambifaria 15, 19, 28, 29]. They mediate cell-density-dependent BC-F was isolated from maize rhizosphere by W. Mao [16]. Wild-type gene expression through their interaction with AHL- and mutant strains of B. ambifaria BC-F were grown at 37¡C in inorganic salts medium [3] supplemented with 1% (wt/vol) yeast ex- binding transcriptional activator proteins belonging to tract (YE medium). Escherichia coli strains used in construction of bafI the LuxR family [6, 9] and play a key role in the and bafR mutant derivatives of strain BC-F, as well as the AHL- interaction of Gram-negative bacteria with plant and responsive reporter strain Chromobacterium violaceum CV026, were animal hosts [2, 6, 9, 13, 23]. N-Acyl homoserine lac- grown in the same medium. For growth of mutants in which bafI or tones have been shown to have a role in pathogenesis by bafR genes were replaced by inactive alleles containing a trimethoprim (Tp)-resistance cassette, the medium was supplemented with 100 ␮g/ml of Tp. For growth of Saccharomyces cerevisiae D273, the Correspondence to: D.P. Roberts; E-mail: [email protected] medium was supplemented with 1% sucrose. H. Zhou et al.: Antifungal Activity of AHL-Deficient Mutants of B. ambifaria 175

PCR amplification and nucleotide sequence analysis of the bafIR locus of B. ambifaria BC-F. PCR reactions were performed according to the manufacturer’s instructions in a Model 2400 thermocycler (Ap- plied Biosystems, Foster City, CA) with RedTaq and JumpStart Ready- Mix kits (Sigma Chem. Co., St. Louis, MO) supplemented with 5% dimethyl sulfoxide and the appropriate DNA templates and primers. DNA primers were obtained from Integrated DNA Technologies (Cor- alville, IA). Nucleotide sequence analysis was performed at the Uni- Fig. 1. Organization of genes related to quorum sensing in Burkhold- versity of Massachusetts Automated DNA Sequencing Facility, by eria ambifaria BC-F. The 2191-nt region shown includes the diver- using an ABI Prism 377XL DNA Sequencer in conjunction with the gently oriented genes bafI and bafR specifying, respectively, AHL ABI Prism BigDye Terminator reaction kit. DNA sequences were synthase and AHL-binding transcriptional activator, as well as an open analyzed with Biology Workbench programs from the University of reading frame of unknown function, ORFX. California San Diego Supercomputer Center (URL: http://work- bench.sdsc.edu/). The nucleotide sequence of the bafIR locus has been deposited in GenBank under Accession No. AY028921. more 76 by P. ultimum were conducted in a soilless potting mix (Redi-Earth, Peat-Lite Mix, Scott’s Horticultural Products, Marysville, Pulsed-field gel electrophoresis and Southern hybridization exper- OH) as described earlier [14]. Bacteria were applied at 108 CFU/seed iments. Preparation of DNA samples and PFGE resolution of randomly in 3% (final concentration) sterile gelatin (Sigma Chem. Co.). Plant linearized replicons and macrorestriction fragments were accomplished stand per pot was determined 14 days after planting seeds treated with as reported earlier [3]. DNA hybridization protocols were essentially strain BC-F and its AHL-deficient derivatives, as well as seeds receiv- those described by Sambrook and Russell [24], with positively charged ing no treatment. Analysis of variance was carried out, and differences nylon membranes and Digoxigenin (DIG)-labeled probe DNA. DIG- among means were estimated by using a least significant difference test labeled probe DNA was synthesized by using bafIR template DNA and in SAS (SAS Institute, Cary, NC). a PCR DIG Probe Synthesis kit from Roche Molecular Biochemicals (Indianapolis, IN). Hybrid DNA was visualized by using a DIG Lumi- nescent Detection kit from the same supplier. Results Construction of bafI and bafR insertion mutants of B. ambifaria BC-F. Derivatives of E. coli DH5␣ containing the suicide plasmids Characterization of the B. ambifaria BC-F bafIR lo- pEXCEPI and pEXCEPR [13] that carried, respectively, cepI and cepR cus. We used PCR primers based on the nucleotide genes inactivated by insertion of a trimethoprim resistance cassette, as sequence of the cepIR locus of the genomovar III isolate well as a counterselectable sacB gene, were provided by Pam Sokol. B. cepacia K56-2 (GenBank Accession No. AF019654) The suicide plasmids were transferred to B. ambifaria BC-F by tripa- [13] to amplify a 2191-nt fragment of the genome of B. rental matings essentially as described by Lewenza et al. [13]. Recom- binants in which pEXCEPI or pEXCEPR had integrated into the B. ambifaria BC-F containing genes specifying an AHL ambifaria genome were isolated by plating the mating mixtures to synthase (BafI) and an AHL-binding transcriptional ac- inorganic salts medium [3] supplemented with Tp (100 ␮g/ml), and tivator protein (BafR). As in the case of the cepIR locus 0.5% sodium citrate or L-histidine as carbon source to counterselect the of strain K56-2, the 606-nt bafI gene (nucleotides 625- E. coli strains. Mutants in which the active I or R gene allele and 17) and 717-nt bafR gene (nucleotides 1350Ð2069) were sacB-gene-containing plasmid DNA had been eliminated were ob- tained by plating the first set of recombinants to the same medium divergently oriented and separated by ca. 700 nt (Fig. 1). supplemented with 5% sucrose essentially as described by Lewenza et The bafI and bafR genes of strain BC-F exhibited a al. [13]. Expression of the sucrose-inducible sacB gene in Gram- high degree of homology with the corresponding cepI negative bacteria was lethal. Thus, the procedure selected for recom- and cepR genes of B. cepacia strain K56-2. The derived binants in which the sacB gene and flanking DNA was deleted. amino acid sequence of BafI (202 amino acids) was 96% Phenotypic analyses of B. ambifaria BC-F and derivative strains. identical to CepI, and that of BafR (239 amino acids) was Formation of AHLs was scored by streaking wild-type and mutant 94% identical to CepR. The bafI promoter region con- bacteria side-by-side with C. violaceum CV026 on YE medium or tained a “lux-box-like” palindromic sequence similar to applying 1Љ diameter filter paper disks saturated with the test strain (ca. 2 ϫ 108 bacteria) on plates seeded with the reporter strain and scoring that in the corresponding promoters of the bmuI and cepI for AHL-dependent formation of the purple pigment violacein owing to genes of B. multivorans 17616 [28] and B. cepacia cross-feeding of AHLs [17, 28]. AHLs from culture supernatants were K56-2, respectively [13]. The 724-nt region between bafI concentrated on columns containing a copolymer of divinyl benzene and bafR contained a 417-nt open reading frame (ORFX) and N-vinyl pyrrolidone, eluted with methanol, and resolved by thin- which terminated 78-nt upstream of the bafR gene (see layer chromatography (TLC) as described earlier [28]. AHLs were ␴70 detected by using agarose overlays seeded with strain CV026 (ca. 108 Fig. 1). There was a -like promoter sequence 66 nt bacteria/mL agarose) [28]. Anti-yeast activity was tested by applying upstream of ORFX and 561 nt upstream of bafR. This filter paper disks seeded with strain BC-F or its AHL-deficient mutants putative promoter may be responsible for co-transcrip- on YE/sucrose plates spread with S. cerevisiae and scoring for zones of tion of ORFX and bafR. clearing after 24Ð48 h of incubation at 25¡C. In vitro activity against B. ambifaria BC-F contained three replicons of 3.5, various plant-pathogenic fungi was measured as described previously [14]. 2.8, and 1.3 Mb (see Table 1). Both bafI-specific and Assays to determine the ability of strain BC-F and its AHL- bafR-specific probes exhibited homology with the deficient derivatives to suppress damping-off of cucumber cv Market- 2.8-Mb replicon. Additional hybridization experiments 176 CURRENT MICROBIOLOGY Vol. 47 (2003)

Table 1. Estimation of the size of the genome of strain BC-F by PFGE resolution of macrorestriction fragments and randomly linearized replicons

Macrorestriction fragments (Mb)

SwaI CeuI Linearized replicons (Mb)

1.8 2.8 3.5 1.4 1.9 2.8 1.3 1.3 1.3 0.9 1.1 0.6 0.3 0.5 0.2 0.45 0.3 0.2 0.05 Totals: 7.5 Mb 7.6 Mb 7.6 Mb

indicated that the bafIR locus was located on the 0.5-Mb SwaI fragment on the 2.8-Mb replicon (results not Fig. 2. The ability of B. ambifaria to cross-feed AHLs to C. violaceum shown). is lost in AHL-deficient mutants of strain BC-F. Filter paper disks saturated with B. ambifaria BC-F, the bafI mutant BC-F I4, and the Inactivation of bafI and bafR by allelic exchange. bafR mutant BC-F R8 were applied to a plate that had been spread with Lewenza et al. constructed cepI- and cepR-mutant strains C. violaceum CV026. of B. cepacia K56-2, using the suicide plasmids pEX- CEPI and pEXCEPR, bearing, respectively, cepI and For example, in a cross with pEXCEPI, selection for cepR genes inactivated by insertion of a Tp-resistance resistance to both Tp and sucrose yielded the represen- cassette [13]. The high degree of homology of the bafI tative AHL-negative recombinant BC-F I4 in which the and bafR genes of B. ambifaria BC-F with cepI and cepR wild-type bafI-gene PCR product of 0.3-kb had been and the availability of suicide plasmids pEXCEPI and replaced by one of 0.9-kb containing a 0.6-kb Tp- resis- pEXCEPR facilitated the construction of similar mutant tance cassette (compare lanes 3 and 4 of Fig. 3). derivatives of strain BC-F in which the bafI and bafR The predominant AHL species in concentrated cul- genes had been replaced with inactive alleles. We trans- ture supernatants of strain BC-F comigrated with oc- ferred plasmids pEXCEPI or pEXCEPR, which both tanoyl homoserine lactone (OHL) on TLC plates. Con- carry a counterselectable sacB gene, to strain BC-F by centrated BC-F culture supernatants also contained conjugation and selected for recombinants able to grow smaller quantities of AHL that comigrated with hexanoyl in the presence of Tp and sucrose. Generation of Tp- homoserine lactone (HHL) (data not shown). Lesser resistant recombinants required chromosomal integration amounts of species that migrated between OHL and of pEXCEPI (or pEXCEPR) as a consequence of recom- HHL were also detected. These possibly represent oxo- bination between the active bafI (or bafR) gene on the and/or hydroxy-derivatives of OHL. The mutant strains chromosome and the insertionally inactivated cepI (or BC-F I4 and BC-F R8 were deficient in all AHL species cepR) gene on the plasmid. Generation of recombinants detected in culture supernatants of the wild-type strain, resistant to both Tp and sucrose required a second re- BC-F. These data confirm that bafI and bafR were re- combination event between the inactive and active I-or quired for production of AHLs by strain BC-F. R-gene alleles that eliminated the sacB gene. The result- ing double recombinants, which carried only an inser- Reduced antifungal activity of AHL-deficient mu- tionally inactivated I or R gene, were unable to cross- tants of strain BC-F. Unlike wild-type strain BC-F, the feed AHLs to strain CV026 and unable to restore its AHL-deficient mutant strains BC-F I4 and BC-F R8 ability to form the purple pigment violacein (see Fig. 2). failed to inhibit growth of S. cerevisiae (see Fig. 4). PCR analysis of the AHL-negative derivatives of There was also a marked reduction of in vitro inhibition strain BC-F using bafI- and bafR-gene-specific primers of the important soilborne fungal plant pathogens P. confirmed that the representative strains BC-F I4 and ultimum, R. solani, and F. oxysporum f.sp. lycopersici BC-F R8 contained inactive bafI or bafR gene alleles. (see Table 2). In addition, strains BC-F I4 and BC-F R8 H. Zhou et al.: Antifungal Activity of AHL-Deficient Mutants of B. ambifaria 177

Fig. 4. Anti-yeast activity of B. ambifaria BC-F is lost in AHL-deficient mutants. Filter paper disks saturated with B. ambifaria BC-F, the bafI mutant BC-F I4, and the bafR mutant BC-F R8 were applied to a plate Fig. 3. PCR analyses showing replacement of the active bafI gene of that had been spread with S. cerevisiae D273. strain BC-F with an insertionally inactivated allele in the AHL-defi- cient mutant BC-F I4. Lanes 1 and 2 contained, respectively, 1 kb and 100 bp DNA markers. Lane 3 contained the expected 300-bp fragment of bafI from wild-type strain BC-F. Lane 4 contained the corresponding such broad-spectrum activity against fungi and nema- 900-bp DNA fragment from the trimethoprim- and sucrose-resistant todes. However, the means by which this versatile bio- recombinant BC-F I4. control agent suppresses these diverse plant pathogens is unknown. Quorum sensing, mediated by AHLs, has been lost the ability to suppress damping-off of cucumber shown to play a key role in interactions of Gram-nega- caused by P. ultimum (see Table 3). Mean cucumber tive bacteria with plants and animals [2, 6, 9, 23]. In the stand in pots challenged with P. ultimum was lower (P Յ present study, we found that B. ambifaria BC-F produces 0.05) when seeds were treated with strains BC-F I4 or high levels of octanoyl homoserine lactone, and demon- BC-F R8 than when seeds were treated with strain BC-F. strated by allelic exchange that inactivation of the genes Mean cucumber stand per cup was similar to the no- bafI or bafR, which encode respectively AHL-synthase bacteria control in the presence of P. ultimum when seeds and AHL-binding transcriptional activator protein, re- were treated with the AHL-deficient strains BC-F I4 or sults in loss of both AHL production and antifungal BC-F R8, but greater than the no-bacteria control when activity, including the ability to suppress damping-off of treated with strain BC-F. Results of a duplicate experi- cucumber caused by P. ultimum. It should be noted that ment were similar. the AHL-deficient mutants of strain BC-F, like AHL- deficient strains of B. cepacia K56-2 [13], were deficient Discussion in exoprotease and appeared to form larger amounts of siderophores (results not shown). These data indicate that The maize rhizosphere isolate B. ambifaria BC-F, a bafI and bafR and quorum sensing have a role in sup- strain able to colonize and persist on the roots of a variety pression of certain fungal plant diseases by BC-F. of crop plants, exhibited broad-spectrum antifungal ac- Efforts are under way in our labs to define the nature tivity against important soilborne pathogens and sup- of the antifungal and anti-nematode compound(s) pro- pressed disease caused by fungal pathogens on a number duced by strain BC-F. Interestingly, the bafI and bafR of important crop plants [14, 16]. Recently, we have mutants BC-F I4 and BC-F R8 were unaffected in sup- noted that strain BC-F also exhibits activity against nem- pression of the nematode M. incognita in vitro (Roberts atodes [14, 18]. To our knowledge, other members of the and Meyer, unpublished observations). Results to date B. cepacia complex have not been reported to exhibit indicate that the anti-nematode activity is associated with 178 CURRENT MICROBIOLOGY Vol. 47 (2003)

Table 2. Pertinent phenotypes of Burkholderia ambifaria strains

In vitro zone of inhibition for pathogensb

Strain AHL productiona PU RS FOL

B. ambifaria BC-F ϩ 9.4 Ϯ 0.7 18.0 Ϯ 1.3 18.8 Ϯ 1.1 B. ambifaria BC-F I4 Ϫ 0.0 Ϯ 0.0 2.6 Ϯ 1.5 4.2 Ϯ 1.2 B. ambifaria BC-F R8 Ϫ 0.1 Ϯ 0.3 0.4 Ϯ 0.7 0.0 Ϯ 0.0 E. coli DH5␣Ϫ0.0 Ϯ 0.0 0.0 Ϯ 0.0 0.0 Ϯ 0.0 a Ability to cross-feed N-acyl homoserine lactones to C. violaceum CV026 and restore violacein formation. b Values represent the mean zone of inhibition (mm) on nutrient agar plates of five replicates Ϯ standard deviation. PU, Pythium ultimum; RS, Rhizoctonia solani; FOL, Fusarium oxysporum f.sp. lycopersici.

Table 3. Biocontrol of damping-off on cucumber by Burkholderia phere population of Burkholderia cepacia: effect of seed treatment ambifaria strains on disease suppression and growth promotion of maize. FEMS Microbiol Ecol 27:225Ð237 Mean plant stand per pota 2. Cha C, Gao P, Chen YC, Shaw PD, Farrand SK (1998) Production Log10 Treatment CFU/seedb 0c 40c 60c of acyl-homoserine lactone quorum-sensing signals by Gram-neg- ative plant-associated bacteria. Mol Plant-Microbe Interact 11: BC-F 8.9 5.0 A 2.6 BC 3.8 AB 1119Ð1129 BC-F I4 8.7 5.0 A 1.0 DE 1.0 DE 3. Cheng H, Lessie TG (1994) Multiple replicons constituting the BC-F R8 8.7 4.8 A 0.8 DE 1.4 CD genome of Pseudomonas cepacia 17616. J Bacteriol 176:4034Ð No bacteria 4.3 5.0 A 0.0 E 1.3 DE 4042 4. Coenye T, Mahenthiralingam E, Henry D, LiPuma JJ, Laevens S, a Values are means of eight replicates, each containing five seeds Gillis M, Speert DP, Vandamme P (2001a) Burkholderia ambi- carried out in soilless mix and expressed as plant stand per pot. faria sp. nov., a novel member of the Burkholderia cepacia com- Numbers followed by the same letter are not statistically different plex comprising biocontrol and cystic fibrosis-related isolates. Int (P Յ 0.05). Least significant difference (P Յ 0.05) was 1.3. J Syst Evol Microbiol 51:1481Ð1490 b Log10 CFU/seed; number of bacteria on seeds as determined by 5. Coenye T, Vandamme P, Govan JR, LiPuma JJ (2001b) dilution plating onto nutrient agar. and identification of the Burkholderia cepacia complex. J Clin cQuantity of Pythium ultimum sporangia per milliliter soilless potting Microbiol 39:3427Ð3436 mix inoculum. 6. Eberl L (1999) N-Acyl homoserine lactone-mediated gene regula- tion in Gram-negative bacteria. Syst Appl Microbiol 22:493Ð506 the low molecular weight (Ͻ 4 kDa) fraction. Other B. 7. El-Banna N, Winkelmann G (1998) Pyrrolnitrin from Burkhold- eria cepacia: antibiotic activity against fungi and novel activities cepacia strains have been shown to produce inhibitory against streptomycetes. J Appl Microbiol 85:69Ð78 metabolites including pyrrolnitrin and other phenylpyr- 8. Fiore A, Laevens S, Bevivino A, Dalmastri C, Tabacchioni S, roles [7, 22]. However, these compounds have not been Vandamme P, Chiarini L (2001) Burkholderia cepacia complex: shown to inhibit nematodes. Since our bafI and bafR distribution of genomovars among isolates from the maize rhizo- mutants still exhibited anti-nematode activity against M. sphere in Italy. Environ Microbiol 3:137Ð143 incognita, it is likely that different inhibitors are respon- 9. Fuqua C, Winans SC, Greenberg EP (1996) Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing sible for the antifungal and anti-nematode activity of transcriptional regulators. Annu Rev Microbiol 50:727Ð751 strain BC-F. 10. Gillis G, Van TV, Bardin R, Goor M, Hebbar P, Willems A, Sergers P, Kersters K, Heulin T, Hernandez MP (1995) Polyphasic ACKNOWLEDGMENTS taxonomy in the genus Burkholderia leading to an emended de- scription of the genus and proposition of Burkholderia vietnam- This research was supported by an award from the Electric Power iensis sp. nov. for N2-fixing isolates from rice in Vietnam. Int J Research Institute, Palo Alto, CA, and by funding from the Cooperative Syst Bacteriol 45:274Ð289 State Research Extension, Education Service, U.S. Department of 11. Gotschlich A, Huber B, Geisenberger O, Togl A, Steidle A, Riedel Agriculture, Massachusetts Agricultural Experiment Station and the K, Hill P, Tummler B, Vandamme P, Middleton B, Camara M, Department of Microbiology, under Project No. MAS00781 of the Williams P, Hardman A, Eberl L (2001) Synthesis of multiple USDA Hatch Grant program. We thank Dr. Pam Sokol at the Univer- N-acylhomoserine lactones is wide-spread among members of the sity of Calgary for providing pEXCEPI and pEXCEPR. We thank Burkholderia cepacia complex. Syst Appl Microbiol 24:1Ð14 Pierre Dery, Wei Li, and Scott Lohrke for assistance with the assays of 12. Govan J, Deretic V (1996) Microbial pathogenesis in cystic fibro- antifungal activity. sis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 60:539Ð574 Literature Cited 13. Lewenza S, Conway B, Greenberg E, Sokol PA (1999) Quorum 1. Bevivino A, Sarrocco S, Dalmastri C, Tabacchioni S, Cantale C, sensing in Burkholderia cepacia: identification of the LuxRI ho- Chicarini L (1998) Characterization of a free-living maize-rhizos- mologs CepRI. J Bacteriol 181:748Ð756 H. Zhou et al.: Antifungal Activity of AHL-Deficient Mutants of B. ambifaria 179

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