Bacterial strains and growth conditions. All strains used in this study are listed in Table 1. Unless otherwise stated, Escherichia coli and B. cenocepacia H111 strains were routinely cultured aerobically in Luria-Bertoni (LB) Lennox broth (Difco) medium at 37°C. Culture media were solidified with 1.5% (w/v) agar and supplemented with the following antibiotics where appropriate: ampicillin, 100 µg/ml; chloramphenicol, 20 µg/ml; gentamycin, 20 µg/ml; kanamycin, 50 µg/ml; and trimethoprim, 50 µg/ml.
Table 1
Bacterial strains used in this study.
Strains | Characteristics | Source/Reference |
Burkholderia cenocepacia | | |
H111 | Clinical isolate from CF patient | 3 |
∆rpfF | H111 with in-frame deletion of rpfF | This study |
∆rpfR | H111 with in-frame deletion of rpfR | This study |
∆rpfF ∆rpfR | H111 with in-frame deletion of rpfF and rpfR | This study |
∆bceC | H111 with in-frame deletion of bceC | This study |
∆gtaB | H111 with in-frame deletion of gtaB | This study |
∆bepB | H111 with in-frame deletion of bepB | This study |
∆berB | H111 with in-frame deletion of berB | 16 |
∆berA | H111 with in-frame deletion of berA | This study |
∆bceC ∆gtaB | H111 with in-frame deletion of bceC and gtaB | This study |
∆rpfR ∆rpoN | H111 with in-frame deletion of rpfR and rpoN | This study |
∆rpfR ∆bceC | H111 with in-frame deletion of rpfR and bceC | This study |
∆rpfR ∆gtaB | H111 with in-frame deletion of rpfR and gtaB | This study |
∆rpfR ∆bepB | H111 with in-frame deletion of rpfR and bepB | This study |
∆rpfR ∆berB | H111 with in-frame deletion of rpfR and berB | This study |
∆rpfR ∆berA | H111 with in-frame deletion of rpfR and berA | This study |
∆rpfR ∆bceC ∆gtaB | H111 with in-frame deletion of rpfR, bceC and gtaB | This study |
∆cdpA | H111 with in-frame deletion of cdpA | 29 |
∆bcal0430 | H111 within-frame deletion of bcal0430 | 29 |
∆bcal1635 | H111 within-frame deletion of bcal1635 | 29 |
∆bcal1975 | H111 within-frame deletion of bcal1975 | 29 |
∆bcal2852 | H111 within-frame deletion of bcal2852 | 29 |
∆bcam0748 | H111 within-frame deletion of bcam0748 | 29 |
∆bca1161 | H111 within-frame deletion of bca1161 | 29 |
∆bcam1554 | H111 within-frame deletion of bcam1554 | 29 |
∆bcam1670 | H111 within-frame deletion of bcam1670 | 29 |
∆bcam2256 | H111 within-frame deletion of bcam2256 | 29 |
∆bcam2822 | H111 within-frame deletion of bcam2822 | 29 |
∆bcam2836 | H111 within-frame deletion of bcam2836 | 29 |
∆bcas0398 | H111 within-frame deletion of bcas0398 | 29 |
∆bcal0621 | H111 within-frame deletion of bcal0621 | 29 |
∆bcal2449 | H111 within-frame deletion of bcal2449 | 29 |
∆bcam1160 | H111 within-frame deletion of bcam1160 | 29 |
∆bcal0652 | H111 within-frame deletion of bcal0652 | 29 |
∆bcal1100 | H111 within-frame deletion of bcal1100 | 29 |
∆bcal2749 | H111 within-frame deletion of bcal1100 | 29 |
∆bcal3188 | H111 within-frame deletion of bcal3188 | 29 |
∆bcam0158 | H111 within-frame deletion of bcam0158 | 29 |
∆bcam2426 | H111 within-frame deletion of bcam2426 | 29 |
∆bcas0263 | H111 within-frame deletion of bcas0263 | 29 |
∆bcas0378 | H111 within-frame deletion of bcas0378 | 29 |
bepB::lacZ | H111 wild type with lacZ inserted downstream of bepB | This study |
∆rpfR bepB::lacZ | H111 ∆rpfR with lacZ inserted downstream of bepB | This study |
∆berA bepB::lacZ | H111 ∆berA with lacZ inserted downstream of bepB | This study |
∆rpfR::rpfRwt | H111 ∆rpfR reverted back to wild-type genotype | This study |
∆rpfR::rpfRGGAAF | H111 carrying rpfR allele with mutated GGDEF domain | This study |
∆rpfR::rpfRAAL | H111 carrying rpfR allele with mutated EAL domain | This study |
Escherichia coli | | |
DH5α | Strain for standard cloning applications | Laboratory stock |
Top10 | Strain for standard cloning applications | Laboratory stock |
E. coli M15[pREP4] | Expression host for recombinant proteins | Qiagen |
SY327 | Maintenance of pGPI-SceI based vectors | 18 |
DMH1 | Bacterial two-hybrid strain | 25 |
Transformation of E. coli and B. cenocepacia H111 strains. E. coli strains were transformed by standard electroporation procedures or the classical CaCl2 method. B. cenocepacia H111 strains were transformed by tri-parental mating18. Briefly, donor, helper and recipient strains were grown overnight in LB at 37°C with shaking. Two ml of each strain were harvested, washed and resupended in 500 µl (donor and helper) or 1 ml (recipient) LB Lennox broth. Donor and helper cells (100 µl each) were mixed and incubated for 20 min at room temperature. Recipient cells (200 µl) were added and the mixture was spot-inoculated onto the surface of prewarmed LB agar plates. After incubation at 37°C for 6 hours, cells were scraped off, resuspended in 500 µl 0.9% NaCl and plated on Pseudomonas Isolation Agar (Difco) supplemented with the appropriate antibiotics for counter-selection.
DNA manipulation. Plasmid DNA was isolated with the QIAprep Spin Miniprep Kit (Qiagen), chromosomal DNA was prepared with the DNeasy Blood & Tissue Kit (Qiagen), and DNA fragments were purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research) where required. For cloning purposes, DNA was amplified with Phusion High-Fidelity DNA Polymerase (New England Biolabs), to confirm vector constructs and mutants, GoTaq DNA Polymerase (Promega) was employed. Oligonucleotides used in this study are listed in Table S1.
Construction of expression vectors. Plasmids employed in this study are listed in Table S2. Coding sequences for rpfR, rpfRGGAAF, rpfRAAL, berA, and yedQ were subcloned into the broad-host-range cloning vector pBBR1MCS-519 as follows: rpfR, rpfRGGAAF, and rpfRAAL as XbaI-HindIII fragments from pBBR-rpfR, pBBR-rpfRGGAAF, and pBBR-rpfRAAL, respectively, generating pRpfRwt, pRpfRGGAAF, and pRpfRAAL; berA as a BamHI-XbaI fragment from pBBR2-Bcam1349, generating pBerA; and yedQ as a HindIII-BamHI fragment from pYhck, generating pYedQ. To generate pRpoN and pBerB, coding sequences together with the putative promoter region were PCR-amplified from genomic DNA using primer pairs P103/P104 and P280/P281, respectively, and cloned as XbaI-HindIII fragments into pBBR1MCS-5.
For heterologous expression, the rpfR gene was PCR-amplified using primers pQE-rpfR-F and pQE-rpfR-R, digested with BamHI and HindIII and cloned into pQE-32 digested with the same enzymes, giving rise to plasmid pQE-RpfR, which was transformed into E. coli M15[pREP4]. Point mutations were inserted into pQE32-rpfR by site-directed mutagenesis, using the QuikChange site directed mutagenesis system (Agilent) as described previously10, generating plasmids pQE-RpfRGGAAF and pQE-RpfRAAL.
Construction of B. cenocepacia H111 mutants. To generate unmarked gene deletions or introduce rpfR variants into the chromosome, we used the method described by Flannagan et al.18, which is based on the homing nuclease I-SceI and allows for markerless gene deletion (knock out) as well as gene insertion (knock in). Since B. cenocepacia H111 is not very sensitive to tetracycline, the XhoI-SalI fragment of pDAI-SceI containing the tetA and tetR genes was replaced with the PCR-amplified (primers GmR_F and GmR_R) gentamicin-3-acetyltransferase gene from pBBR1MCS-5, generating the I-SceI expression plasmid pDAIGm-SceI.
Knock-out plasmids were generated by PCR amplification of the flanking regions of target genes (~ 1200 bp for rpfR, ~ 600 bp for all other target genes) from genomic DNA of B. cenocepacia H111, incorporating EcoRI/NcoI and NcoI/KpnI recognition sites into left and right homology arms, respectively, using the following primer pairs: P14/P15 and P16/P20 for pGPI_∆rpfR; P47/P48b and P49/P50b for pGPI_∆rpfF; P46/P47 and P14/P15 for pGPI_∆rpfFR; P66/P67 and P68/P69 for pGPI_∆bepB; P73/P74 and P75/P76 for pGPI_∆bceC; P80/P81 and P82/P83 for pGPI_∆gtaB; P86/87 and P88/89 for pGPI-ΔrpoN; P93/P94 and P95/P96 for pGPI_∆berA. PCR fragments were digested with the respective enzymes and cloned into the EcoRI/KpnI-linearized pGPI-SceI plasmid in a three-way ligation, thereby joining the two homology arms through the NcoI site. All knock-out plasmids were verified by sequencing.
The multiple cloning site of plasmid pGPI-SceI was modified by insertion of annealed oligonucleotides MCS-GPI_F and MCS-GPI_R into the EcoRI/XbaI-linearized vector, generating pGPI-SceI-ΔXbaI. The unique PstI site in the vector backbone was removed by linearizing pGPI-SceI-ΔXbaI with PstI, blunting ends with T4 DNA polymerase and re-ligation, giving rise to pGPI-SceI-ΔXbaI-ΔPstI. Annealed ologonucleotides MCS2-GPI_F and MCS2-GPI_R were ligated into the NcoI/KpnI-linearised pGPI-SceI-ΔXbaI-ΔPstI vector, thus generating plasmid pGPI2-SceI, which carries unique restriction sites for EcoRI, BglII, EcoRV, NcoI, SpeI, XbaI, XhoI, SphI, KpnI in its multiple cloning site.
To introduce point mutations into the GGDEF and EAL domain of chromosomally encoded RpfR, the respective rpfR variants were excised as XbaI-KpnI fragments from plasmids pRpfRGGAAF, pRpfRAAL, and pRpfRwt and subcloned into pGPI2-SceI cleaved with the same enzymes, yielding plasmids pGPI-rpfRGGAAF, pGPI-rpfRAAL, and pGPI-rpfRWT. All knock-in plasmids were confirmed by sequencing.
B. cenocepacia H111 locus tags, orthologs in the closely related B. cenocepacia J2315, and products of genes investigated in this study are summarized in Table 2.
Table 2
Genes/gene clusters inactivated in this study.
Locus Tag a | Ortholog in J2315 b | Product description c | Gene/cluster name |
I35_2858 | BCAL0813 | RNA polymerase factor sigma-54 | rpoN |
I35_4474 | BCAM0580 | Cis-2-dodecenoic acid receptor RpfR | rpfR |
I35_4475 | BCAM0581 | BDSF synthase rpfF | rpfF |
I35_4769 | BCAM0855 | UDP-glucose dehydrogenase | bceC |
I35_4929 | BCAM1010 | UTP–glucose-1-phosphate uridylyltransferase | gtaB |
I35_5181 | BCAM1331 | Tyrosine-protein kinase Wzc | bepB |
I35_5193 | BCAM1342 | Sigma-54 dependent transcriptional regulator | berB |
I35_5200 | BCAM1349 | CRP/FNR family transcriptional regulator (10) | berA |
I35_4767-I35_4777 | BCAM0854-BCAM0864 | Cepacian | bce-I |
I35_4922-I35_4930 | BCAM1003-BCAM1011 | Cepacian | bce-II |
I35_5180-I35_5191 | BCAM1330-BCAM341 | Burkholderia exopolysaccharide | bepA-N |
a Nomenclature according to GenBank accession numbers HG938370, HG938371, and HG938372. |
b Orthologs in B. cenocepacia J2315 were identified by BLAST search. |
c Product description according to GenBank accession numbers HG938370, HG938371, and HG938372. |
Construction of the bepB-lacZ reporter strains. To generate pGPI-bepB::lacZ, a pGPI-SceI based plasmid to introduce the lacZ gene along with its ribosome binding site (RBS) downstream of the bepB gene, left and right homology arms were amplified from genomic DNA of B. cenocepacia H111. The right homology arm (RHA) was PCR-amplified using primer pair P69/P122, the left homology arm (LHA) was amplified with primer pair P120/P121. The XbaI/KpnI-digested RHA was first cloned into pBluescript SK(+) linearized with the same enzymes, resulting in pBluescript-RHA. The EcoRI/PstI-digested LHA was cloned into pSUP3535 linearized with the same enzymes, giving rise to pSUP3535-LHA-lacZ. The KpnI-XbaI fragment of pBluescript-RHA comprising the RHA, and the EcoRI-XbaI fragment from pSUP3535-LHA-lacZ, comprising the LHA and the lacZ gene, were cloned into the EcoRI/KpnI-linearized pGPI-SceI plasmid, generating pGPI-bepB::lacZ.
Macrocolony morphology and cepacian production assays. Strains were precultured overnight in LB Lennox broth under aeration at 37°C. To assay macrocolony morphology, 3 µl of precultures were spotted on NYG agar plates (0.5% peptone, 0.3% yeast extract, 2% (w/v) glycerol, and 1.5% agar) and incubated for 3 days at 37°C, followed by at least three days at room temperature. Cepacian production was assayed by streaking precultures on YEM agar plates (0.05% yeast extract, 0.4% mannitol, and 1.5% agar) and incubation at 37°C for 2 days. All strains to be compared were grown in parallel on single square petri dishes (120 mm, Greiner Bio-One).
Determination of β-galactosidase activity. To assay β-galactosidase activity within macrocolonies, NYG agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal; 40 µg/ml) were spot inoculated as described above. For quantification of β-galactosidase activity, strains to be tested were grown overnight in LB Lennox broth at 37°C and used to inoculate 100-ml Erlenmeyer flasks containing 20 ml of, unless otherwise stated, AB minimal medium20 supplemented with 1.5% glycerol as carbon source to an OD600 of 0.05. Cultures were incubated with agitation (220 rpm) for 24 h at 37°C. β-galactosidase activity was quantified as described by Stachel et al.21 with slight modifications. Briefly, 50–200 µl of cell culture were harvested and resuspended in 500 µl Z-buffer. After addition of 25 µl of CHCl3 and 25 µl of 0.05% SDS, the cell suspension was vortexed for 10 seconds and then incubated at 30°C for 15 min. The reaction was started by adding 200 µl of o-nitrophenyl-β-D-galactopyranosid (ONPG; 4 mg/ml) and incubated at 30°C. The reaction was stopped by addition of 250 µl of 1 M Na2CO3. Cell debris was removed by centrifugation and absorbance was recorded at 420 nm and 550 nm. β-galactosidase activity was calculated as Miller Units, using the formula Miller Units = 1000*(OD420-(1.75*OD550))/(time[min]*V[ml]*OD600).
DGC/PDE assays. To assess the enzymatic activity of RpfR, a DGC/PDE assay was performed as previously described22. Briefly, 2 µM of purified RpfR or its variants were resuspended in 50 mM Tris-Cl pH 7.8, 500 mM NaCl, 2 mM MgCl2. The reaction was started by adding GTP (Sigma) or c-di-GMP (Biolog) as substrate to a final concentration of 100 µM and incubating the reaction mix at 37°C. After 180 min of incubation, the protein was denatured by heating to 99°C for 5 min, followed by centrifugation at 20.000 g for 15 min. Supernatants were analysed on an Äkta FPLC system, using 1 ml ResourceQ columns (GE Healthcare) and a linear gradient from 0.5%-100% of 1 M NH4HCO3, pH 8. GTP, c-di-GMP and pGpG (Biolog) were used as standards at a concentration of 100 µM.
Extraction and quantification of c-di-GMP. To quantify intracellular c-di-GMP levels, strains to be analysed were grown in LB Lennox broth to an OD600 of ~ 1.8 at 37°C with aeration. Extraction and quantification of c-di-GMP was performed as described by Burhenne and Kaever23.
Bioassay for BDSF production. To test H111 strains for BDSF production, cross-streak experiments with the H111-rpfFBc / pAN-L15 biosensor strain were performed as previously described24.
Bacterial two hybrid analysis. For bacterial two hybrid analysis, we used the system described by Karimova, 199825 using a split adenylate cyclase. Primer pairs P301/P302 and P303/304 were used to PCR amplify the berB. The resulting PCR fragments and the plasmids pUT18 and pUT18C were then digested with the restriction enzymes BamHI and HindIII before being ligated together to create pUT18-BerB and pUT18C-BerB. The following primer pairs were used to create PCR fragments of the full length rpfF gene, full length rpfR gene, and specific domains of the rpfR gene: P272/P273 for full length rpfF; P214/P215 for full length rpfR; P214/P238 for rpfR PAS-GGDEF; P237/P215 for rpfR GGDEF-EAL; P214/P236 for rpfR PAS; P237/P238 for rpfR GGDEF; and P239/P215 for rpfR EAL. The resulting PCR fragments were digested with the restriction enzymes XbaI and KpnI alongside the plasmids pKT25 and pKNT25. Then each fragment was ligated into each plasmid to create pKT25-RpfF, pKNT25-RpfF, pKT25-RpfR and pKNT25-RpfR and the various derivatives. The plasmids were introduced into E. coli DHM1 by electroporation and selected for on first single selection plates then double selection plates to create DHM1 strains containing one pUT18-derivative or pUT18C-derivative with one pKT25-derivative or pKNT25-derivative. Pairs of the parental plasmids were used as a negative control, and a Zip domain plasmid pair25 was used as a positive control. 5µl of the DHM1 strains were grown on MacConkey agar supplemented with 1% maltose at 30°C for 72 hours before being imaged.
Determining the β-galactosidase activity of the bacterial two hybrid mutants. DMH1 strains were grown overnight at 30°C in LB cultures with the appropriate antibiotics before inoculating 100µl of this culture into agitated conical flasks (220 rpm) containing 10ml LB. Cultures were grown overnight at 30°C and 200µl samples were used for β-galactosidase assays after permeabilization of the cells with chloroform-sodium dodecyl sulfate26.