Vol. 165, No. 3JOURNAL OF BACTERIOLOGY, Mar. 1986, p. 696-7030021-9193/86/030696-08$02.00/0Copyright 0 1986, American Society for Microbiology

Molecular Cloning of Genetic Determinants for Inhibition of FungalGrowth by a Fluorescent Pseudomonad

NEAL I. GUTTERSON,* TAMARA J. LAYTON, JANET S. ZIEGLE, AND GARETH J. WARREN

Advanced Genetic Sciences, Inc., Oakland, California 94608

Received 18 October 1985/Accepted 3 December 1985

Pseudomonasfluorescens HV37a inhibits growth of the fungus Pythium ultimum vitro. Optimal inhibitionis observed on potato dextrose agar, a rich medium. Mutations eliminating fungal inhibition were obtainedafter mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Mutants were dassified by cosynthesis and threegroups were distinguished, indicating that a minimum of three genes are required for fuigal inhibition.Cosmids that contain wild-type alleles of the genes were identified in an HV37a genomic library bycomplementation of the respective mutants. This analysis indicated that three distinct genomic regions wererequired for fungal inhibition. The cosmids contining these loci were mapped by transpsoso insertionmutagenesis. Two of the cosmids were found to contaifn at least two genes each. Therefore, at least five genesin HV37a function as determinants of fungal inhibition.

Fluorescent pseudomonads are common soil microorga-nisms, often found on plant root surfaces (the rhizosphere).A number of fungi also inhabit the rhizosphere, and some ofthem are pathogenic to plants (i, 5). Fungal damage to plantscan be reduced by specific fluorescent pseudomonads thatinhibit the growth of one or more phytopathogenic fungi (3,8, 11, 12, 14, 15, 20). This work is directed toward under-standing the mechanisms of bacterial inhibition of fungalgrowth in the rhizosphere.A number of the pseudomonads that protect plants from

fungal diseases also produce antibiotics on agar media. It issometimes postulated that antibiotic production is responsi-ble for disease protection in the soil (5, 14, 20), but such arelationship has yet to be thoroughly explored. To clarifythis relationship, we began a genetic study of the biosynthe-sis of antifungal substances by a fluorescent pseudomonad.The subject of this study is a fluorescent pseudomonad thatprotects cotton seedlings from Pythium ultimuim-induceddamping-off disease and that also inhibits the growth of P.ultimum on agar media. We isolated mutants deficient infungal inhibition and classified them to infer the number ofgenes involved. Using a broad host range cosmid vector, weconstructed a library of HV37a and used it to complementthe mutants for identification of the corresponding genes.

MATERIALS AND METHODSMedia, strains, and plasmids. L-broth and L-agar (LA) (18)

and potato dextrose agar (PDA) were prepared as describedpreviously (23). Antibiotics were used at the foXlowingconcentrations (,ug/ml): kanamycin, 50; chloramphenicol, 34;streptomycin, 100; ampicillin, 75; tetracycline, 12.5. Pseu-domonas fluorescens strains were grown at 28°C withaeration, and Escherichia coli strains were grown at 37C withaeration.

E. coli strain MM294R F- endAl hsdRJ7 (HsdR-HsdM')supE44 thi-l recA was obtained from Barry Bochner andstrain HB101 was F- hsdS20 HsdR- HsdM+) recA13 ara-14proA2 lacYl galK2 rpsL20 (Sm) xyl-5 mtl-l supE44.A number of fluorescent pseudomonads were isolated by

T. Suslow from barley root tips (this laboratory). Manyisolates inhibited P. ultimum on LA, but only two isolates

* Corresponding author.

inhibited P. ultimum on PDA. HV37a was the more effectivePythium-antagonist on PDA. It has been classified as Pseu-domonasfluorescens and has natural resistance to ampicillinand chloramphenicoL The strains derived from HV37a aredescribed in Table 1.

P. ultimum was- obtained from T. Suslow. It was main-tained on PDA, with periodic growth on water agar supple-mented with streptomycin and tetracycline to prevent bac-terial contamination.The plasmids used in this work are described in Table 2.Pythium inhibition assays. Fungal inhibition by living bac-

terial inocula was performed by streaking one to six singlebacterial colonies around the edge of a 1(Xmm diameterpetri plate and incubating it at 28C for 1 to 2 days. An agarplug inoculum of P. ultimum (ca. 4-mm square) was thentransferred to the center ofthe plate from a source plate ofP.ultimnum maintained on PDA for 2 to 7 days. After incubationfor 2 additional days at 280C, P. ultimum grew to the edge ofthe plate, and inhibition zones were readily observed.For assay of antifungal activity in culture filtrates, bacte-

rial cultures were grown 2 days at 280C with aeration. Thisallowed 24 h for incubation at stationary phase. Cells wereremoved by centrifugation for 10 min at 12,000 x g. Theculture supernatant was then filtered aseptically through0.45-pm-pore-size Gelman acrodiscs. The resulting filtratewas stored at 4°C. Small petri plates (35 mm in diameter)were fiHed with culture filtrate incorporated into molten PDAor LA. After the plates were cooled, P. ultimum inoculumwas placed on the agar surface, and the plates were incu-bated for 20 to 36 h. The diameter ofthe Pythium colony wasrecorded, and percent inhibition was calculated relative to acontrol without incorporated culture filtrate.

Mutagenesis. N-methyl-N'-nitro-N-nitrosoguanidine mu-tagenesis was performed as described (9). Mutagenesis wasperformed for 10 min, resulting in less than 1% survival andapproximately 5% auxotrophy. Mutants were plated on LA,and then screened for Pythium inhibition on LA or PDA.

Cosynthesis experiments. Mutants and the wild type weregrown overnight in L broth at 280C. UIsing a cotton-tippedapplicator, we streaked two mutants onto opposite quad-rants of a PDA plate. The wild type was streaked ontoanother quadrant, and the two mutants were streaked ontothe same location of the remaining quadrant, and thus mixed

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TABLE 1. Pseudomonas strainsStrain Parent, properties Derivationa

HV37a Wild type, Ampr CamrTS100 HV37a, NaY Spcr SpontaneousNP65 HV3Ia, Aful afu-l NTG mutagenesisNP68 HV37a, AfulI afu4 NTG mutagenesisNP69 HV37a, Afull afu-S NTG mutagenesisNP72 HV37a, Aful afu-8 NTG mutagenesisNP91 HV37a, AfIII afu-16 NTG mutagenesisNP92 HV37a, AfullI afu-17 NTG mutagenesisNP121 HV37a, AfuIllI afu-28 NTG mutagenesisNP134 HV37a, Aful afu-1905 Marker exchangeNP135 HV37a, AfuIII af-1801 Marker exchangeNP137 TSIOO, AfuIl afu-2008 Marker exchangeNP138 TS100, AfuIl afu-2016 Marker exchangeNP142 TS100, AfulI afu-20202 Marker exchangeNP180 TS100, Aful afu-1912 Marker exchangeNP182 TS1IO, Afull afu-2004 Marker exchange

a For mutants obtained by marker exchange, allele numbers refer first to thecosmid (first two digits are cosmid number, see Table 2} and then to theTn3503 insertion number. For example, NP137 was obtained by markerexchange of the pNG20::Tn3503-8 insertion into TS100, giving allele 2008.NTG, Nitrosoguanidine.

there. Growth periods were as described for the Pythiumgrowth inhibition test. If an inhibition zone was generated bythe mutants streaked together, but not by the individualmutants, a positive cosynthesis result was scored.Mating experiments. Standard triparentat matings with the

helper plasmid pRK2013 were performed as described (7).For matings in situ, a grid of 70 donor strains per plate wasestablished on selective LA plates. The grid was replicatedand grown overnight on fresh plates. Cultures of helper andrecipient were grown to mid-log phase, and 0.1 ml of eachculture was spread onto LA plates. Using a 70-prong metalapplicator, we transferred the bacterial grid onto the matingplate. After overnight incubation, replicas of the plate weretransferred to selective medium. After incubation for 2 days,bacterial growth was obtained only where donors werepresent in the grid.

Library construction. A cosmid library of HV37a wasconstructed in the broad host range cosmid pRK7813 (Fig. 1)by the strategy of Ish-Horowicz and Burke (13). In brief,chromosomal DNA ofHV37a was digested partially with therestriction endonuclease Sau3A and ligated with pRK7813arms digested with either BamHI and EcoRI or BamHI andHindIII. HB101 was transfected with the resulting recombi-nant molecules after their packaging into lambda particles(2).

TABLE 2. Plasmids

Plasmid Relevant properties Source or- ~~~~~~reference

pRK7813 Cosmid vector, 12.5 kb, Tcr J. Jones, thislaboratory

pRK2013 Mobilization helper, Kmr 7pNG8 Tn3503 source, TnpR- Apr Cmr Kmr This workpNG16 Tn3503 source, TnpR+ Apr Cmr Kmr This workpNG18 AfuIII, complements NP91 This workpNG19 AMI, complements NP65 This workpNG20 AfuIl, complements NP68 This workpNG35 Partial complementation of mutants This work

(see text)pNG57 AfuII, complements NP68 This work

Transposon mutagenesis. The Tn3 transposition systemwas used here because of its high efficiency (10). Sincetransposon insertion mutants of cosmids would be used toconstruct mutants in HV37a by marker exchange, it wasnecessary to use a transposon that encoded resistance to anantibiotic other than ampicillin or chloramphenicol. There-fore, a derivative of Tn3 was constructed which encodedneomycin phosphotransferase; the Tn3 resolvase functionwas inactivated by this construction. A source plasmid wassubsequently constructed containing an intact resolvasegene (see below). The scheme for construction of a suitabletransposon source plasmid is shown in Fig. 2.A Tn3 insertion was obtained in the nontransmissible

ColEl derivative pAT273 (24) by transposition from a Tn3derivative of R64drdll (17), giving pNG5. The neomycinphosphotransferase of Tn903, present in pUC4K (25), wascloned into the BamHI site of Tn3 in pNG5, giving pNG8.The resulting modified Tn3 was designated Tn3503. Theresolvase gene of Tn3 was cloned into pNG12 on a 2.8-kilobase (kb) PstI fragment, giving pNG13. As an interme-diate in the transposition of Tn3503 from pNGS to pNG13, acosmid clone, pA62, was used. Cosmid pA62::Tn3503 wasmobilized into HB101 harboring pNG13, transposition wasallowed to occur, and total plasmid DNA was isolated. Aftertreatment with restriction endonuclease BglII, which cleavespA62 but not Tn3503 or pNG13, HB101 was transformed,and colonies resistant to chloramphenicol and ampicillinwere obtained. Several colonies were shown to have Tn3503insertions in pNG13. One was retained as pNG16.Tn3503 mutagenesis was routinely performed by mobiliz-

ing a cosmid into an E. coli recA- mutant harboring pNG16(10). The resulting colonies were grown at 28°C to allowtransposition to occur, and the cosmid was then mobilizedinto HB101, selecting for transmission of both the ampicillinresistance of Tn3503 and for the tetracycline resistanceencoded by the cosmid.Transplacement experiments. Plasmid pRK404 and its de-

rivatives are unstable in many gram-negative bacteria (6).Since pRK7813 (a derivative of pRK404) is lost from HV37aand its mutants at a frequency ranging from 20 to 90% afterovernight culture, it seemed likely that pRK7813-derivedcosmids would frequently become integrated in the hostchromosome. Thus, we employed a two-step procedure forthe replacement of chromosomal alleles with cosmid alleles,based on the strategy of Scherer and Davis in yeast (21).Cointegrates were obtained (data not shown) by mobilizingcosmids with Tn3503 insertions into HV37a and selecting forchloramphenicol and tetracycline resistance. After purifyingsingle colonies, such strains were grown in L-broth contain-ing kanamycin but no tetracycline to allow resolution of thecointegrated cosmid. Single colonies were isolated on LA

Sma EcoRIFIG. 1. Restriction map of cosmid pRK7813.

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R64drdll-.Tn3

transpositionpNG9 kb

BamH I ligate

BamH I

pA62

10 5 kb transposition

Kmr

nt end

,ligate

FIG. 2. Construction of Tn3503 and its donor plasmid. Insertion of the Tn9Q3 neomycin phosphotransferase gene inactivates the Tn3resolvase gene (tnpR). The donor plasmid, pNG16, carries an intact copy of tnpR and the internal resolution site (IRS) outside of thetransposon. For further details, see the text.

plates containing kanamycin, and the colonies were thentested for tetracycline sensitivity. Kanamycin-resistant, tet-racycline-sensitive colonies were retained as the desiredinsertion mutants.DNA manipulation. Plasmid DNA isolations from E. coli

were performed by the method of Birnboim and Doly (4).Restriction enzyme reactions were performed according tothe recommendation of the manufacturers (Bethesda Re-search Laboratories or Boehringer Mannheim) or as de-scribed by Maniatis et al. (16). All other recombinant DNAtechniques were performed by the method of Maniatis et al.(16).

RESULTSMutational analysis. P. fluorescens HV37a was found to

inhibit Pythium ultimum most effectively on PDA. Mutantsproducing no zone of inhibition (Fig. 3) were obtained aftermutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine.Approximately 5% auxotrophy was found in each mutagen-esis performed; at this frequency of auxotrophy, each sur-viving cell is expected to contain multiple mutations. Inaddition, we observed mutants that produced larger inhibi-tion zones; these are presumed to overproduce antifungalactivity(ies) and will not be discussed further here. Overall,more than 30 prototrophic Afu- (antifungal activity) mutantswere obtained.Mutants were initially classified by cosynthesis tests.

Cosynthesis analysis has been used in streptomycete antibi-otic characterization and is similar to cross-feeding analysisof auxotrophic mutants (9). When two mutants were blockedat different points in a biosynthetic pathway, different inter-mediates accumulated. If their cell walls were freely perme-able to these intermediates, one mutant was able to utilizethe intermediate accumulated by the other mutant to synthe-size antibiotic. Cosynthesis was detected visually in thestandard Pythium inhibition test on agar plates. On eachplate, two mutants were incubated both separately andtogether. NP134 and NP92 gave cosynthesis, whereas mu-tants NP135 and NP92 did not (Fig. 4). Thus, even though a

biosynthetic pathway is not known to be involved here,cosynthesis tests were still useful in genetic classification.Three cosynthesis groups were found (Aful, Afull, and

AfuIII) (Table 3). For example, mutant NP68 (AfuII) cancosynthesize antifungal activity in combination with theother mutants shown, with the exception of the other AfuIImutant, NP69. These results indicated that at least threegenes are involved in fungal inhibition. This is only aminimum estimate since the absence of cosynthesis could bedue to nondiffusibility of intermediates or to a regulatorymutation which prevented the production of intermediates,etc. Mutants with defects in distinct genes could thereforefall into the same cosynthesis group.

Identification of genes. A library of the HV37a chromo-some was constructed in the wide host range cosmidpRK7813, as described above. Since pRK7813 is only 12.5kb, cosmid packaging selects an average insert size of about35 kb. The entire chromosome would therefore be includedin less than 150 nonoverlapping clones. 1,540 library cloneswere retained on grids of 70 per plate.

Library cosmids containing genetic determinants for fun-gal inhibition were identified by complementation of mu-tants. To detect complementation, library cosmids weremobilized into a mutant strain, and the resulting strain wastested for fungal inhibition as described above using theassay shown in Fig. 3. In the initial complementation exper-iments, only a subsection of the library (250 clones) wasmobilized into one member of each cosynthesis group: NP65(MuI), NP68 (AfuII), and NP91 (AfufII). From among these,we found two different complementing cosmids for each ofNP65 and NP68, and six cosmids for NP91. Plasmid DNAwas prepared from the library members harboring thesecosmids, and restriction analysis was used to determinewhether these cosmids were structurally related. We ob-served that clones within each class were similar to eachother in restriction pattern but that there were no apparentsimilarities between classes. One cosmid from each classwas subjected to further restriction mapping (Fig. 5). Theresulting maps indicate that there is no overlap between the

pla

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P. FLUORESCENS FUNGAL INHIBITION 699

FIG. 3. Mutant phenotypes. The standard fungal inhibition assay, as described in the text, was used for the wild type and four mutants.

cosmid classes. Thus, the three genetic loci were not clus-tered; a minimum of 20 kb must separate them in thechromosome of HV37a.A single cosynthesis group may contain more than one

unlinked gene. To ensure that all the genes involved inantibiotic biosynthesis had been isolated, every availabletight Afu- mutant was tested for complementation by thethree cosmids mapped above (pNG18, pNG19, and pNG20).All 16 mutants tested were complemented, in each case byonly one of these cosmids. This indicated that there are onlythree genomic loci responsible for fungal inhibition.Recombinational rescue of mutant phenotypes. During li-

brary complementation, an additional cosmid (pNG35) wasidentified that partially restores fungal inhibition to membersof two cosynthesis groups (e.g., NP121, an AfuIII mutant).One possible explanation for such a result is that cosmidpNG35 encodes functions responsible for production ofanother antifungal activity that is not normally producedunder test conditions (growth on PDA). Since pNG35 mayrestore fungal inhibition without providing a wild-type copyof the defective gene, the mechanism of restoration of fungalinhibition with the other cosmids must be suspect as well. Todetermine whether cosmids pNG18 (AfuIII), pNG19 (AfuI),and pNG57 (AFuII) contain wild-type DNA sequences cor-responding to those mutated to cause loss of fungal inhibi-tion, the ability of each cosmid to recombinationally restorewild-type levels of fungal inhibition was tested. A cosmidcan only recombinationally restore fungal inhibition to amutant if the wild-type allele of the mutated gene has beencloned.

The four cosmids indicated above were tested forrecombinational restoration of the wild-type phenotype to amutant from each cosynthesis group, as well as NP121. Eachmutant was mated with each cosmid, selecting for thevector-encoded tetracycline resistance. Subsequently, a sin-gle colony was inoculated into L-broth lacking tetracycline,and colonies obtained from the resulting culture were nolonger resistant to tetracycline. These colonies were thenscreened for inhibition of P. ultimum on PDA plates.pNG18, pNG19, and pNG57 recombinationally restoredfungal inhibition to mutants which each plasmid initiallycomplemented, but they did not restore inhibition to othermutants (Table 4). In contrast, pNG35 did not restore fungalinhibition to any of the mutants tested. Thus, sequenceshave been cloned in pNG18, pNG19, and pNG57 corre-sponding to the mutated genes in NP91, NP65, and NP68.Cosmid pNG35 does not contain cloned genes correspondingto any Afu mutant.Mapping Afu loci. Genetic organization of the Afu loci was

investigated by correlating genetic maps of the representa-tive cosmids with their physical maps. Cosmids weremutagenized with transposon Tn3503, encoding kanamycin-resistance (see above). Each cosmid insertion was mappedby using three different restriction enzymes, giving mappositions accurate to +0.5 kb. Each mutated cosmid wasthen tested for the ability to complement the prototypemutants, as described for complementation tests with librarycosmids. The resulting maps (Fig. 5) can be used to infer thedegree of complexity of the genetic organization.The AfuI locus (in pNG19) and the AfuIII locus (in

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wt

NP92 + NP134wt

NP 134

1........

NP92

NP92

NP92 + NP135FIG. 4. Cosynthesis analysis of Afu- mutants. The wild-type and two mutants were streaked onto PDA plates, and the two mutants were

streaked together at the bottom. In the assay at the bottom, a negative cosynthesis result was observed, whereas a positive result is shownat the top.

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P. FLUORESCENS FUNGAL INHIBITION 701

TABLE 3. Classification of mutants by cosyntheSiSaMutant NP65 NP72 NP68 NP69 NP91 NP92

NP65 (I) - - + + + + + +NP72 (I) - - + + + + + +NP68 (II) + + - - + +NP69 (II) + + - - + +NP91 (III) + + + + + + - -NP92 (III) + + + + + + - -

a Cosynthesis experiments were performed as described in the text. Sym-bols: -, no zone of inhibition; +, zone of inhibition smaller than thatproduced by the wild type; + +, zone of inhibition approximately equivalentto that produced by the wild type.

pNG18) each map as contiguous regions, about 3.5 and 5 kblong, respectively. These regions are relatively large toencode individual genes, suggesting that multiple genes withrelated functions may be present. Insertion pNG19::Tn3503-152 gives partial complementation ofNP65, as do three otherinsertions which were mapped to the same location. Theseinsertions probably affect the promoter region or the 3' endof a gene. A more complex pattern is observed for the AfuIIlocus. Insertional inactivation of complementation occursalong a 12 kb region, with an intervening region of about 1.5kb where insertions do not prevent complementation. Thesize of the region where insertion inactivated complementa-tion suggests that the AfuII locus may contain multiple genesinvolved in fungal inhibition.

Analysis by transplacement. We wanted to extend ourmutational analysis of the Afu genes by transferring theTn3503 insertion mutations (described above) into the chro-mosome of HV37a. First, this would confirm that thosecosmid insertions which inactivate complementation do in-deed lie in Afu genes. Second, it is possible that the cosmidscarry Afu genes which are not necessary for complementingthe particular Afu- mutants chosen; insertions in thesegenes would still complement the mutants but would gener-ate an Afu- phenotype when introduced into the chromo-some. For the actual process of marker exchange, we were

R HK C HK KlJ I 11 I

pNG18 (AfuM)

TABLE 4. Recombinational restoration of Afu I phenotypeaCosmid

MutantpNG18 pNG19 pNG57 pNG35

NP65 (I) 0/48 21/68 0/42 0/43NP68 (II) 0/39 0/24 12/49 0/24NP91 (III) 10/103 0/24 0/22 0/82NP121 (III) 28/69 0/25 0/24 0/60

a Each cosmid (pNG18, etc.) was mobilized into each mutant (NP65, etc.)selecting for tetracycline resistance encoded by the vector portion of thecosmid. These strains were then grown in the absence of tetracycline, andafter plating for single colonies on LA, colonies were picked that were nolonger resistant to tetracycline. These colonies, which had lost the originalcosmid, were tested for inhibition of P. ultimum. At each intercept, we reporttwo values, x/y, where x represents the number of isolates with a wild-typephenotype, and y represents the total number of tetracycline-sensitive colo-nies tested.

able to use the unstable replication property of the vectorreplicon in pseudomonads, as described above.

Characterization of genomic DNA from several of theresulting insertion mutants is shown in Fig. 6. As an exam-ple, in Fig. 6C, the structure expected for the insertionmutant derived from pNG19::Tn3503-109 is shown. TwoBamHI fragments with sequences homologous to Tn3503were expected, with sizes of 4.2 and 2.9 kb. When chromo-somal DNA isolated from this insertion mutant was digestedwith BamHI, and then probed with pNG8 which contains theintact Tn3503 (see Fig. 2), bands of the expected size wereobserved. (It should be noted that only one BstEII junctionfragment had sufficient Tn3503 sequences to give hybridiza-tion.) The insertion mutants had the expected structures,except for the one derived from pNG19::Tn3503-174. Thechromosomal structure of this mutant in the Aful region hasnot been elucidated. This aberrant event is the only onedetected from greater than 25 marker exchange experiments.Genomic insertion mutants throughout each Afu region

were then tested for their antifungal phenotypes. First, allinsertions that inactivate complementation of Afu mutantsby a cosmid were also found to give rise to Afu- phenotypes

H HXK X HKH R CXRI III I W IL 1 I

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FIG. 5. Correlated physical and genetic maps of representative complementing cosmids. Restriction maps were determined by doubledigests of cosmid DNA. The Tn3S03 transposon insertions were mapped by using three different restriction endonucleases. Symbols: R,EcoRI; H, HindIII; K, KpnI; C, ClaI; X, XhoI; B, BamHI; Bs, BstEII; l, positive complementation; f , negative complementation; 1, partialcomplementation; _, vector DNA; -, cloned HV37a DNA.

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702 GUTTERSON ET AL.

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FIG. 6. Southern blots of Tn3503 homogenotes. All homogenates are derived from pNG19::Tn3503 insertions, as represented in Fig. 5.Each lane represents total DNA isolated from each homogenote digested with (A) BamHI or (B) BstEII. The probe used was pNG8, a plasmidwhich contains Tn3503. (C) Expected structure for insertion mutant obtained from pNG19::Tn3503-109. Restriction sites are (A) BamHI and(B) BstEII. Symbols: -, pRK7813; _, cloned HV37a DNA; -----, noncloned HV37a DNA; , Tn3503.

when present in the chromosome. This result was expected;it confirmed the results of the complementation experiments.Next, insertion mutants were derived from insertions map-ping near the region where complementation was inacti-vated. On the premise that genes of similar function arelikely to be clustered, this procedure was intended to dis-cover any further Afu genes for which we did not alreadyhave chromosomal mutations. Genomic mutants, derivedfrom insertions near the region of AfuIII complementationinactivation all had an Afu+ phenotype. Some of the mutantsin the AfuI region (derived from pNG19 insertion alleles 23,24, and 109) also had an Afu+ phenotype. However, oneinsertion mutant (derived from pNG19 insertion allele 12)

had an Afu- phenotype. This suggests that a second Afugene is present at the AfuI locus and that it is still functionalin the prototypical AfuI mutant NP65.A similar situation was observed for the AfulI region.

Most insertion mutants (those derived from pNG20 insertionalleles 11, 14, 28, 30, 120, and 209) had a wild-type pheno-type, whereas one insertion mutant (derived from pNG20insertion allele 16) had an Afu- phenotype. The AfuIl locuswas further analyzed by complementation between genomicinsertion mutations and Tn3503 insertions in the Afullcosmid. Cosmid pNG20 insertion allele 19 did not comple-ment insertion mutants derived from pNG20 insertion alleles4 or 202 but did complement the insertion mutant derived

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P. FLUORESCENS FUNGAL INHIBITION 703

from pNG20 insertion allele 16. Thus, insertion allele 16defines a second gene in the AfuII region, while all otherinsertion alleles behave as though they are affecting the samegene that is mutant in the Afull prototype, NP68. Since polarchromosomal insertions in one region complement polarcosmid-borne insertions in another region, the two genes ofthe AfuIl region must be in different operons.

DISCUSSIONIt is known that Pseudomonasfluorescens HV37a protects

cotton seedlings from root disease caused by Pythiumultimum. To evaluate the role of antifungal substances indisease protection, an understanding of the pathway forsynthesis of antifungal substances and their regulation isessential. Little is known, however, about the genetics offungal inhibition in pseudomonads. Here, we have shownthat HV37a is amenable to genetic analysis and have begunto study the synthesis of antifungal substances at the molec-ular level.Mutants deficient in fungal inhibition have been isolated.

The mutants were classified on the basis of cosynthesis andcomplementation properties and by analysis of cloned genes.

We can distinguish five mutant classes and thus we infer thata minimum of five genes is involved in fungal inhibition. Thisconclusion is reached by combining three inferences.

(i) Cosynthesis tests provided evidence for three groups ofAfu mutants. Every member of a group is complemented bya cosmid that complements any one member. Thus, threeseparate genomic loci provide genetic determinants for fun-gal inhibition.

(ii) Analysis of genomic insertion mutants, derived bymarker exchange from Tn3503 insertions in the AfuI cosmid,indicated that two genes are present at the Aful locus, one

(AfuIa) defined by insertion alleles 5 and 25 and the other(AfuIb) defined by insertion allele 12.

(iii) Similar analysis of the AfuII locus indicated that atleast two genes are present, giving a total of at least fivegenes. The AfuIIa locus is defined by AfulI insertion alleles4, 19, and 202, while the AfuIlb locus is defined by Afullinsertion allele 16.

Restriction mapping of the three Afu cosmids shows thatat least 20 kb separates these biosynthetic loci from eachother. The AfuIla and AfuIlb loci are within 10 kb of eachother, but they are probably not within a single operon.Three of the currently recognized Afu loci may constitutepolycistronic operons, based on the target size for inser-tional inactivation of complementation. Therefore, we may

expect to distinguish additional Afu genes after furtheranalysis.

ACKNOWLEDGMENTSWe thank Paul Gill, Trevor Suslow, and Jonathan Jones for helpful

discussions. The cosmid library was constructed in collaborationwith Jonathan Jones.

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