NUCLEIC ACID
manipulation Cloning, purification, electrophoresis and other manipula- tions of nucleic
acid fragments and constructs were per- formed using standard techniques (Sambrook et
al., 1989). PCR was performed under standard conditions as suggested by Invitrogen Life
Technologies data sheets supplied with their Taq polymerase.
Tn5-OT182 transposon mutagenesis
Bacterial conjugations were performed to introduce Tn5-OT182 into P. chlororaphis PA23
by biparental mating following the method of Lewenza et al. (1999). For each mating, 5–
10 Tc R colonies were screened by PCR to ensure that transconjugants contained a Tn5
insertion using TNP5- Forward and TNP5-Reverse primers (Table 1). To determine the site
of Tn5-OT182 insertion, rescue cloning was per- formed following previously described
methods (Lewenza et al., 1999).
Generation of PA23 PRN-deficient strains
The PRN biosynthetic gene cluster was PCR amplified from PA23 genomic DNA using
primers prnF and prnR (Table 1). The 5.6-kb PCR product was cloned into pCR2.1-TOPO
and the resulting plasmid, pTOPO-prn, was digested with ClaI and BspE1 to remove a 785-
bp fragment containing a portion of prnB and prnC. The ends were polished with Klenow
and ligated to an 840-bp SmaI-cut Gm R cassette isolated from pUCGm. The interrupted
prn operon was released from pTOPO-prn with XbaI and BamH1 and cloned into the same
sites of suicide vector pEX18Ap, yielding pEX18Ap-prn (Table 1). Plasmid pEX18Ap-prn was
mobilized into PA23 and PA23-63 by triparental mat- ing. Putative PRN-deficient mutants
PA23-8 and PA23-63-1 were screened by PCR analysis to verify that a double- crossover
event had occurred primers and primer walking. The PA23 prnABCD sequence was
submitted to GenBank (accession number EU188755). Sequence analysis of the Tn5 XhoI
rescue clones was performed using oligonucleotide primer Tn5-ON82 (Table 1), which
anneals to the 5 0 end of Tn5-OT182. Sequencing was performed at the University of
Calgary Core DNA Services facility, and sequences were analyzed with BLASTN and BLASTX
databases
Antifungal assays
Radial diffusion assays to assess fungal inhibition in vitro were performed according to
previously described methods (Poritsanos et al., 2006). Six replicates were analyzed for
each strain and assays were repeated four times.
PHZ and PRN gene expression
To monitor PHZ gene expression, a phzA–lacZ transcrip- tional fusion was created using
PCR primers phzAR-F and phzAR-R (Table 1). The phzA promoter region was PCR amplified
and cloned into pCR2.1-TOPO. A 1.1-kb HindIII and EcoRV fragment was removed and
cloned into HindIII–SmaI-digested pLP170, yielding pPHZA-lacZ. To generate a prn (PRN)
transcriptional fusion, PCR primers prnA-F and prnA-R (Table 1) were used to amplify a
1.5-kb fragment corresponding to the prnA promoter region of PA23. The PCR product
was first cloned into pCR2.1- TOPO, and then excised using HindIII and XbaI and ligated
into the same sites of pLP170, yielding pPRNA-lacZ. The phzA–lacZ and prnA–lacZ
transcriptional fusions were mo- bilized into PA23, PA23-63 (phzE), PA23-8 (prnBC) and
PA23-63-1 (phzE;prnBC). Cultures harboring these plasmids were grown in PTSB and b-
galactosidase assays were per- formed at various points throughout growth (Miller, 1972).
Samples were analyzed in triplicate and the experiment was repeated three times.
Quantification of PRN
The amount of PRN produced by PA23, PA23-8, PA23-63 and PA23-63-1 was quantified
by HPLC. Each strain was grown in 10 mL of 523 media on a rotary shaker for 4 days. The
10 mL volume was extracted with 50 mL ethyl acetate according to the method of Hwang
et al. (2002). Samples were dissolved in 500 mL of methanol and 500-mL aliquots of each
sample were injected into a Gemini C 18 column (100 4.6 mm; 5-mm particle diameter)
(Phenomenex Inc, Torrance, CA) and analyzed in an isocratic solution of 45%
acetonitrile/35% water/20% methanol. The eluent flow rate was 1.0 mL min 1 . Peaks
were detected by UV absorption at 225 nm using a Dionex AD20 Detector (Dionex,
Sunnyvale, CA). The concentration of PRN in each sample was based on standard curves
prepared from purified PRN
