CURRENT MICROBIOLOGY Vol. 47 (2003), pp. 174–179 DOI: 10.1007/s00284-002-3926-z Current Microbiology An International Journal © Springer-Verlag New York Inc. 2003 AHL-Deficient Mutants of Burkholderia 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 bacteria [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 1350–2069) 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.