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 ⌬rpoT 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 ⌬rpoT 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 ⌬rpoT strain, expression of the toluene efflux pump ttgGHI genes is severalfold lower than in the parental strain.In natural environments, microorganisms are exposed to changing conditions and have therefore developed a series of strategies to cope with these stressors (17, 48). With world industrialization, humans have synthesized a large number of chemicals, many of which reach the biosphere and constitute a new burden for the environment. Among the most toxic chemicals are organic solvents, such as toluene, xylenes, and styrene, which dissolve in the cell membrane, disorganize it, cause the loss of lipids and proteins, and eventually lead to cell death (8,45,56). Microorganisms have developed various mechanisms to resist the lethal effects of these chemicals. One of the most relevant of these mechanisms is the active reduction of their entry into the cells through the action of membrane efflux pumps, which belong to the group of multidrug resistance pumps (45). These efflux pumps extrude a broad range of structurally unrelated synthetic and natural chemicals and thus constitute an effective barrier against toxic chemicals.Pseudomonas putida DOT-T1E exhibits the unusual property of being highly tolerant of organic solvents (39, 47). Solvent tolerance in this strain is ultimately the result of an interplay between three efflux systems known as TtgABC, TtgDEF, and TtgGHI, which have been assigned to the root nodulation cell division family (15,45,46,55). Of these three efflux pumps, the TtgGHI pump seems to be the most critical for the removal of solvents from a quantitative point of view (51). The ttgGHI and the ttgDEF operons ...

A Two-Component Regulatory System Integrates Redox State and Population Density Sensing in Pseudomonas putida

“…While comparing the transcriptional profiling data obtained here with data from previous studies, we noticed that the roxSR operon was among the genes that showed increased expression in an rpoT mutant of P. putida (8). Consistently, a subset of genes with reduced expression in EU58 is upregulated in the rpoT background, as shown in Table 2.…”

“…Consistently, a subset of genes with reduced expression in EU58 is upregulated in the rpoT background, as shown in Table 2. RpoT belongs to the family of extracytoplasmic sigma factors and plays a role in tolerance to toluene, mainly through increased expression of the ttgGHI efflux pump genes (8). The significance of this observation in the physiology and stress responses of P. putida deserves further investigation.…”

A Set of Activators and Repressors Control Peripheral Glucose Pathways in Pseudomonas putida To Yield a Common Central Intermediate

“…The genome-wide DNA chip used in this work (printed by Progenika Biopharma) was described in detail previously (7,44). It consists of an array of 5,539 oligonucleotides (50-mer) spotted in duplicate onto ␥-aminosilane-treated slides and covalently linked with UV light and heat.…”

“…Labeling efficiency was checked with a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies). Hybridization conditions were as described before (7,44). Images were acquired at 10-m resolution, and the background-subtracted median spot intensities were determined with GenePix Pro 5.1 image analysis software (Axon Instruments, Inc.).…”

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“…one of the most relevant of these mechanisms is the active reduction of their entry into the cell through the action of membrane efflux pumps, which belong to the group of multidrug resistance pumps (40). These efflux pumps extrude a broad range of structurally unrelated synthetic and natural chemicals and thus constitute an effective barrier against organic solvents (10). to determine whether efflux pumps of RND (resistance, nodulation, and cell division) family were present in Shewanella putrefaciens iBB ct4 and Pseudomonas aeruginosa iBB ct5 cells it was performed an amplification reaction with oligonucleotides A24f2 and A577r2 (Fig.…”