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Supplementary material
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Appendix S1.
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Total RNA isolation
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Total RNA (totRNA) was isolated from cultures of B. cepacia strain 2a and its mutants
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grown in MSM supplemented with 2 mM succinate and 2.26 mM 2,4-D using a silica-
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magnetite magnetisable solid-phase support, produced at the University of Kent.
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Frozen cell pellets were thawed on ice, resuspended in 100µl TE buffer containing
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lysozyme (400µg/ml) and incubated for 5 minutes at 20°C. Then, 200µl of lysis buffer (50
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mM Tris-HCl pH8; 25 mM EDTA pH8; 4 M guanidine thiocyanate; 0.01% Triton-X-100; 1%
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β-mercaptoethanol) was added and the mixtures vortexed for 5 seconds. Subsequently,
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300 µl of isopropanol was added and the resultant volume transferred to a sterile 1.5 ml
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Eppendorf tube, containing 2 mg of silica-magnetite. The mixture was incubated with
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gentle agitation for 5 minutes at 20°C, the silica-magnetite immobilised using a magnetic
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stand (Promega Ltd.) and the supernatant removed and discarded. The silica-magnetite
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was then washed with 200 μl of cold 80% (v/v) aqueous ethanol, immobilised
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‘magnetically’ the supernatant removed and the silica-magnetite left to air dry for 5
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minutes. RNA was then eluted from the support by addition of 70 μl of nuclease free water
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followed by incubation with gentle agitation for 1 minute at room temperature. The support
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was then immobilised and the RNA-containing supernatant transferred to a sterile 1.5 ml
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Eppendorf tube and the elution process repeated and the eluates combined. 1 µl of the
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product was removed for quantification and purity measurement (OD260/280). RNasin®
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(Promega Ltd.) was added to the remaining sample to a final concentration of 1U/μl and 5
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μl of the solution mixed with 2 μl of 10X RNA loading buffer (Sigma Ltd) and loaded onto a
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2% (w/v) agarose gel (Hi-PureTM low EEO agarose) for physical quality assurance.
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Undegraded totRNA was confirmed by the state of intactness of the 23 and 16S rRNA
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bands.
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Prior to quantitative reverse transcription real time-PCR, totRNA samples were treated
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with DNase I to remove any contamining DNA in the following manner: 2 μg of RNasin ®
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containing totRNA was placed in a sterile 0.5 ml Eppendorf tube containing 1μl of 10X
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DNase I reaction buffer (200 mM Tris-HCl pH 8.4, 20mM MgCl2, 500 mM KCl), 1 U DNase
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I (Promega Ltd.) and the volume made up to 10 μl with nuclease free water. The reaction
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mixture was incubated for 30 minutes at 37°C and stopped by addition of 1 μl of RQ1
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DNase stop solution (20 mM EGTA pH 8) and heating to 65°C for 10 minutes.
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Subsequently, 2 μl of DNase I treated total RNA was mixed with 2 μl of RNA loading buffer
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(50% (v/v) glycerol; 1 mM EDTA pH 8; 0.5% bromophenol blue; 1 μg/ml ethidium bromide)
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and loaded onto a 2 % (w/v) agarose gel to check for contaminating DNA. Successful
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removal of DNA was also confirmed by PCR amplification of DNase I-treated totRNA. The
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remaining sample was stored at -196°C until required.
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Table 2. Primers used to construct open- and closed-channel B. cepacia strain 2a mutants
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Mutant name
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(genotype)
Sequences (5’ – 3’)
Product size
(bp)
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Open-channel
Fw- CGGAATTCCGTTCGGCTTACTGCGGCAAAG
651
EcoRI
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(Glu162Ala)
Rw- CGGATCCCGTCGCGACGAACTTGAGTAAG
BamHI
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Mut- TCGATTGGGCGCGCTGGCCCTTCAGTGC
GAG
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Closed-channel
Fw- CGGAATTCCGTTCGGCTTACTGCGGCAAAG
651
EcoRI
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(Glu162Asp)
Rw- CGGATCCCGTCGCGACGAACTTGAGTAAG
BamHI
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Mut- CGATTGGGCGCGGATGGCCCTTCAGTGC
GAG
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Mutations were generated by amplification of pRP15c with primers designed using
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PrimerX (available at http://bioinformatics.org/primerx/) according to Ke and Madison
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(1997) and were modified so as to possess EcoRI and BamHI restriction sites (underlined)
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allowing products to be used in subsequent cloning. The nucleotide changes to introduce
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alanine (Ala) and aspartate (Asp) in place of a glutamate (Glu) are underlined in the
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mutagenic primers (Mut). GAG, GCT and GAT represented the trinucleotides encoding
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Glu, Ala and Asp, respectively.
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