The RpoT regulon of Pseudomonas putida DOT-T1E and its role in stress endurance against solvents

Abstract

Pseudomonas putida encodes 20 extracytoplasmic sigma factors (ECFs). In this study, we show that one of these ECFs, known as ECF-Pp12 (PP3006), plays a role in tolerance of toluene and other organic solvents. Based on this finding, we have called the gene that encodes this new ECF rpoT. The rpoT gene forms an operon with the preceding gene and with the gene located downstream. The translated gene product of the open reading frame PP3005 is an inner membrane protein, whereas the PP3007 protein is periplasmic. A nonpolar DeltarpoT mutant was generated by homologous recombination, and survival of the mutant was tested under various stress conditions. The mutant strain was hypersensitive to toluene and other solvents but just as tolerant as the wild type of stress imposed by heat, antibiotics, NaCl, paraquat, sodium dodecyl sulfate, H(2)O(2), and benzoate. In the DeltarpoT mutant background, expression of around 50 transcriptional units was affected: 31 cistrons were upregulated, and 23 cistrons were downregulated. This indicates that about 1% of all P. putida genes are under the direct or indirect influence of RpoT. The rpoT gene controls the expression of a number of membrane proteins, including components of the respiratory chains, porins, transporters, and multidrug efflux pumps. Hypersensitivity of the P. putida RpoT-deficient mutant to organic solvents can be attributed to the fact that in the DeltarpoT strain, expression of the toluene efflux pump ttgGHI genes is severalfold lower than in the parental strain.

FIG. 1.

Physical map of the area around ORF3006 and RT-PCR assays. (A) Physical organization of the DNA region containing ORFs that encode PP3005, RpoT, and PP3007 of P. putida DOT-T1E. (B) Proof of operon structure of rpoT with adjacent genes. Total RNA was isolated from the wild-type strain P. putida DOT-T1E, and oligonucleotides 14 (5′-CGCAGAACAAAGGTGCCAGGC-3′) and 13 (5′-CGTGATCGTAGAGCGCCTGC-3′), as well as 12 (5′-GCAGCCACGCCGTGATAGCC-3′) and 11 (5′-TGCGGGGTAGACAGCGCGG-3′), were used to generate cDNA, which was separated on agarose gels. Lanes 2 and 4 correspond to the control, which contained the same amounts of RNA, primers, and Taq polymerase as the other samples but no reverse transcriptase. The positions of the molecular size markers (in bp) are indicated.

FIG. 2.

Subcellular localization of P. putida DOT-T1E PP3007. (A) Hydrophobicity (Kyte-Doolittle) plot of the PP3007 protein showing the putative signal peptide (window size, 9). The average hydrophobicity of the amino acids included in the window is plotted in the midpoint of the window. (B) Schematic view of the PP3007′-′phoA fusion under the control of the P_lac promoter in plasmid pB3007AP (positive for alkaline phosphatase activity). (C) Immunoblot detection of PP3007′-′PhoA in P. putida DOT-T1E(pP3007AP) cell fractions (lanes 5 to 8). P. putida DOT-T1E(pBBRphoA) was used as a negative control (lanes 1 to 4). Protein samples were subjected to electrophoresis (12.5% [wt/vol] SDS-PAGE), followed by Western blotting with an anti-PhoA antibody. Lanes 1 and 5, whole-cell lysate (W); lanes 2 and 6, periplasmic fraction (P); lanes 3 and 7, cytoplasmic fraction (C); and lanes 4 and 8, membrane fraction (M). The positions of molecular mass markers are indicated (in kilodaltons) on the left.

FIG. 3.

Subcellular localization of P. putida DOT-T1E(PP3005). (A) Hydrophobicity (Kyte-Doolittle) plot of the PP3005 protein showing a potential transmembrane segment (window size, 19; peaks with scores greater than 1.8 indicate putative transmembrane α-helices). (B) Schematic view of two different PP3005′-′phoA fusions under the control of the P_lac promoter in plasmids pB3005AP-S and pB3005AP-L, indicating the expression of alkaline phosphatase activity. The numbers indicate the positions of the first and last amino acid residues of the proposed transmembrane domain. (C) Immunoblot detection of the PP3005′-′PhoA fusions in P. putida DOT-T1E cell fractions. Protein samples were subjected to electrophoresis (12.5% [wt/vol] SDS-PAGE), followed by Western blotting with an anti-PhoA antibody. Lanes 1 through 4, P. putida DOT-T1E(pB3005AP-S); lanes 5 through 8, P. putida DOT-T1E(pP3005AP-L). Lanes 1 and 5, whole-cell lysate (W); lanes 2 and 6, periplasmic fraction (P); lanes 3 and 7, cytoplasmic fraction (C); and lanes 4 and 8, membrane protein fraction (M).

FIG. 4.

Survival of P. putida and its ΔrpoT mutant upon sudden temperature shock. (A) Wild-type DOT-T1E and (B) DOT-T1E-ΔrpoT. Cells were grown in 40 ml LB medium at 30°C, and when indicated, four aliquots were made and incubated at 30°C (⧫), 37°C (•), 42°C (▴), and 50°C (▪). The viable cells were counted at the indicated times. The data are the averages of six determinations, with standard deviations below 5% of the given values.

FIG. 5.

Survival of P. putida DOT-T1E and its ΔrpoT mutant upon solvent shock. The strains used were P. putida DOT-T1E (A), DOT-T1E-ΔrpoT (B), and DOT-T1E-PS28 (C). Cells were grown in 30 ml LB medium (circles) or LB medium with toluene in the gas phase (triangles) until the culture reached a turbidity of about 0.8 at 660 nm. The culture was divided into two halves, and to one we added 0.3% (vol/vol) toluene (closed symbols) while the other was kept as a control (open symbols). The numbers of viable cells were determined at the indicated times. The data are the averages of 6 to 10 determinations, with standard deviations in the range of 3 to 8% of the given values.