Mechanisms of Resistance to Chloramphenicol in Pseudomonas putida KT2440

ABSTRACTPseudomonas putidaKT2440 is a chloramphenicol-resistant bacterium that is able to grow in the presence of this antibiotic at a concentration of up to 25 μg/ml. Transcriptomic analyses revealed that the expression profile of 102 genes changed in response to this concentration of chloramphenicol in the culture medium. The genes that showed altered expression include those involved in general metabolism, cellular stress response, gene regulation, efflux pump transporters, and protein biosynthesis. Analysis of a genome-wide collection of mutants showed that survival of a knockout mutant in the TtgABC resistance-nodulation-division (RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline (PQQ) were compromised in the presence of chloramphenicol. The analysis also revealed that an ABC extrusion system (PP2669/PP2668/PP2667) and the AgmR regulator (PP2665) were needed for full resistance toward chloramphenicol. Transcriptional arrays revealed that AgmR controls the expression of thepqqgenes and the operon encoding the ABC extrusion pump from the promoter upstream of open reading frame (ORF) PP2669.

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Knockout of Extracytoplasmic Function Sigma Factor ECF-10 Affects Stress Resistance and Biofilm Formation in Pseudomonas putida KT2440

Applied and Environmental Microbiology ◽

10.1128/aem.01291-14 ◽

2014 ◽

Vol 80 (16) ◽

pp. 4911-4919 ◽

Cited By ~ 17

Author(s):

Beatrix Tettmann ◽

Andreas Dötsch ◽

Olivier Armant ◽

Christopher D. Fjell ◽

Joerg Overhage

Keyword(s):

Antibiotic Resistance ◽

Pseudomonas Putida ◽

Mutant Strain ◽

Stress Resistance ◽

Sigma Factor ◽

Biofilm Formation ◽

Efflux Pump ◽

Pseudomonas Putida Kt2440 ◽

Content Type ◽

Extracytoplasmic Function Sigma Factor

ABSTRACTPseudomonas putidais a Gram-negative soil bacterium which is well-known for its versatile lifestyle, controlled by a large repertoire of transcriptional regulators. Besides one- and two-component regulatory systems, the genome ofP. putidareveals 19 extracytoplasmic function (ECF) sigma factors involved in the adaptation to changing environmental conditions. In this study, we demonstrate that knockout of extracytoplasmic function sigma factor ECF-10, encoded by open reading frame PP4553, resulted in 2- to 4-fold increased antibiotic resistance to quinolone, β-lactam, sulfonamide, and chloramphenicol antibiotics. In addition, the ECF-10 mutant exhibited enhanced formation of biofilms after 24 h of incubation. Transcriptome analysis using Illumina sequencing technology resulted in the detection of 12 genes differentially expressed (>2-fold) in the ECF-10 knockout mutant strain compared to their levels of expression in wild-type cells. Among the upregulated genes werettgA,ttgB, andttgC, which code for the major multidrug efflux pump TtgABC inP. putidaKT2440. Investigation of an ECF-10 andttgAdouble-knockout strain and attgABC-overexpressing strain demonstrated the involvement of efflux pump TtgABC in the stress resistance and biofilm formation phenotypes of the ECF-10 mutant strain, indicating a new role for this efflux pump beyond simple antibiotic resistance inP. putidaKT2440.

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Light Response of Pseudomonas putida KT2440 Mediated by Class II LitR, a Photosensor Homolog

Journal of Bacteriology ◽

10.1128/jb.00146-20 ◽

2020 ◽

Vol 202 (20) ◽

Author(s):

Satoru Sumi ◽

Naotaka Mutaguchi ◽

Teppei Ebuchi ◽

Hiroaki Tsuchida ◽

Takahiro Yamamoto ◽

...

Keyword(s):

Pseudomonas Putida ◽

Transcriptional Control ◽

Fatty Acid Synthase ◽

Direct Repeat ◽

Class Ii ◽

Wide Distribution ◽

Transcriptional Analysis ◽

Pseudomonas Putida Kt2440 ◽

Content Type ◽

A Genome

ABSTRACT Pseudomonas putida KT2440 retains three homologs (PplR1 to PplR3) of the LitR/CarH family, an adenosyl B12-dependent light-sensitive MerR family transcriptional regulator. Transcriptome analysis revealed the existence of a number of photoinducible genes, including pplR1, phrB (encoding DNA photolyase), ufaM (furan-containing fatty acid synthase), folE (GTP cyclohydrolase I), cryB (cryptochrome-like protein), and multiple genes without annotated/known function. Transcriptional analysis by quantitative reverse transcription-PCR with knockout mutants of pplR1 to pplR3 showed that a triple knockout completely abolished the light-inducible transcription in P. putida, which indicates the occurrence of ternary regulation of PplR proteins. A DNase I footprint assay showed that PplR1 protein specifically binds to the promoter regions of light-inducible genes, suggesting a consensus PplR1-binding direct repeat, 5′-T(G/A)TACAN12TGTA(C/T)A-3′. The disruption of B12 biosynthesis cluster did not affect the light-inducible transcription; however, disruption of ppSB1-LOV (where LOV indicates “light, oxygen, or voltage”) and ppSB2-LOV, encoding blue light photoreceptors adjacently located to pplR3 and pplR2, respectively, led to the complete loss of light-inducible transcription. Overall, the results suggest that the three PplRs and two PpSB-LOVs cooperatively regulate the light-inducible gene expression. The wide distribution of the pplR/ppSB-LOV cognate pair homologs in Pseudomonas spp. and related bacteria suggests that the response and adaptation to light are similarly regulated in the group of nonphototrophic bacteria. IMPORTANCE The LitR/CarH family is a new group of photosensor homologous to MerR-type transcriptional regulators. Proteins of this family are distributed to various nonphototrophic bacteria and grouped into at least five classes (I to V). Pseudomonas putida retaining three class II LitR proteins exhibited a genome-wide response to light. All three paralogs were functional and mediated photodependent activation of promoters directing the transcription of light-induced genes or operons. Two LOV (light, oxygen, or voltage) domain proteins, adjacently encoded by two litR genes, were also essential for the photodependent transcriptional control. Despite the difference in light-sensing mechanisms, the DNA binding consensus of class II LitR [T(G/A)TA(C/T)A] was the same as that of class I. This is the first study showing the actual involvement of class II LitR in light-induced transcription.

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CovRS-Regulated Transcriptome Analysis of a Hypervirulent M23 Strain of Group A Streptococcus pyogenes Provides New Insights into Virulence Determinants

Journal of Bacteriology ◽

10.1128/jb.00511-15 ◽

2015 ◽

Vol 197 (19) ◽

pp. 3191-3205 ◽

Cited By ~ 13

Author(s):

Yun-Juan Bao ◽

Zhong Liang ◽

Jeffrey A. Mayfield ◽

Shaun W. Lee ◽

Victoria A. Ploplis ◽

...

Keyword(s):

Streptococcus Pyogenes ◽

Broad Spectrum ◽

Virulence Genes ◽

Expression Profiles ◽

Altered Expression ◽

Content Type ◽

Metabolic Genes ◽

A Genome ◽

Group A ◽

Regulated Gene Expression

ABSTRACTThe two-componentcontrolofvirulence (Cov) regulator (R)-sensor (S) (CovRS) regulates the virulence ofStreptococcus pyogenes(group AStreptococcus[GAS]). Inactivation of CovS during infection switches the pathogenicity of GAS to a more invasive form by regulating transcription of diverse virulence genes via CovR. However, the manner in which CovRS controls virulence through expression of extended gene families has not been fully determined. In the current study, the CovS-regulated gene expression profiles of a hypervirulentemm23GAS strain (M23ND/CovS negative [M23ND/CovS−]) and a noninvasive isogenic strain (M23ND/CovS+), under different growth conditions, were investigated. RNA sequencing identified altered expression of ∼349 genes (18% of the chromosome). The data demonstrated that M23ND/CovS−achieved hypervirulence by allowing enhanced expression of genes responsible for antiphagocytosis (e.g.,hasABC), by abrogating expression of toxin genes (e.g.,speB), and by compromising gene products with dispensable functions (e.g.,sfb1). Among these genes, several (e.g.,parEandparC) were not previously reported to be regulated by CovRS. Furthermore, the study revealed that CovS also modulated the expression of a broad spectrum of metabolic genes that maximized nutrient utilization and energy metabolism during growth and dissemination, where the bacteria encounter large variations in available nutrients, thus restructuring metabolism of GAS for adaption to diverse growth environments. From constructing a genome-scale metabolic model, we identified 16 nonredundant metabolic gene modules that constitute unique nutrient sources. These genes were proposed to be essential for pathogen growth and are likely associated with GAS virulence. The genome-wide prediction of genes associated with virulence identifies new candidate genes that potentially contribute to GAS virulence.IMPORTANCEThe CovRS system modulates transcription of ∼18% of the genes in theStreptococcus pyogenesgenome. Mutations that inactivate CovR or CovS enhance the virulence of this bacterium. We determined complete transcriptomes of a naturally CovS-inactivated invasive deep tissue isolate of anemm23strain ofS. pyogenes(M23ND) and its complemented avirulent variant (CovS+). We identified diverse virulence genes whose altered expression revealed a genetic switching of a nonvirulent form of M23ND to a highly virulent strain. Furthermore, we also systematically uncovered for the first time the comparative levels of expression of a broad spectrum of metabolic genes, which reflected different metabolic needs of the bacterium as it invaded deeper tissue of the human host.

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Involvement of MexS and MexEF-OprN in Resistance to Toxic Ion Chelators in Pseudomonas putida KT2440

Microorganisms ◽

10.3390/microorganisms8111782 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1782

Author(s):

Tania Henriquez ◽

Tom Baldow ◽

Yat Kei Lo ◽

Dina Weydert ◽

Andreas Brachmann ◽

...

Keyword(s):

Pseudomonas Putida ◽

Efflux Pump ◽

Toxic Metal ◽

Growth Defect ◽

Metal Chelators ◽

Pseudomonas Putida Kt2440 ◽

Pseudomonas Species ◽

Resistance Nodulation Division ◽

Rnd Efflux Pump

Bacteria must be able to cope with harsh environments to survive. In Gram-negative bacteria like Pseudomonas species, resistance-nodulation-division (RND) transporters contribute to this task by pumping toxic compounds out of cells. Previously, we found that the RND system TtgABC of Pseudomonas putida KT2440 confers resistance to toxic metal chelators of the bipyridyl group. Here, we report that the incubation of a ttgB mutant in medium containing 2,2’-bipyridyl generated revertant strains able to grow in the presence of this compound. This trait was related to alterations in the pp_2827 locus (homolog of mexS in Pseudomonas aeruginosa). The deletion and complementation of pp_2827 confirmed the importance of the locus for the revertant phenotype. Furthermore, alteration in the pp_2827 locus stimulated expression of the mexEF-oprN operon encoding an RND efflux pump. Deletion and complementation of mexF confirmed that the latter system can compensate the growth defect of the ttgB mutant in the presence of 2,2’-bipyridyl. To our knowledge, this is the first report on a role of pp_2827 (mexS) in the regulation of mexEF-oprN in P. putida KT2440. The results expand the information about the significance of MexEF-OprN in the stress response of P. putida KT2440 and the mechanisms for coping with bipyridyl toxicity.

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Stability of a Pseudomonas putida KT2440 Bacteriophage-Carried Genomic Island and Its Impact on Rhizosphere Fitness

Applied and Environmental Microbiology ◽

10.1128/aem.00901-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 6963-6974 ◽

Cited By ~ 10

Author(s):

Jose M. Quesada ◽

María Isabel Soriano ◽

Manuel Espinosa-Urgel

Keyword(s):

Pseudomonas Putida ◽

Genomic Island ◽

Mobile Element ◽

Direct Repeat ◽

Promoter Sequence ◽

Repeat Sequence ◽

Genomic Islands ◽

Pseudomonas Putida Kt2440 ◽

Bacterial Populations ◽

Content Type

ABSTRACTThe stability of seven genomic islands ofPseudomonas putidaKT2440 with predicted potential for mobilization was studied in bacterial populations associated with the rhizosphere of corn plants by multiplex PCR. DNA rearrangements were detected for only one of them (GI28), which was lost at high frequency. This genomic island of 39.4 kb, with 53 open reading frames, shows the characteristic organization of genes belonging to tailed phages. We present evidence indicating that it corresponds to the lysogenic state of a functional bacteriophage that we have designated Pspu28. Integrated and rarely excised forms of Pspu28 coexist in KT2440 populations. Pspu28 is self-transmissible, and an excisionase is essential for its removal from the bacterial chromosome. The excised Pspu28 forms a circular element that can integrate into the chromosome at a specific location,attsites containing a 17-bp direct repeat sequence. Excision/insertion of Pspu28 alters the promoter sequence and changes the expression level of PP_1531, which encodes a predicted arsenate reductase. Finally, we show that the presence of Pspu28 in the lysogenic state has a negative effect on bacterial fitness in the rhizosphere under conditions of intraspecific competition, thus explaining why clones having lost this mobile element are recovered from that environment.

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Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

Infection and Immunity ◽

10.1128/iai.01453-12 ◽

2013 ◽

Vol 81 (4) ◽

pp. 1121-1128 ◽

Cited By ~ 9

Author(s):

Cristine G. Campos ◽

Luke Borst ◽

Peggy A. Cotter

Keyword(s):

Burkholderia Pseudomallei ◽

Efflux Pump ◽

Outer Membrane Proteins ◽

Lung Epithelial Cells ◽

Wild Type ◽

Passenger Domain ◽

Content Type ◽

Reading Frame ◽

Type V

ABSTRACTBurkholderia pseudomalleiis a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) inB. pseudomalleistrain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3′ tobcaAis Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations ofbcaAandbcaBwere constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaAand Bp340ΔbcaBmutants to wild-typeB. pseudomalleiin vitrodemonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

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SatR Is a Repressor of Fluoroquinolone Efflux Pump SatAB

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00515-13 ◽

2013 ◽

Vol 57 (7) ◽

pp. 3430-3433 ◽

Cited By ~ 3

Author(s):

Jose Antonio Escudero ◽

Alvaro San Millan ◽

Natalia Montero ◽

Belen Gutierrez ◽

Cristina Martinez Ovejero ◽

...

Keyword(s):

Abc Transporter ◽

High Mortality ◽

Human Population ◽

Efflux Pump ◽

Streptococcus Suis ◽

Open Reading Frame ◽

Fluoroquinolone Resistance ◽

Content Type ◽

Reading Frame

ABSTRACTStreptococcus suisis an emerging zoonotic agent responsible for high-mortality outbreaks among the human population in China. In this species, the ABC transporter SatAB mediates fluoroquinolone resistance when overexpressed. Here, we describe and characterizesatR, an open reading frame (ORF) encoding a MarR superfamily regulator that acts as a repressor ofsatAB. satRis cotranscribed withsatAB, and its interruption entails the overexpression of the pump, leading to a clinically relevant increase in resistance to fluoroquinolones.

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Multiple Roles for Two Efflux Pumps in the Polycyclic Aromatic Hydrocarbon-Degrading Pseudomonas putida Strain B6-2 (DSM 28064)

Applied and Environmental Microbiology ◽

10.1128/aem.01882-17 ◽

2017 ◽

Vol 83 (24) ◽

Cited By ~ 6

Author(s):

Xuemei Yao ◽

Fei Tao ◽

Kunzhi Zhang ◽

Hongzhi Tang ◽

Ping Xu

Keyword(s):

Antibiotic Resistance ◽

Polycyclic Aromatic Hydrocarbon ◽

Aromatic Hydrocarbon ◽

Pseudomonas Putida ◽

Mutant Strain ◽

Efflux Pump ◽

Efflux Pumps ◽

Content Type ◽

Pah Degradation ◽

Polycyclic Aromatic

ABSTRACTMicrobial bioremediation is a promising approach for the removal of polycyclic aromatic hydrocarbon (PAH) contaminants. Many degraders of PAHs possess efflux pump genes in their genomes; however, their specific roles in the degradation of PAHs have not been clearly elucidated. In this study, two efflux pumps, TtgABC and SrpABC, were systematically investigated to determine their functions in a PAH-degradingPseudomonas putidastrain B6-2 (DSM 28064). The disruption of genesttgABCorsrpABCresulted in a defect in organic solvent tolerance. TtgABC was found to contribute to antibiotic resistance; SrpABC only contributed to antibiotic resistance under an artificial overproduced condition. Moreover, a mutant strain withoutsrpABCdid not maintain its activity in long-term biphenyl (BP) degradation, which correlated with the loss of cell viability. The expression of SrpABC was significantly upregulated in the course of BP degradation. BP, 2-hydroxybiphenyl, 3-hydroxybiphenyl, and 2,3-dihydroxybiphenyl (2,3-DHBP) were revealed to be the inducers ofsrpABC. 2,3-DHBP was verified to be a substrate of pump SrpABC; SrpABC can enhance the tolerance to 2,3-DHBP by pumping it out. The mutant strain B6-2ΔsrpSprolonged BP degradation with the increase ofsrpABCexpression. These results suggest that the pump SrpABC of strain B6-2 plays a positive role in BP biodegradation by pumping out metabolized toxic substances such as 2,3-DHBP. This study provides insights into the versatile physiological functions of the widely distributed efflux pumps in the biodegradation of PAHs.IMPORTANCEPolycyclic aromatic hydrocarbons (PAHs) are notorious for their recalcitrance to degradation in the environment. A high frequency of the occurrence of the efflux pump genes was observed in the genomes of effective PAH degraders; however, their specific roles in the degradation of PAHs are still obscure. The significance of our study is in the identification of the function and mechanism of the efflux pump SrpABC ofPseudomonas putidastrain B6-2 (DSM 28064) in the biphenyl degradation process. SrpABC is crucial for releasing the toxicity caused by intermediates that are unavoidably produced in PAH degradation, which enables an understanding of how cells maintain the intracellular balance of materials. The findings from this study provide a new perspective on PAH recalcitrance and shed light on enhancing PAH degradation by genetic engineering.

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Paralogous Regulators ArsR1 and ArsR2 of Pseudomonas putida KT2440 as a Basis for Arsenic Biosensor Development

Applied and Environmental Microbiology ◽

10.1128/aem.00606-16 ◽

2016 ◽

Vol 82 (14) ◽

pp. 4133-4144 ◽

Cited By ~ 15

Author(s):

Matilde Fernández ◽

Bertrand Morel ◽

Juan L. Ramos ◽

Tino Krell

Keyword(s):

Pseudomonas Putida ◽

Inorganic Phosphate ◽

Efflux Pump ◽

Metal Resistance ◽

Valuable Insight ◽

Background Signal ◽

Priority List ◽

Arsenic Resistance ◽

Content Type

ABSTRACTThe remarkable metal resistance of many microorganisms is related to the presence of multiple metal resistance operons.Pseudomonas putidaKT2440 can be considered a model for these microorganisms since its arsenic resistance is due to the action of proteins encoded by the two paralogous arsenic resistance operons ARS1 and ARS2. Both operons contain the genes encoding the transcriptional regulators ArsR1 and ArsR2 that control operon expression. We show here that purified ArsR1 and ArsR2 bind the trivalent salt of arsenic (arsenite) with similar affinities (~30 μM), whereas no binding is observed for the pentavalent salt (arsenate). Furthermore, trivalent salts of bismuth and antimony showed binding to both paralogues. The positions of cysteines, found to bind arsenic in other homologues, indicate that ArsR1 and ArsR2 employ different modes of arsenite recognition. Both paralogues are dimeric and possess significant thermal stability. Both proteins were used to construct whole-cell,lacZ-based biosensors. Whereas responses to bismuth were negligible, significant responses were observed for arsenite, arsenate, and antimony. Biosensors based on theP. putidaarsB1 arsB2arsenic efflux pump double mutant were significantly more sensitive than biosensors based on the wild-type strain. This sensitivity enhancement by pump mutation may be a convenient strategy for the construction of other biosensors. A frequent limitation found for other arsenic biosensors was their elevated background signal and interference by inorganic phosphate. The constructed biosensors show no interference by inorganic phosphate, are characterized by a very low background signal, and were found to be suitable to analyze environmental samples.IMPORTANCEArsenic is at the top of the priority list of hazardous compounds issued by the U.S. Agency for Toxic Substances and Disease. The reason for the stunning arsenic resistance of many microorganisms is the existence of paralogous arsenic resistance operons.Pseudomonas putidaKT2440 is a model organism for such bacteria, and their duplicatedarsoperons and in particular their ArsR transcription regulators have been studied in depth byin vivoapproaches. Here we present an analysis of both purified ArsR paralogues by different biophysical techniques, and data obtained provide valuable insight into their structure and function. Particularly insightful was the comparison of ArsR effector profiles determined byin vitroandin vivoexperimentation. We also report the use of both paralogues to construct robust and highly sensitive arsenic biosensors. Our finding that the deletion of both arsenic efflux pumps significantly increases biosensor sensitivity is of general relevance in the biosensor field.

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The RpoT Regulon of Pseudomonas putida DOT-T1E and Its Role in Stress Endurance against Solvents

Journal of Bacteriology ◽

10.1128/jb.00950-06 ◽

2006 ◽

Vol 189 (1) ◽

pp. 207-219 ◽

Cited By ~ 40

Author(s):

Estrella Duque ◽

José-Juan Rodríguez-Herva ◽

Jesús de la Torre ◽

Patricia Domínguez-Cuevas ◽

Jesús Muñoz-Rojas ◽

...

Keyword(s):

Pseudomonas Putida ◽

Organic Solvents ◽

Efflux Pump ◽

Sodium Dodecyl ◽

Multidrug Efflux ◽

Reading Frame ◽

Multidrug Efflux Pumps ◽

Respiratory Chains ◽

Transcriptional Units ◽

Inner Membrane Protein

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 ΔrpoT 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, H2O2, and benzoate. In the ΔrpoT 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 ΔrpoT strain, expression of the toluene efflux pump ttgGHI genes is severalfold lower than in the parental strain.

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