Article Figures & Data
Figures
- FIG. 1.
TOL plasmid promoters under control of the XylR protein. The xylR gene, expressed from two overlapping PR promoters, yielded an inactive XylR protein (□) which, in the presence of toluene, became active (▪) and stimulated transcription (+) from Pu and Ps1 while repressing (−) its own synthesis. The alternative RpoN sigma factor participating in transcription of Pu and Ps1 is indicated. IHF has a positive role in the transcription of Pu (37).
- FIG. 2.
Integration of glucose and toluene metabolism into central metabolism in P. putida KT2440. The pathways are based on experimental data reported by Worsey and Williams (49), Velázquez et al. (49), and del Castillo et al. (9). Abbreviations: GLC, glucose; GLT, gluconate; TKG, 2-ketogluconate; 6PG, 6-phosphogluconate; 2K3D6PG, 2-dehydro-3-deoxy-6-phosphogluconate; PYR, pyruvate; PEP, phosphoenolpyruvate; G3P, glyceraldehyde-3-phosphate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; TOL toluene; BEN, benzoate; CAT, catechol; AcCoA, acetyl coenzyme A; CIT, citrate; ICIT, isocitrate; 2KG, 2-ketoglutarate; MAL, malate; OAA oxaloacetate.
- FIG. 3.
13C-labeling patterns in different amino acids derived from [13C]glucose. Bacterial cultures were fed with 20% (wt/wt) [13C]glucose, and 13C levels in serine (hatched bars), glycine (open bars), proline (stippled bars, white dots on black background), and glutamate (stippled bars, black dots on white background) were determined. The strains used are indicated as follows: KT, parental strain; glk pWWO, mutant deficient in glk; and gcd pWWO, mutant deficient in gcd. −tol indicates that toluene was absent, and +tol indicates that toluene was present. The y axis indicates the percentages of 13C/12C in the amino acids.
- FIG. 4.
Induction of Pu in parental strain P. putida KT2440 and edd and eda mutants of this strain. Bacterial cells were grown on citrate as the sole C source, and when cultures reached the mid-exponential phase (OD660, 0.7 ± 0.1), cells were harvested, washed in M9 minimal medium without a C source, and divided into three aliquots that were supplemented with glucose, toluene, or glucose plus toluene. β-Galactosidase levels were determined 30 min later. The data are the averages and standard deviations of three independent assays. Open bars, P. putida KT2440(pS10); gray bars, edd mutant containing pS10; dotted bars, eda mutant.
Tables
- TABLE 1.
Strains and plasmids used in this study
Strain or plasmid Genotype or relevant characteristicsa Reference(s) P. putida strains KT2440 Wild type, prototroph; Cmr Rifr 1, 32 M1044b edd:mini-Tn5-Km Kmr Rifr 14 M1128b eda:mini-Tn5-Km Kmr Rifr 14 M438b gcd:mini-Tn5-Km Kmr Rifr 14 PSC278b glk::pCHESIΩ-Km Rifr 9 KT2440 ptsN Kmr; P. putida KT2440 with a kanamycin resistance cassette interrupting the ptsN gene 3 KT2440 crc Gmr; P. putida KT2440 with a gentamicin resistance cassette interrupting the crc gene 3 KT2440 crp Kmr; P. putida KT2440 with a kanamycin resistance cassette interrupting the crp gene 3 KT2440 cyoB Tcr; P. putida KT2440 with a tetracycline resistance cassette interrupting the cyoB gene 38 KT2440 relA Kmr; P. putida KT2440 with a kanamycin resistance cassette interrupting the relA gene 44 KT2440(pWW0r PmΩ Sm) Smr; P. putida KT2440 with a kanamycin resistance cassette interrupting the Pm gene 26 KT2440(pWW0::μ 21) Kmr; P. putida KT2440 with a kanamycin resistance cassette interrupting the xylE gene 26 Plasmids pRK600 Helper plasmid; tra + mob+ Cmr 5, 19 pS10 IncP1 Smr xylR; transcriptional Pu::lacZ::tet fusion 3 pWW0 IncP9 mob+ tra+ 3MB+ 49 - TABLE 2.
Growth rates and physiological parameters of P. putida KT2440(pWW0) and isogenic mutants of this strain growing with different carbon sources
Strain Growth rate (h−1) with: qGlu (μmol/mg [cell dry wt] h−1)a qtol (μmol/mg [cell dry wt] h−1)b Glucose Toluene Glucose + toluene Without toluene With toluene Without glucose With glucose KT2440(pWW0) 0.73 ± 0.03c 0.72 ± 0.02 0.74 ± 0.07 13.1 ± 0.8 5.7 ± 0.5 11.9 ± 0.5 6.4 ± 0.2 KT2440 gcd(pWW0) 0.42 ± 0.01 0.73 ± 0.04 0.45 ± 0.01 5.0 ± 0.5 2.4 ± 0.1 12.4 ± 0.1 11.4 ± 0.4 KT2440 glk(pWW0) 0.38 ± 0.06 0.72 ± 0.01 0.66 ± 0.01 5.1 ± 0.5 5.9 ± 0.7 11.3 ± 0.2 5.5 ± 0.3 KT2440 ptsN(pWW0) 0.67 ± 0.01 0.70 ± 0.01 0.71 ± 0.18 NDd ND ND ND KT2440 crc(pWW0) 0.51 ± 0.1 0.65 ± 0.1 0.65 ± 0.04 ND ND ND ND ↵ a Glucose consumption was determined in cells growing in the absence and in the presence of toluene.
↵ b Toluene consumption was determined in cells growing in the absence and in the presence of glucose.
↵ c The data are averages ± standard deviations of three to six independent assays, each done in duplicate.
↵ d ND, not determined.
- TABLE 3.
P. putida KT2440 upregulated genes in cells growing on glucose plus toluene compared to cells growing on glucose alone
Open reading frame and/or gene Family Fold changea xylU Probable toluene porin 13.43 xylW Probable toluene porin 6.31 xylA Toluene monooxygenase 6.05 xylB Benzyl alcohol dehydrogenase 3.05 xylC Benzaldehyde dehydrogenase 2.93 xylX Toluate 1,2-dioxygenase 2.86 xylZ Toluate 1,2-dioxygenase 4.68 xylL 1,2-Dihydroxy-3,5-cyclohexadiene-1-1-carboxylate 4.11 xylE Catechol 2,3-dioxygenase 2.70 xylF Hydrolase semialdehyde 2-hydroxymuconic 2.02 xylG Dehydrogenase semialdehyde 2-hydroxymuconic 3.44 xylH 4-Oxalocrotonate tautomerase 3.74 xylI 4-Oxalocrotonate decarboxylase 2.78 xylK 4-Hydroxy-2-oxovalerate hydrolase 3.42 xylM Toluene monooxygenase 3.84 xylN Unknown function 2.61 xylQ Acetaldehyde dehydrogenase 2.48 xylT Ferredoxine 2.43 PP0210 Putative phycobiliprotein 2.86 PP1074 (glpR) Glycerol-3-phosphate regulon repressor 2.79 PP1897 DNA internalization-related competence protein 4.36 PP2268 Phage endodeoxyribonuclease 2.19 PP2589 Aldehyde dehydrogenase family protein 2.52 PP2805 Monooxygenase flavin-binding family 2.27 PP3243 Acetyltransferase GNAT family 7.38 PP3413 Sensor histidine kinase/response regulator 2.38 PP3717 Transcriptional regulator LuxR family 2.94 PP3998 Glutathione S-transferase domain protein 4.05 PP4538 Putative acyl carrier protein phosphodiesterase 3.87 PP4983 Flavin-containing monamine oxidase family protein 2.23 PP5340 Acetylpolyamine aminohydrolase 2.09 pcaC 4-Carboxymuconolactone decarboxylase 2.13 pcaJ 3-Oxoadipate coenzyme A-transferase, subunit B 2.15 PP3726 (ech) Enoyl coenzyme A hydratase/isomerase family protein 4.91 PP5248 Hydrolase isochorismatase family 2.46 PP5255 Hydrolase isochorismatase family 2.49 PP1982 (ibpA) IbpA heat shock protein IbpA 2.00 PP3735 ABC transporter ATP-binding protein 2.00 PP3961 Putative transporter 2.00 PP1687 Hypothetical protein 2.94 PP2644 Hypothetical protein 2.97 PP3353 Conserved hypothetical protein 7.04 PP4561 Conserved hypothetical protein 2.73 PP4901 Conserved hypothetical protein 2.72 PP4981 Conserved hypothetical protein 2.02 PP4982 Conserved hypothetical protein 2.64 Hypothetical protein pWW0 c57031-56282 2.70 Hypothetical protein pWW0 c65777-65391 2.08 Hypothetical protein pWW0 c94962-94570 3.65 Hypothetical protein pWW0 c66769-66416 3.97 ↵ a The changes are averages of at least two assays. The P values were ≤0.05.
- TABLE 4.
Glucokinase activities in wild-type and mutant cells
Strain Substrate(s) Glucokinase activity (nmol/min/mg protein)a KT2440(pWW0) Glucose 27.3 ± 1.9 KT2440(pWW0) Glucose + toluene 16.4 ± 1.9 KT2440(pWW0) Toluene 1.3 ± 0.3 KT2440 pstN(pWW0) Glucose 30.2 ± 1.4 KT2440 pstN(pWW0) Glucose + toluene 16.2 ± 2.7 KT2440 pstN(pWW0) Toluene 0.9 ± 0.1 KT2440 crc(pWW0) Glucose 27.3 ± 5.4 KT2440 crc(pWW0) Glucose + toluene 27.6 ± 1.7 KT2440 crc(pWW0) Toluene 1.4 ± 0.2 KT2440(pWW0 Δ Pm) Glucose 26.3 ± 0.3 KT2440(pWW0 Δ Pm) Glucose + toluene 26.7 ± 0.4 KT2440(pWW0 Δ Pm) Toluene 1.5 ± 0.2 ↵ a The data are the averages ± standard deviations of at least three independent determinations done in triplicate.
- TABLE 5.
Expression of the Pu promoter fused to lacZ in the wild type and glucose mutants growing under different conditionsa
Strain β-Galactosidase activity (Miller units) with: Glucose Toluene Glucose + toluene KT2440(pWW0) 310 ± 20 6,620 ± 150 2,060 ± 40 KT2440 gcd(pWW0) 185 ± 15 6,035 ± 125 4,710 ± 700 KT2440 glk(pWW0) 400 ± 30 6,775 ± 100 3,870 ± 140 KT2440 ptsN(pWW0) 450 ± 20 8,100 ± 200 8,550 ± 190 KT2440 crc(pWW0) 380 ± 40 7,700 ± 500 5,680 ± 300 ↵ a The strains used were transformed with pS10, which carries a Pu:′lacZ fusion and the xylR gene. Assays were done in duplicate, and the data are the averages ± standard deviations of at least three independent assays.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Tables S1 and S2, List of downregulated P. putida genes and change in expression of genes encoding glucose metabolism proteins, respectively, in cells growing on glucose plus
toluene compared to cells grown on glucose alone
MS Word document, 57K.
- Supplemental file 1
-
Tables S1 and S2, List of downregulated P. putida genes and change in expression of genes encoding glucose metabolism proteins, respectively, in cells growing on glucose plus
toluene compared to cells grown on glucose alone
