INTRODUCTION

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Each year, fungal diseases cause millions of dollars worth of crop damage all over the world despite the extensive use of pesticides (11). Also, environmental concerns and development of resistance in target populations have reduced the availability of effective fungicides. Nonetheless, the vast array of antimicrobial molecules produced by diverse soil microbes remains as a reservoir of new and potentially safer biopesticides. For these and other reasons, a heightened interest in so-called biological control or biocontrol (i.e., the use of natural microorganisms or their products to limit attack and damage by phytopathogens) has arisen (2, 4, 19, 63, 65).

There are many well-documented cases (reviewed in references 19 and 65) where the efficacy of a biological control microbe in the greenhouse and in the field depends on the production of single or multiple fungal growth antagonists (antifungal compounds [AFCs]). Many cases involve fluorescent pseudomonads that produce antibiotics such as phenazines (64), 2,4-diacetylphloroglucinol (12, 28), pyrrolnitrin (21, 22), pyoluteorin (23, 36, 43), or siderophores (10). Recently, gene clusters encoding biosynthetic pathways for many of these antibiotics have been cloned and characterized (18, 21, 36, 44; reviewed in reference 63). Mutants overexpressing the biosynthetic genes, and hence overproducing the AFCs, have shown increased efficacy and potential in biological control (40, 57).

In contrast, much less work has been done with Burkholderia (Pseudomonas) cepacia (3, 67), even though B. cepacia is a ubiquitous soil organism (5, 38) and various strains have been reported to produce a large variety of AFCs such as cepacin (49), altericidins (32), pyrrolnitrin (26, 27), xylocandins (also called cepacidines) (6, 37, 46), and siderophores (60). B. cepacia is one of the most nutrionally diverse bacteria known (i.e., it can use >200 different organic compounds as its carbon source [38]), a trait that probably contributes to its ability to compete for root exudates and very effectively colonize roots and the rhizosphere (5, 31, 47). Studies suggest that B. cepacia can be an effective biocontrol agent for Pythium-induced damping off and Aphanomyces-induced root rot of pea (30, 47, 48), Botrytis-induced gray mold of apple (25), Rhizoctonia solani-induced root rot of Poinsettia (7), and other fungal diseases (13). However, in all these cases very little is known about the genetics or biochemistry of the biocontrol ability of B. cepacia.

A major limitation for molecular studies of the biological control ability of B. cepacia is that unlike many other pseudomonads, tools for its genetic manipulation and analysis are much less well developed, largely due to its inherently high levels of resistance to many antibiotics and low frequencies of electroporation and conjugative plasmid transfer. Furthermore, the B. cepacia genome is very large (>7 Mb), is composed of multiple replicons, and contains a large number of insertion sequences, resulting in extensive genomic and physiological variability and heterogeneity (39). However, as we report here, it is possible to apply some standard molecular genetic techniques to B. cepacia, which, in conjunction with classical biochemical approaches, can provide new insights into the physiology and ecology of B. cepacia. We describe the discovery of a new antifungal metabolite that inhibits the growth of numerous fungi. We also demonstrate that this AFC (herein called AFC-BC11) is largely responsible for the ability of B. cepacia BC11 to effectively control a damping-off disease caused by R. solani. Purification and characterization of this AFC, as well as analysis of several genes required for its biosynthesis, provided good evidence that it is a novel lipopeptide antibiotic which is synthesized nonribosomally.

	MATERIALS AND METHODS

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Microorganisms, growth conditions, and media. The bacterial strains used in this study are listed in Table 1. B. cepacia was routinely grown at 30°C on plates containing either nutrient agar (Difco) supplemented with 0.5% glucose (NAG) or potato dextrose agar (PDA [pH 5.6]; Difco). Escherichia coli was grown at 37°C in Luria-Bertani (LB) medium (42). BSM minimal medium was prepared as described previously (54). Fungi were obtained from G. Michaels (Microbiology Department, University of Georgia) and D. Sumner and R. Roncadori (Plant Pathology Department, University of Georgia) and cultured at 30°C on PDA plates. Pseudomonas aeruginosa PAO1, Pseudomonas putida ATCC 12633, and Acinetobacter calcoaceticus BND1 were from our laboratory collection. The antibiotic levels used to select for B. cepacia constructs were 350 µg/ml for kanamycin, 100 µg/ml for rifampin, and 200 µg/ml for tetracycline. The levels used to select for E. coli constructs were 50 µg/ml for kanamycin, 20 µg/ml for tetracycline, and 100 µg/ml for ampicillin.

Bioassay for AFC activity. Samples to be assayed were dried in a vacuum centrifuge, and the residue was dissolved in acidic methanol (85% methanol adjusted to pH 3 with concentrated HCl). Serial dilutions (1:3) were made in acidic methanol, and 10-µl aliquots were added to wells of 96-well-format microtiter dishes containing 0.3 ml of solidified PDA. After being dried for 30 min at 37°C, the wells were inoculated with 10 µl of a hyphal suspension of R. solani AG4 prepared by scraping aerial hyphae from a 4-day-old PDA plate culture with a loop and resuspending them in sterile water. After incubation for 2 to 3 days at 30°C, the greatest dilution giving complete growth inhibition was recorded and defined as containing 1 U of activity; 10 µl of acidic methanol alone had no observable effect on the growth of R. solani. For a qualitative assay of AFC production by growing cells, the hyphal suspension was spread onto PDA plates and dried for 15 min before being subjected to spot inoculation with a loopful of test bacteria. After 3 days at 30°C, the size of the fungal growth inhibition zone around each bacterial patch was used as a measure of AFC production.

Purification of AFC-BC11. A 5-ml volume of water containing 10⁹ cells of B. cepacia BC11 was spread onto 500 ml of PDA in each of 10 disposable aluminum baking trays (40 by 25 cm). After 48 h at 30°C, the cells were scraped from the surface by using a glass plate spreader and 500 ml of water. Cells (ca 15 g [wet weight]) were collected by centrifugation and extracted twice by vigorous vortexing for 1 min with 50 ml of 80% acetone. Insoluble material was removed by centrifugation at 2,700 × g for 15 min and discarded. Acetone was evaporated with a stream of nitrogen at 50°C, and the remaining aqueous solution was centrifuged at 2,700 × g for 15 min. The pelleted insoluble material was washed with water three times and then dried in a vacuum centrifuge. The dried residue was extracted three times each with 50 ml of 100% acetone and then 1-butanol. After being dried, the residue was dissolved in 5 ml of dimethyl sulfoxide (DMSO) and applied to a C₁₈ reverse-phase high-pressure liquid chromatography (HPLC) column (Z18TP54; Vydac Corp.). The column was eluted for 5 min with methanol-water-DMSO (50:45:5), after which the methanol concentration was increased to 75% over a 10-min period. Elution with methanol-water-DMSO (75:22:3) was continued until the AFC peak had completely eluted, usually after 10 to 20 min. Elution of BC11 AFC was monitored by measurement of the absorbance at 320 nm (A₃₂₀) and/or bioassay.

Structural analyses of AFC-BC11. Positive-mode fast atom bombardment mass spectrometry (FAB-MS) was performed with a JEOL SX/SX 102A tandem mass spectrometer at an accelerating potential of 10 kV (53). Equal volumes of sample and thioglycerol FAB matrix were mixed on the probe tip and then bombarded with ions generated with xenon and a JEOL FAB gun at 6 kV. The spectra consisted of averaged profile data of three scans recorded by a JEOL XMS data system and acquired from 200 to 2,000 m/z at a rate that would scan the mass range from 0 to 2,500 in 1 min; the filtering rate was 100 Hz. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed on an LDI 1700XP spectrometer at ~10⁶ torr (accelerating voltage, 30 kV; extractor voltage, 9 kV) (1, 53). A sample-2,4-dihydroxybenzoic acid matrix mixture was serially vacuum dried on the probe and then ionized with a nitrogen laser ( = 337 nm) by using a 3-ns, 6-mJ pulse. Spectra were recorded from m/z 400 to 10,000 and are the averages of ~150 acquisitions.

Assay for biological control of R. solani-induced damping-off of cotton. Cotton seeds (Nucotin 35B) were immersed in 5% bleach for 10 min and then washed three times with sterile water. They were then immersed in bacterial suspensions of various concentrations (10⁴ to 10⁷ cells/ml) for 15 min and dried at 25°C for 30 min. The number of cells coating the seeds at each concentration was determined by vortexing portions of seed lots in sterile water and determining the number of viable cells in supernatants by dilution plating. Seed treatment with BC11 AFC was conducted by multiple applications of 10-µl aliquots of purified antibiotic dissolved at 0.3 mg/ml in methanol. After being dried, the seeds were planted in 9- by 8-cm pots containing R. solani-infested vermiculite (made by resuspending the aerial hyphae of a 5-day old PDA plate culture of R. solani in 50 ml of water and pouring the suspension onto the vermiculite). Disease development was monitored at 30°C for 3 weeks by scoring germination, seedling emergence, and survival.

Tn5 mutagenesis and screening for AFC-deficient B. cepacia mutants. E. coli SM10, containing the Tn5 delivery plasmid pSUP2021 (58), and B. cepacia BC11R were grown to an A₆₀₀ of 0.5 by shaking in 18 ml of LB medium or nutrient broth with 0.5% glucose (NBG), respectively. The cells were washed twice with sterile water by centrifugation, resuspended in 0.3 ml of sterile water, and combined, and 30 0.02-ml aliquots were spotted on NAG plates. After 24 h at 30°C, the cells were scraped up, suspended in water, and adjusted to an A₆₀₀ of 2, and 0.1-ml aliquots were spread on NAG plates plus kanamycin and rifampin. After incubation at 37°C for 2 days, Km^r Rif^r colonies arose at a frequency of ~10⁶ per donor. These were individually transferred into 96-well microtiter dishes filled with LB medium-7% glycerol-7% DMSO, incubated for 16 h at 37°C, and frozen at 80°C or transferred en masse with a 96-well format replicator array to a large (15- by 150-mm) PDA plate previously spread with a suspension of R. solani hyphae. After incubation for 2 to 3 days at 30°C, BC11 cells from patches showing reduced fungal growth inhibition were picked and purified to single colonies.

Cloning of genes involved in the production of BC11 AFC. The plasmids used are listed in Table 1; the physical and genetic maps of some of these are shown in Fig. 1. Genomic DNA from AFC666 (afc666::Tn5) was isolated as described previously (54), digested with EcoRI, ligated with EcoRI-digested pTZ19U (45), and transformed into E. coli DH5. A plasmid from one of the resultant Amp^r Km^r transformants was designated pKW666 and further characterized. pKW666-1 was constructed by digesting pKW666 with BamHI, ligating under dilute conditions, and transforming E. coli to Amp^r. Analogously, pKW666-1 was digested with PstI, ligated, and transformed into E. coli to obtain pKW666-2. pKW666-4 was constructed by digesting pKW666-1 with HpaI and BamHI, filling in cohesive ends with deoxynucleoside triphosphates (dNTPs) and Klenow polymerase, and ligating under dilute conditions. pKW666-5 was constructed by ligating the 0.9-kb SalI fragment of pSB315 (containing nptI [14]) into XhoI-digested pKW666-4. pT666-1 was constructed by ligating the 3.3-kb XbaI-EcoRI fragment of pKW666-5 into EcoRI- and XbaI-digested pTOK2 (33). pR666 was constructed by ligating the 2.2-kb StyI-EcoRI fragment of pKW666-1 into EcoRI- and XbaI-digested pRK415 (29). pT666 was made by ligating a 10-kb EcoRI fragment of pKW666 into EcoRI-digested pTOK2.

View larger version (38K): [in this window] [in a new window]   FIG. 1.   (A) Physical and genetic map of plasmids containing afc genes of B. cepacia BC11. The solid circle shows the position of the Tn5 insertion in the genome of strain AFC666. Dotted arrows show putative transcripts of afc genes, and the solid box represents the putative divergent promoter between afcA and afcC (see below). Open boxes represent the afc ORFs detected by DNA sequence analysis. The open circle shows location of the nonpolar nptI insertion in pT666-1 that was marker-exchanged into the BC11 genome to generate strain AFC666M2. Shaded boxes represent polylinkers of vectors; Plac, lac promoter of the pRK415 vector. Restriction sites: A, HpaI; B, BamHI; C, HincII; H, HindIII; N, NotI; P, PstI; R, EcoRI; S, StyI; X, XhoI; Xb, XbaI. The BamHI site in tn5 used to construct pKW666-1 is not shown. (B) DNA sequence of the divergent promoter region between afcA and afcC. The region between afcA and afcC is shown (nucleotides 1925 to 2300 from GenBank accession no. AF076477). Sequences resembling 35 and 10 E. coli consensus promoter sequences (overlined and italicized) are shown. +1->, putative transcription start sites; , RBS. Pairs of segments of the promoter region that are underlined and labelled I, II, and III are highly homologous in the DNA sequence.

B. cepacia strain constructions. Derivatives of pTOK2 and pRK415 were mobilized from E. coli DH5 into B. cepacia by triparental mating with E. coli HB101 containing the helper plasmid pRK2013 (9). B. cepacia AFC666M1 was constructed by mobilizing the pTOK2 derivative pT666 into BC11R (a spontaneous rifampin-resistant mutant of BC11); since pTOK2 is unable to replicate in B. cepacia, selection for Km^r, Rif^r, and Tc^s yields allelic-exchange recombinants. Strain AFC666M2 was similarly constructed by transferring pT666-1 into BC11R and selecting Km^r Rif^r Tc^s allelic-exchange recombinants.

Recombinant DNA techniques and DNA sequence analysis. Transformation of E. coli, restriction digests, ligations, electrophoresis, and DNA isolation were performed by standard methods as described previously (42, 54). Southern blots were prepared and hybridized with DNA labeled by random priming with [-³²P]dATP (42). Double-stranded plasmid DNAs were sequenced with commercial and custom primers on an ABI 380 sequencer. DNA sequence analysis and comparisons with sequences contained in the GenBank and EMBL databases were performed with Genetics Computer Group programs and the BLAST algorithm.

Nucleotide sequence accession number. The DNA sequence of the 4.5-kb B. cepacia genomic DNA on pKW666 (Fig. 1) has been deposited in GenBank under accession no. AF076477.

	RESULTS

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Characterization of AFC-BC11. Strain BC11 was isolated from agricultural soil in south Georgia and identified as B. cepacia by both fatty acid analysis and polyphasic taxonomic analysis (16). When grown on PDA plates next to R. solani, it produced a small, impenetrable growth inhibition zone. When fivefold-concentrated culture supernatants of BC11 were spotted on PDA plates, a similarly compact fungal growth inhibition zone was observed, suggesting limited aqueous solubility and diffusion. The levels of AFC activity found in preparations of filter-sterilized culture supernatants varied widely, but extraction of PDA-grown whole cells with 80% acetone consistently produced an organic-solvent-soluble cell-free preparation with high antifungal activity. When the cells were disrupted by sonication and centrifuged at 100,000 × g to yield a cytosolic fraction (supernatant) and membrane fraction (pellet), >80% of the AFC activity was found in the membrane fraction, suggesting that the AFC is lipophilic.

Purification and structural analyses of AFC-BC11. A dried 80% acetone extract from 15 g of BC11 cells (58 mg) was sequentially extracted with various solvents (see Materials and Methods), removing 85% of the mass but only 10% of the AFC activity. Fractionation of the residue (8 mg) by HPLC gave a single peak of AFC activity that contained ca. half of the loaded mass. This purified AFC, like the AFC activity in concentrated culture supernatants, was reasonably soluble in DMSO, 80% acetone, or 70 to 95% methanol but poorly soluble in water, butanol, chloroform, or hexane. Similarly, it was also resistant to overnight treatment with protease or 30 min of treatment with 0.1 M acid or 0.1 M base or by boiling.

Selectivity of AFC-BC11: determination of MICs for various fungi. Using the microtiter dish dilution assay, we quantified the potency (MICs) of purified AFC-BC11 against various fungi and bacteria. Concentrations of <1 µg/ml strongly inhibited the growth of some soil-borne phytopathogenic fungi, especially Pythium ultimum and Colletotrichium sp. (Table 2). In contrast, it had little effect on Candida albicans, several other yeasts, and some pathogenic and nonpathogenic filamentous fungi (Table 2). The sensitivity pattern of various fungi to growth inhibition by purified AFC-BC11 was identical to that exhibited by growing B. cepacia BC11 cells on PDA plates, suggesting that AFC-BC11 is a primary factor responsible for fungal growth inhibition by this bacterium on agar plates.

Efficacy of B. cepacia BC11 and AFC-BC11 in biological control of damping-off of cotton caused by R. solani. Cotton seeds were coated with various numbers of BC11 cells and then planted into vermiculite infested with R. solani. After 3 weeks, we found that as few as 100 BC11 cells per seed dramatically increased germination rates (Table 3). At 10⁵ cells per seed, 100% survival and complete disease protection were observed. To evaluate the importance of AFC-BC11 in the observed biocontrol, different amounts of purified AFC-BC11 were applied to cotton seeds, which were subsequently planted into R. solani-infested vermiculite. As little as 5 µg of purified AFC-BC11 dramatically increased cotton seedling emergence and survival (Table 3). The disease control provided by 10 µg of AFC was the same as that provided by 10⁴ BC11 cells. Taken together, these results show that B. cepacia BC11 can be very effective in controlling damping-off and imply that the AFC it produces is a primary determinant of its biological control ability.

Generation and characterization of AFC-deficient mutants of B. cepacia BC11. To confirm the primary importance of AFC-BC11 in biocontrol and gain insight into its biosynthesis, we isolated and characterized Tn5 insertion mutants defective in AFC-BC11 production. Matings between B. cepacia BC11R and E. coli SM10(pSUP2021) produced 2,000 putative Tn5 insertion mutants of B. cepacia, which were picked and replica plated onto PDA plates spread with R. solani hyphae. After 3 days, 20 mutants showed reduced fungal growth inhibition. However, after rigorous retesting, only four mutants (AFC143, AFC145, AFC202, and AFC666 [Table 1]) showed both a wild-type growth rate and a bona fide loss of ability to inhibit growth of R. solani. AFC activity in 80% acetone extracts of PDA-grown cells of all four mutants was less than 1% of that in extracts from wild-type cells. When extracts from mutants AFC202 and AFC666 were fractionated by HPLC, the major peak associated with AFC activity was missing (data not shown). Moreover, both extracts showed 10-fold less A₃₂₂, the absorbance maximum of AFC-BC11. These results strongly suggested that these B. cepacia mutants have Tn5 inserted in genes required for production of AFC-BC11.

Cloning of genes involved in biosynthesis of AFC-BC11. In previous genetic studies of biocontrol bacteria, DNA sequence analysis of cloned genomic regions harboring Tn5 insertions that reduce the biocontrol activity has helped elucidate AFC biosynthetic pathways and their regulation (65). Thus, we cloned the EcoRI fragment of the AFC666 genome that had Tn5 inserted into an AFC-BC11 production gene. Plasmid DNA from a Km^r transformant (designated pKW666) harbored a 10-kb insert whose restriction endonuclease cleavage map indicated that it contained a 4.5-kb genomic DNA fragment with Tn5 inserted 1.5 kb from one end (Fig. 1).

9

DNA sequence analysis of a genomic region involved in AFC production. The nucleotide sequence of the 4.5-kb genomic DNA on pKW666 was determined. Based on analysis of codon usage, putative ribosome-binding sites (RBS), and BLAST searches, four genes (afcA, afcB, afcC, and afcD) containing three complete and one partial open reading frame (ORF) were tentatively identified (Fig. 1). afcA and afcB appeared to be transcribed from one strand, while afcC and afcD were seemingly transcribed from the other. The ca. 200-bp intergenic region between afcA and afcC did not appear to contain an ORF and had a G+C content 20% lower than that of the flanking coding regions. Further inspection identified a putative divergent promoter region having two very similar sets of 35 and 10 consensus sequences separated by 150 bp (Fig. 1B). In fact, the DNA sequence upstream of the two putative 35 sequences showed extensive (60%) dyad symmetry centered around position 2130, suggesting that it may have arisen by duplication.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Amino acid sequence similarities between afc gene products and proteins of known function. (A) Alignment of a portion of AfcA with the amino acid activating domains of several peptide synthetases. Residues similar or identical in at least five are marked #; residues identical or similar in at least three are marked * and +, respectively. I, ATP-binding domain; II, ATPase domain. SafB1, saframycin Mx1 synthetase (U24657); SnbC, pristinamycin synthetase (X98690); Bsu1, polyketide synthetase of Bacillus subtilis (U00024); EntF, enterobactin synthetase component F (P11454); GrsB, gramicidin S synthetase (P14688). (B) Alignment of the predicted amino acid sequences of two portions of AfcC with two segments of the -3 fatty acid desaturase (-desat) of Brassica napus (L22963). Conserved histidine boxes believed to be associated with the active site are marked ().

Evidence for a direct role of the afc gene cluster in AFC-BC11 production. In strain AFC666, Tn5 is in afcC, suggesting a role for the putative AfcC desaturase in AFC production, but possible polar effects by Tn5 complicate the interpretation. Based on its sequence homology to enzymes involved in synthesis of peptide antibiotics, it is plausible that AfcA is involved in synthesis of a peptide moiety of AFC-BC11. To directly assess this possibility, we inserted a nonpolar nptI cartridge (14) into the single XhoI site of afcA and in the same transcriptional orientation (Fig. 1). The resultant afcA::nptI fragment was recloned into the pTOK2 suicide vector, and the resultant plasmid (pT666-1) was transferred into BC11R by conjugative mobilization. Double-crossover marker-exchanged recombinants were selected by their Km^r Tc^s phenotype and arose at a frequency of 10⁹ per donor. Bioassay of 80% acetone extracts of cells from four such mutants (e.g., AFC666M2 [Table 1]) showed that they produced >300-fold-reduced levels of AFC-BC11 (data not shown). When afcA alone (i.e., the StyI-EcoRI fragment of pKW666-1) was cloned downstream of the lac promoter of pRK415 and the resultant plasmid (pR666 [Fig. 1]) was transferred into the afcA::nptI BC11 mutant, AFC production was increased >40-fold, arguing against strong polar effects by the nptI insertion. These results prove that AfcA is directly involved in the biosynthesis of AFC-BC11.

	DISCUSSION

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We previously screened 15 different soil isolates with biocontrol potential for consistent production of extracellular AFCs and amenability to genetic manipulation (56). One promising strain, B. cepacia BC11, strongly inhibited the growth of R. solani and Sclerotium rolfsii in artificial soils and, in later field tests, appeared to enhance the yield of peanuts (55). However, the inconsistency of the performance of BC11, and biocontrol systems in general (19, 65), prompted us to seek a more detailed understanding of B. cepacia BC11 and its production of AFCs. We found that the major antifungal antibiotic activity of BC11 resided in a relatively insoluble, membrane-associated compound, although sometimes it was detected in the culture supernatant. After purification of this AFC by differential solvent extraction and reverse-phase HPLC, MS analysis showed that it had a molecular mass of 733 Da. NMR suggested that it contains an aromatic component and a fatty acyl substituent with a cis double bond. Analysis of acid hydrolysates detected four amino acids, suggesting the presence of a peptide moiety. These characteristics imply that this AFC produced by B. cepacia BC11 (called AFC-BC11) is a lipopeptide different from most other previously purified and structurally characterized antibiotics from biocontrol pseudomonads (19, 65). While some of its characteristics are reminiscent of members of the cepacidine (xylocandin) family of lipopeptide antibiotics from some B. cepacia strains (6, 37, 46), AFC-BC11 is much smaller (733 versus 1100 Da) and lacks xylose. Moreover, cepacidines are very active against Candida spp., Saccharomyces cerevesiae, Aspergillus niger, and several other fungal dermatophytes whereas AFC-BC11 was not (Table 2). Nonetheless, AFC-BC11 was very active against many other fungi, most of which were soil isolates and plant pathogens. The in vitro growth of many fungi was completely inhibited by as little as 1 µg/ml, similar to the potency of amphotericin B (37), pyrrolnitrin (22), and pyoluteorin (23) but more potent than at least one phenazine derivative (64).

Production of a potent AFC does not always guarantee good biological control performance (35, 43). However, with B. cepacia BC11, we found that in the presence of R. solani as few as 10⁴ cells (or 5 µg of purified AFC-BC11) per seed increased the germination and survival rates of cotton seedlings from 0 to >50%. In comparison to what has been reported for many biocontrol strains (21, 23, 30, 35, 40, 43, 57), the minimum number of BC11 cells per seed required for full disease control is 2 orders of magnitude lower than that for most strains. However, unlike some other tests, our biocontrol assays were not done in natural soils, and so the efficacy of BC11 relative to other strains remains to be clarified.

Using Tn5 tagging, we isolated a genomic fragment encoding proteins essential for biological control by B. cepacia BC11. DNA sequence analysis of this fragment provided insight into the structure and biosynthesis of AFC-BC11. Part of the putative amino acid sequence of the product of one of the resident ORFs, AfcA, showed high similarity to a domain conserved in proteins which use ATP to convert amino acids into aminoacyl adenylates (34, 61), in particular to the amino acid activation domains of two large multifunctional peptide synthetases, SafB1 (51, 52) and SnbC (8, 62), that are involved in nonribosomal peptide synthesis of the antibiotics saframycin (M. xanthus) and pristinamycin (S. pristinaespiralis). However, AfcA is much smaller than peptide synthetases, since it has only one activation domain instead of the more typical three or four and lacks domains for elongation and attachment of the 4-phosphopantetheinyl arm (62). This architecture is reminiscent of SnbA, the 3-OH picolinic acid:AMP ligase that catalyzes the adenylation and activation of 3-OH picolinic acid during the synthesis of pristinamycin but does not subsequently bind it as a thioester. Instead, SnbA is thought to "deliver" activated 3-OH picolinic acid to a peptide synthetase for incorporation into pristinamycin (8, 62). Since AfcA shows such high homology to these amino acid activation domains and since a nonpolar insertion of nptI in afcA completely knocked out AFC production, it is plausible that AfcA activates an amino acid before its incorporation into a nonribosomally synthesized peptide component of AFC-BC11, consistent with physiochemical data suggesting that AFC-BC11 is a lipopeptide.

Unlike most other reports about B. cepacia, we were able to use reverse genetics to confirm the function of a cloned DNA sequence in a physiological process. Insertion of a nonpolar nptI cartridge into the genomic copy of afcA of B. cepacia BC11 completely eliminated the production of AFC-BC11, the ability to inhibit growth of R. solani in vitro, and the biocontrol activity. These data strongly suggest that BC11 produces only one major AFC that is a primary determinant of its biocontrol ability for R. solani-induced damping-off of cotton. This is in contrast to many other biological control pseudomonads, which commonly produce multiple AFCs that additively contribute to their biocontrol activity (65).

Although we did not unambiguously demonstrate a role in biosynthesis of AFC-BC11 for the two other complete ORFs on the cloned fragment, the fact that the initial Tn5-generated AFC-deficient mutant AFC666 had Tn5 inserted in afcC implies that one or both of these genes are required for AFC-BC11 biosynthesis. However, potential polar effects on downstream genes could also cause AFC deficiency. On the other hand, the similarity of the predicted amino acid sequences of segments of AfcC and AfcD to domains of desaturase enzymes which introduce cis double bonds into aliphatic chains (68), coupled with our NMR data indicating the presence of an acyl substituent with a cis double bond in AFC-BC11, further argues for a direct role for AfcC and/or AfcD in the biosynthesis of AFC-BC11. Genetic and biochemical experiments to confirm this are in progress.

For more effective field use of biocontrol microbes, we need a clearer knowledge of the field conditions that affect the biosynthesis of AFCs and hence biocontrol. The availability of cloned AFC-BC11 biosynthetic genes should allow the construction of reporter strains to study in situ rhizosphere conditions that increase expression of the biosynthetic genes and possibly the consistency of biocontrol. This type of approach has already been proven to be valuable (15, 36). Alternatively, addition of cloned AFC-BC11 biosynthetic genes to other biocontrol strains could lead to improved biocontrol agents (12, 17, 40, 57).