Article Figures & Data
Figures
- Fig. 1.
Effect of cepR on CAS activity in B. cepacia. CAS activity was monitored throughout growth in succinate medium. ■, Pc715j (wild type); □, Pc715j-R1 (cepR). Growth (turbidity at 600 nm) is shown as solid lines, and CAS activity (OD630/OD600) is shown as dashed lines. All values shown are the means ± standard deviations of values from triplicate experiments. The difference between Pc715j and Pc715jR1 is significant at all time points between 9 and 30 h ( P < 0.05 ; t test for unpaired observations).
- Fig. 2.
TLC of acyl-HSLs. Ethyl acetate extracts were chromatographed on C18 reversed-phase TLC plates developed with methanol-water (60:40, vol/vol). The spots were visualized using the A. tumefaciens reporter strain. Lane 1, K56-2; lane 2, K56-I2; lane 3, K56-R2; lane 4, Pc715j; lane 5, Pc715j-R1; lane 6, synthetic OHL and HHL standards.
- Fig. 3.
Effect of cepR on cepR andcepI expression. β-Galactosidase activity was monitored throughout growth in tryptic soy broth plus 100 μg of trimethoprim per ml. (A) ■, K56-2 (pSLR111, cepR-lacZ); □, K56-R2 (pSLR111, cepR-lacZ); ○, K56-2 (pUCP28T). The difference between K56-2 and K56-R2 is significant at all time points between 4.5 and 29.5 h ( P < 0.02 ; t test for unpaired observations). (B) ■, K56-2 (pSLS222, cepI-lacZ); □, K56-R2 (pSLS222, cepI-lacZ). The difference between K56-2 and K56-R2 is significantly different at all time points between 4.5 and 29.5 h ( P < 0.02 ; t test for unpaired observations). Growth (turbidity at 600 nm) is indicated by solid lines, and β-galactosidase activity (Miller units) is indicated by dashed lines. All values are the means ± standard deviations of values from triplicate experiments.
Tables
- Table 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Descriptiona Source or reference Strains E. coli DH5α φ80dlacZΔM15 (lacZYA-argF) recA1 endA gyrA96 thi-1 hsdR17 supE44 relA1 deoR U169 Life Technologies SM10 Mobilizing strain, RP4 integrated in chromosome, Kmr 34 HB101 supE44 hsdS20 (rBmB) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1 29 B. cepacia K56-2 CF respiratory isolate, genomovar III, BCESM+cblA+ 17, 19 K56-R2 cepR::Tn5-OT182 derivative of K56-2, Tcr 17 K56-I2 cepI::tp derivative of K56-2, Tpr 17 Pc224c CF respiratory isolate (Cleveland, Ohio) 3 Pc224c-R1 cepR::tpderivative of Pc224c, Tpr This study Pc715j CF respiratory isolate, genomovar III 3, 20 Pc715j-R1 cepR::tpderivative of Pc715j, Tpr This study ATCC 17759 Soil isolate, genomovar I 19 ATCC 25416 Onion isolate, genomovar I 19 P109 CF respiratory isolate (Seattle, Wash.) 3 Pc22-20 Plant isolate 3 34192 CF respiratory isolate (Calgary, Alberta, Canada) 3 34930 CF respiratory isolate (Calgary) 3 T10 pvdD::Tn5OT182 derivative of K56-2, Tcr 37 T10-R10 pvdD::Tn5OT182,cepR::tp TcrTpr This study I117 pvdA::Tn5OT182 derivative of K56-2, Tcr 37 I117-R21 pvdA::Tn5OT182,cepR::tp TcrTpr This study A. tumefaciens A136 Ti plasmidless host C. Fuqua Plasmids pNOT19 Modified pUC19 cloning vector, Apr 30 pUCP28T Broad-host-range vector, IncP OriT, pRO1600 ori, Tpr 31 p34E-Tp Source of trimethoprim cassette, Tpr 5 pRK2013 ColE1 Tra (RK2)+ Kmr 7 pEX18Tc Suicide vector, sacB Tcr 13 pUCP26 Broad-host-range vector, IncP pRO1600 ori, Tcr 47 pZ1918G Source of lacZreporter, Apr Gmr H. P. Schweizer pCRR2.1TOPO Cloning vector for PCR products Invitrogen pSLA3.2 pUCP28T with 3.2-kbSphI fragment containing cepIR, Tpr 17 pSLR101 pNOT19 with 1.6-kbKpnI-SphI fragment from pSLA3.2 containingcepR, Apr This study pSLR101-T pSLR101 with tp cassette cloned inPstI site, Apr Tpr This study pSLS200 pUCP28T with 2.3-kb SphI-PstI fragment from pSLA3.2 containing cepI and a part ofcepR, Tpr This study pSLR111 cepR-lacZ transcriptional fusion withlacZ-Gmr cassette cloned in PstI site of pSLS200, Tpr This study pSLS210 pUCP28T with 0.8-kb PstI-BamHI fragment containingcepI promoter, Tpr This study pSLS222 cepI-lacZ transcriptional fusion withlacZ-Gmr cassette cloned in BamHI site of pSLS210, Tpr This study pEXCEPR pEX18Tc with tp-inactivated cepR fragment from pSLR101-T, Tpr Apr This study pCF218 IncPtraR 49 pCF372 traI-lacZ 9 ↵a BCESM, B. cepacia epidemic strain marker.
- Table 2.
Effect of a cepR mutation on siderophore, protease, and autoinducer productiona
Strain Genotype Length of CAS agar zonesb(mm) Ornibactinc (μg/ml/OD600unit) Pyochelind (μg/ml/OD600unit) Salicylic acidd(μg/ml/OD600 unit) Radius of protease zonee (mm) cepI bioassay resultf Pc715j Wild type 7.3 ± 0.3 53.9 ± 20.5 5.4 ± 0.8 0.5 ± 0.2 8.3 ± 0.3 + Pc715j-R1 cepR::tp 10.8 ± 0.3* 99.1 ± 19.7 4.1 ± 2.0 0.3 ± 0.2 6.8 ± 0.3* − ↵a All values are the means of results of triplicate experiments ± standard deviations, unless indicated otherwise. *, value is significantly different from that for the parent Pc715j in an unpaired t test (P < 0.05) .
↵b The CAS zone is the distance from the edge of the orange zone to the edge of the colony after 48 h.
↵c Values are means ± standard deviations of results of duplicate experiments.
↵d Amount of siderophore in CAA cultures grown for 24 h.
↵e The protease zone radius is the distance from the edge of the colony to the edge of the zone of clearance on D-BHI plus 1.5% skim milk agar at 48 h.
↵f Cross-feeds the cepI reporter K56-I2 and restores protease production.
- Table 3.
Effect of cepR on pvdA andpvdD expression in B. cepacia
Strain β-Galactosidase activity (Miller units)a in: TSBD-C medium TSBD-C + FeCl3 medium I117 11,324 ± 548 270 ± 37 I117-R21 13,526 ± 330b 439 ± 69b T10 8,290 ± 113 1,469 ± 200 T10-R10 9,678 ± 357 1,986 ± 112c ↵a β-Galactosidase activity was determined from stationary-phase cultures (22 h) normalized for absorbance at A600. Values are the means ± standard deviations of results of triplicate assays.
↵b Significantly different than the value for I17 ( P < 0.02 ; t test for unpaired observations).
↵c Significantly different than the value for T10 ( P < 0.02 ; t test for unpaired observations).
