Supplementary Experimental Procedures
Bacterial transformation
Competent cells of E. coli were prepared by the standard CaCl2 method and plasmid DNA
was introduced by heat shock method (Sambrook et al., 1989). Competent cells of P.
aeruginosa were prepared by chemical treatment with MgCl2 combined with a 50º C heatpulse (Irani and Rowe, 1997). Competent cells of P. syringae were prepared by treatment with
0. 5 M sucrose and plasmids were introduced by electroporation.
DNA manipulations
Purification of bacterial plasmids and Pseudomonas genomic DNA was conducted using
Plasmid Mini Kit and Genomic Mini Kit, respectively (A&A Biotechnology), according to
the manufacturer’s instructions.
To purify DNA from agarose, gel slices were cut out and processed using the Gel-Out system
(A&A Biotechnology), according to the manufacturer’s instructions. PCR amplification was
performed using high fidelity Pfu DNA polymerase (Promega). Usually 30 cycles of
amplification were performed, at reaction conditions depending on the pairs of primers and
the size of the final amplification product. The PCR reactions were conducted on PTC-200
Engine Cycler (MJ Research), and the PCR products were identified by agarose gel
electrophoresis. The primers are listed in Table 1.
Western blotting
Samples were separated by 12-15% SDS-PAGE and then transferred to PVDF membranes for
20-30 min at 15 V using a BioRad SD device (BioRad Laboratories) . The membrane was
blocked overnight at 4°C in 1×TBST (Tris-buffered saline plus 0.1% Tween-20) containing
5% non-fat dry milk. Primary mouse anti-His antibodies were diluted 1:5000 with TBST
containing 1% nonfat dried milk and incubated at 37°C for 2 h. After washing with TBST, the
membrane was incubated with alkaline phosphatase-conjugated goat anti-mouse antibody at
1:5000 dilution for 1 h at 37°C, and then developed according to the manufacturer’s
instructions.
Introduction of mutant alleles into P. syringae DC3000 or P. aeruginosa PAO1161
The donor E. coli S17-1 with pAKE600 suicide vector derivatives and the appropriate
recipient strain (either P. syringae DC3000KmR or PAO1161RifR) were mixed in ratio 1:1 or
1:2 and plated out on L agar plates and incubated at 37°C overnight. The lawn of bacterial
cells was scraped and resuspended in L broth. Serial dilutions of the suspension were plated
on L agar with carbenicillin and rifampicin for PAO1161RifR conjugants and
chloramphenicol and rifampicin for the DC3000 strain, and allowed to grow for 24-48 h at
37°C or 28°C, respectively. Putative integrants were plated on L agar with rifampicin,
carbenicilin and chloramphenicol (when the CmR cassette was used to disrupt the Ps nudC
gene). After double restreaking of RifRCmR or RifRCbR colonies (for Ps nudC or Pa
nudCΔ13-597 mutant), the cells were used to inoculate L broth containing 10% (w/v) sucrose
and cultured to facilitate removal of the suicide vector insertion from integrants. Survivors
were plated on L agar with 10% (w/v) sucrose and the grown colonies were analyzed by PCR
(using chromosomal DNA as a template) to determine whether allele exchange had occurred.
To confirm the presence of mutations in Ps nudC in the P. syringae DC3000 backbone and in
Pa nudC in the PAO1161RifR backbone RT-PCR analysis of transcripts was conducted.