Analysis of solvent tolerance in Pseudomonas putida DOT-T1E based on its genome sequence and a collection of mutants.

Review Analysis of solvent tolerance in Pseudomonas putida DOT-T1E based on its genome sequence and a collection of mutants Zulema Udaondo a,1 , Estrella Duque a,1 , Matilde Fernández b , Lázaro Molina c , Jesús de la Torre a , Patricia Bernal a , José-Luis Niqui b , Cecilia Pini a , Amalia Roca b , Miguel A. Matilla b , M. Antonia Molina-Henares a , Hortencia Silva-Jiménez a , Gloria Navarro-Avilés a , Andreas Busch a , Jesús Lacal a , Tino Krell a , Ana Segura a , Juan-Luis Ramos a, ⇑ a Consejo Superior de Investigaciones Científicas, EEZ, Department of Environmental Protection, 18008 Granada, Spain b Bio-Iliberis R&D, Polígono Juncaril, Calle Capileira 7, 18210 Granada, Spain c CIDERTA-Universidad de Huelva, Huelva, Spain article info Article history: Received 4 July 2012 Revised 9 July 2012 Accepted 9 July 2012 Available online 20 July 2012 Edited by Miguel De la Rosa, Felix Wieland and Wilhelm Just Keywords: Gene regulation Pseudomonas Efflux pumps Energy generation abstract Pseudomonas putida strains are prevalent in a variety of pristine and polluted environments. The genome of the solvent-tolerant P. putida strain DOT-T1E which thrives in the presence of high concentrations of monoaromatic hydrocarbons, contains a circular 6.3 Mbp chromosome and a 133 kbp plasmid. Omics information has been used to identify the genes and proteins involved in solvent tolerance in this bacterium. This strain uses a multifactorial response that involves fine-tuning of lipid fluidity, activation of a general stress-response system, enhanced energy generation, and induction of specific efflux pumps that extrude solvents to the medium. Local and global transcriptional regulators participate in a complex network of metabolic functions, acting as the decision makers in the response to solvents. 1. Introduction Strains of the species Pseudomonas putida are ubiquitous, metabolically very versatile and adapted to prosper in diverse habitats. These strains can use a wide range of compounds as carbon, nitrogen, phosphate and sulfur sources [1–4]. They exhibit an unusual wealth of determinants for high affinity nutrient transport systems; and a wide variety of monoand di-oxygenases, which are useful for degradation of natural products and xenobiotics, efflux pumps that combat antimicrobial compounds of different origin, an extensive set of extracytoplasmatic function (ECF) sigma factors; and a wide range of regulators and stress response systems that permit these microbes to rapidly respond to environmental changes and challenges [5]. The metabolic flexibility of Pseudomonas derives not only from an array of genetic determinants, but also from the tight regulation of the expression of the different metabolic pathways that allow metabolism of a wide variety of chemicals [6,7]. In addition, as other free-living microorganisms, strains of the genus Pseudomonas have genomes that continuously acquire new DNA and undergo expansion rather than reduction [3,4,8]. The subject of this study is the DOT-T1E (T1E) P. putida strain that was isolated from a wastewater treatment plant as an efficient degrader of benzene, ethylbenzene, toluene and other aromatic hydrocarbons [9]. These aromatic hydrocarbons are oxidized to their corresponding catechols and upon meta cleavage, the resulting alkyl muconic acid semialdehydes are directed towards the Krebs cycle [10], where generation of NADH takes places and electrons are channeled to a wealth of respiratory chains [11]. In addition, this strain is unusually highly resistant to organic solvents such as octanol, decanol, benzene, toluene and others [9,12], which has led the scientific community to consider this microorganism as a model system for the study of the response of gram negative bacteria to toxic organic chemicals. It should be noted that utilization of solvents as C-sources and tolerance to solvents are independent events [10]. Strain T1E has been also used for the development of biotechnological biotransformation processes within two-phase systems for the production of p-hydroxybenzoate, alkylcatechols and other chemical products [12,13]. T1E has also been used for the production of tyrosine-derived chemicals http://dx.doi.org/10.1016/j.febslet.2012.07.031 ⇑ Corresponding author. Address: EEZ-CSIC, C/Prof. Albareda, 1, E-18008 Granada, Spain. E-mail address: [email protected] (J.-L. Ramos). 1 These two co-authors contribute equally to this study. FEBS Letters 586 (2012) 2932–2938 journal homepage: www.FEBSLetters.org Ó2012 Federation of European Biochemical Societies. Published by Elsevier B.V. Open access under CC BY-NC-ND license. 0014-5793 Ó2012 Federation of European Biochemical Societies. Published by Elsevier B.V. Open access under CC BY-NC-ND license. such as p-hydroxyphenylpyruvate, L -DOPA and phenol (Udaondo et al., unpublished results). In this review we used the genomics data and available transcriptomic, proteomic and metabolomic data to reveal a picture of the assortment of genes involved in solvent tolerance. 2. The sequence of the P. putida T1E genome The genomic sequence of T1E was determined to identify potential solvent tolerance clusters (or islands) responsible for the enhanced solvent-tolerance of this strain over other P. putida strains. To this end the T1E genome was sequenced using the 454 technology. Sanger sequencing was used to confirm frameshifts, potential short deletions and closing gaps. The resulting annotated sequence has been submitted to GenBank (number GDSUB19779). The 6.39 Mbp genome of P. putida strain T1E comprises two circular replicons: a single chromosome of 6,260,702 bp (GC content of 63%) and a 133,451 bp self-transmissible plasmid named pGRT1. Based on sequence coverage it is most likely that pGRT1 is present in cells at a copy number of one plasmid per chromosome. The pGRT1 plasmid encodes one hundred and 26 proteins, which represent less than 2% of the total number of encoded proteins [14], and has a slightly lower G + C content (58%) than the chromosome, but a similar coding density [14]. Global alignment of the T1E genome with those of other strains of this species was carried out using BioEdit software package to identify strain-specific regions [8,15]. Whole genome alignment with BioEdit of both nucleotide and deduced protein sequences of GB1, W619, KT2440, F1 and other strains revealed a high chromosomal gene synteny with some genome rearrangements and considerable inverted alignments on both sides of the chromosomal replication origin as compared to the other P. putida strains. Of the 5756 (open reading frames) identified in the genome of T1E, around 84% are shared with the genomes of the other four P. putida strains. The T1E strain has 82, 47, 45 and 108 CDS that are uniquely shared with the strains KT2440, GB-1, W619 and F1, respectively. It should also be noted that T1E contains 170 unique CDS that share no similarity (E value of < 10 5 ) with CDS present among the sequenced Pseudomonas genomes, which suggests that these genes may have originated from microorganisms outside of the genus Pseudomonas. We have previously generated a genome-wide mutant collection for T1E and have screened the collection for solvent-sensitive strains. This collection comprises more than 30000 mini-Tn5insertions. Sequencing of the insertion sites revealed that they are randomly distributed in the genome, with an estimated average insertion every 200 bp. No mutant exhibiting solvent-sensitivity was associated with insertion in any of the 170 unique CDS. Therefore, gene identity alone could not indicate whether solvent-related genes were enriched in T1E-specific regions. Below we provide an overview of the general features of toluene utilization and solvent-tolerance traits of T1E, while highlighting genomic determinants that are relevant to these traits. 3. The toluene catabolic pathway and its regulation To better understand the genetic context of the toluene degradation gene cluster in strain T1E, we examined the chromosomal region in detail. Homologous sequences near the 5 0 and 3 0 ends of a conspicuous 96 kb region are continuous in strain KT2440 but interrupted in strain T1E. Toluene degradation (tod) takes places through the toluene dioxygenase pathway encoded by the todFC1C2BADE operon [16,17], followed in 5 0 by the genes encoding the corresponding two-component regulatory system, todST [18,19]. In addition to its ability to use toluene, ethylbenzene and benzene as a sole carbon source, T1E is able to grow on p-cymene (p-isopropyltoluene) and its acid derivative, p-cumate similarly to F1 strain [20,21]. In T1E as in F1, the cym/cmt and the tod pathways are located less than 3 kbp apart on a putative genomic island, which exhibits lack of synteny with other P. putida strains. Taken together this data and considering that the genes are surrounded by phage-related genes, suggests that this region may have been acquired via transduction. Molina-Henares and Ramos (unpublished results) isolated mutants of the T1E strain capable of growing on n-propylbenzene, n-butylbenzene and isopropylbenzene as a sole carbon source. These strains were derived from the accumulation of mutations in the genes coding for toluene dioxygenase and catechol 2,3-dioxygenase. Similarly to the F1 strain, the wild-type broad substrate toluene dioxygenase of the T1E strain can oxidize trichloroethylene (TCE), indole and other substituted aromatic compounds [22–24]. The TodS and TodT proteins form a highly specific two-component regulatory system that regulates the expression of the genes involved in the degradation of toluene, benzene, and ethylbenzene through the toluene dioxygenase pathway. TodS is a sensor protein that contains two input domains, each of which are followed in sequence by a histidine kinase domain [19]. TodS has basal autophosphorylation activity, which is enhanced by the presence of effectors. We used isothermal titration calorimetry to study the binding of different effector molecules to TodS, and related these findings to their capacity to induce gene expression in vivo. Toluene was found to bind to TodS with high affinity (K d around 700 nM) and a 1:1 stoichiometry, which in turn increased the level of autophosphorylation of TodS. The analysis of the truncated variants of TodS revealed that toluene binds to the N-terminal input domain (K d around 2 l M) but not to the C-terminal half. ortho-Substitutions of toluene reduced or abolished in vivo responses, as exemplified by o-xylene which is recognized by TodS with high affinity, but does not enhance autophosphorylation. Compounds that bind TodS but do not promote autophosphorylation-regulation were considered antagonists, in contrast with agonists that bind and stimulate phosphorylation. Antagonists and agonists compete for binding to TodS both in vitro and in vivo. We propose intramolecular TodS signal transmission, not molecular recognition of compounds by TodS, to be the phenomenon that determines whether a given compound will lead to activation of expression of the tod genes. Molecular modeling identified residues F46, I74, F79 and I114 as potentially involved in the binding of effector molecules. Alanine substitution mutants of these residues showed reduced affinities (2to 345-fold) for both agonistic and antagonistic compounds [25]. In response to toluene TodS transphosphorylates TodT, which binds to target DNA binding sites. Lacal et al. [19] reported that in addition to TodT phosphorylation, integration host factor (IHF) also plays a relevant role in the process of transcriptional activation, since expression from the tod promoter was found to be eight-fold lower in an IHF-deficient background. IHF binds between the TodT boxes and the -10 hexamer region, and a functional model was proposed [19] in which IHF favors the contact between the TodT activator and the a -subunit of RNA polymerase (while bound to the downstream promoter element), leading to efficient transcription from the tod operon. 4. Solvent tolerance determinants The genome of the T1E strain was also examined for features related to adaptation to the presence of solvents, combined with information gathered through a range of physiological, biochemical and genetic analyses carried out in our laboratory and other laboratories that have worked with the solvent-tolerant S12 and Z. Udaondo et al. / FEBS Letters 586 (2012) 2932–2938 2933 18733468, 2012, 18, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1016/j.febslet.2012.07.031 by Csic Organización Central Om (Oficialia Mayor) (Urici), Wiley Online Library on [09/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License the Idaho strains [26–29]. Solvent tolerance is an energy intensive process, and it has been found that growth yields of Pseudomonas in the presence of sublethal toluene concentrations can be reduced by up to 50% of dry cell weight. Physiological and biochemical analyses, together with the profile of proteins and transcripts in P. putida T1E and P. putida S12 upon exposure to sublethal toluene concentrations was analyzed versus cells growing without toluene. Witje et al. [28], van der Werf et al. [29], Segura et al. [30] and others identified almost 90 proteins that were upregulated as a result of an exposure of the S12 or T1E strains to toluene. These studies showed that T1E and S12 respond to toluene by: altering lipid composition to adapt membrane fluidity to the presence of solvents [31,32]; inducing efflux pumps such as ttgDEF and ttgGHI or srpABC to remove these toxic chemicals from cell membranes [12,33,34]; activating the ROS defence system to remove reactive oxygen species; and by synthesizing a number of chaperones to refold proteins denatured by toluene (Fig. 1)[28,30,35]. Induction of Krebs cycle enzymes and another set of enzymes related to energy production indicates a requirement for enhanced metabolism in order to power efflux pumps that remove solvents from the cell membranes a process that seems to be the most important determinant in solvent tolerance. These solvent tolerance responses are both multifactorial and combinatorial, and the synergy between these elements, which include a pool of efflux pump genes and stress defence systems, contribute to solvent-tolerance. 4.1. Modifications at the membrane lipid level Organic solvents accumulate in bacterial membranes increasing membrane fluidity [32,36] and many microorganisms respond to solvents at the membrane level by counteracting the increase in fluidity. In the short-term Pseudomonas responds by implementing isomerisation of the cis unsaturated fatty acids to trans unsaturated fatty acids, a reaction mediated by the cis–trans isomerase [37–39]. The isomerisation is a quick response that provides the bacteria with denser membranes and a selective advantage. The isomerization occurs via the CTI isomerase, which is encoded by the cti gene. Bernal et al. [32] used fluorescence polarization assays to show that mutants deficient in the cti gene exhibited less rigid membranes than the wildtype strain. The cti gene was also found to be monocistronic and expressed constitutively at low basal levels in the log and stationary phase. The CTI enzyme is catalytically inactive in the absence of solvents, but is rapidly activated in the presence of solvents. A mutant deficient in CTI exhibited retarded growth with respect to the parental strain when exposed to toluene [37]. The cyclopropane fatty acids (CFAs) have long been recognized as an important determinant of acid and alcohol resistance in Escherichia coli [40]. Expression of the cfa synthase gene in Pseudomonas is also dependent on the RpoS sigma factor, and expression of the gene occurs when cells enter the stationary phase [40,41]. We identified a role for CFAs in solvent tolerance due to the fact that a P. putida T1E cfaB mutant was more sensitive to toluene shock than the parental strain [42], although, cfaB expression is not enhanced in response to toluene in P. putida [43]. In Gram-negative bacteria, cell membrane fluidity is also influenced by phospholipid head group composition and the length of linked fatty acids [44]. Changes in phospholipid head groups have also been shown to be involved in solvent tolerance [36,45,46]. However, it is not clear if the changes in relative phospholipid conFig. 1. Schematic representation of the main mechanisms involved in the multifactorial solvent tolerance process in P. putida strains microorganisms. 2934 Z. Udaondo et al. / FEBS Letters 586 (2012) 2932–2938 18733468, 2012, 18, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1016/j.febslet.2012.07.031 by Csic Organización Central Om (Oficialia Mayor) (Urici), Wiley Online Library on [09/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License tent corresponded with an increased packing order in the membrane. Based on data on a cardiolipin-deficient mutant of P. putida T1E it has been speculated that changes in the membrane architecture, as a consequence of low cardiolipin content, provokes a decrease in the efficiency of the efflux pumps which is translated into reduced solvent tolerance [32]. The cardiolipin synthase (cls) genes in the solvent-tolerant P. putida T1E strain is located on a monocistronic operon and is expressed from sigma-70 promoters. Expression from the cls promoter is six-fold higher in the stationary phase than in the log phase, and expression of the cls gene is not influenced by solvents. Mutants with a knockout in the cls gene exhibit increased membrane rigidity. 4.2. Chaperones and oxidative stress response The reduced permeability of cells induced via changes in lipid composition is only partial and solvents that enter the periplasmic space and cytoplasm denature proteins. This results in damage to which the cell reacts by refolding proteins and by an activation of the ROS defence system to reduce ROS-mediated damage. Proteomic and transcriptomic assays identified genes annotated as involved in ‘heat stress response’ that were overexpressed in the presence of solvents such as ethanol, butanol, toluene and xylenes. In fact, it has been well established that the rpoH regulon is upregulated in the presence of several alcohols [47,48]. The presence of organic solvents in the cytoplasm and periplasm alter protein folding; thus it is not surprising that production of diverse chaperones is necessary to cope with the presence of the solvent. A series of assays in strains KT2440, S12 and T1E have shown that in response to toluene a number of chaperones are induced. Our bioinformatics analysis revealed that T1E encodes at least 41 chaperones, within this set of chaperones only GroEL, GroS, GrpE, IbpA are expressed at higher levels in the presence of toluene. It should be noted that KT2440, a solvent-sensitive strain, activates more chaperones than T1E and S12 in response to toluene and this may be related to the fact that T1E and S12 are more efficient than KT2440 in toluene removal (See efflux pumps below). Strain T1E was isolated from a wastewater treatment plant, a habitat likely to fluctuate between aerobic, anaerobic and microaerobic conditions. In experiments designed to test the preference of strain T1E for oxygen tension, the fastest growth of strain T1E in soft agar (0.2%) was observed about 2 mm below the agar surface, suggestive of a preference for a microaerophilic lifestyle. P. putida T1E is active at low dissolved O 2 levels in bioreactors. A microaerophilic origin of P. putida T1E would be consistent with the presence of the TCA cycle and the anaerobic cobalamin biosynthesis pathway. Comparative genomics shows that many of the enzymes involved in the oxidative stress response are common among different strains of P. putida. Several studies have demonstrated that alcohols and aromatic compounds activate the response against oxidative agents and even provoke typical oxidative damage in Pseudomonas and other bacteria. This is most likely due to the interference of solvents with the electron transport systems that leads to an increase in the production of hydrogen peroxide and other reactive oxygen species [35,47,49]. In response to oxidative stress several genes of the OxyR regulon are induced by toluene in P. putida KT2440 [35], and genes regulated by OxyR or NrdR are commonly upregulated in E. coli ethanol-tolerant strains [49]. The T1E genome encodes two superoxide dismutases: SodA, a Mn superoxide dismutase (T1E-5070); SodB, a Fe superoxide dismutase (T1E-1925). P. putida T1E is also predicted to contain five, peroxidases, namely: KatA, a putative catalase; two alkyl hydroperoxide reductases; a chloroperoxidase; two thiol peroxidases; and two putative glutathione peroxidases (T1E-1488 and T1E3636). Additionally we found several glutaredoxin genes and six thioredoxin genes that may play a role in thiol redox control in the T1E strain [50]. The Xen-like enzymes are known to be relevant in the response of P. putida to agents that provoke oxidative stress [51]. In this regard it is worth noting the presence of six Xenobiotic-like reductases. 4.3. Energy Metabolism and operation of efflux pumps As mentioned above cultures of T1E and S12 can exhibit lower yields when growing in the presence of solvents, suggesting that high levels of energy are essential for solvent tolerance [9,52]. Indeed, proteomic analysis revealed that a number of proteins related to energy metabolism were upregulated upon toluene exposure [28]. As described below the main solvent defense mechanism is mediated by efflux pumps that extrude toxic chemicals, which is an energetically intensive process. In strain S12 it was found that proteins associated with the storage of sugars, such as GlgX and GlgP, were downregulated in the presence of toluene, whereas proteins involved in the conversion of glucose, glucokinase (Glk) and glucose-6-phosphate 1-dehydrogenase (Zwf1) were upregulated, indicating increased production of 6-phosphogluconate – the key intermediate of the Entner–Doudoroff pathway [53]. Conversely, in S12 gluconeogenesis appears to be suppressed upon toluene exposure as evidenced by the downregulation of both Fbp, which converts fructose 1,6-biphosphate to fructose 6phosphate; and Pgi2, which converts fructose-6-phosphate into glucose-6-phosphate and vice versa. These observations strongly suggest an increased rate of glucose consumption and decreased metabolic generation and storage of glucose under solvent stress. The increased production of 6-phosphogluconate leads to higher pyruvate levels, which is converted to acetyl-CoA – the primary substrate of the TCA cycle. Moreover, the increased acetate uptake and conversion to acetyl-CoA suggests increased activation of the TCA cycle by an alternative means to glucose conversion. Several proteins of the TCA cycle were also found to be upregulated in the presence of toluene, including Mqo-1, SdhD and SucD, as well as several NADH dehydrogenase and ATP synthase subunits [28,30]. The upregulation of proteins involved in energy production suggests that P. putida S12 and T1E have the ability to respond to high energy demands due to toluene exposure by increasing energy metabolism, which is in agreement with various fluxomics, proteomics and transcriptomics studies [20,30]. Several terminal oxidase genes were found to be upregulated in the presence of solvents (putative cytochrome aa 3 -type oxidase, cytochrome cbb 3 -type oxidase and cytochrome bd-type quinol oxidase), suggesting adaptation by strain T1E to variable aerobic and microaerobic conditions as well as to solvents, a situation that demands energy consumption due to the high activity of efflux pumps [11].InP. putida T1E complex I consists of an NADH ubiquinone oxidoreductase (EC 1.6.5.3) with 14 subunits coded for by the nuo genes (A through N) that are grouped in a gene cluster. The cytochrome bc1 complex, or quinol:cytochrome coxidoreductase (EC: 1.10.2.2); and a NADH dehydrogenase (EC: 1.6.99.3), made of three independent subunits that are FAD-dependent and are unlinked in the genome (nda, ORF 00102 and ORF 00044 and 1.6.99.5 F2) are used to optimize the NADH/NAD + balance under changing environmental conditions. Complex II is a succinate dehydrogenase (EC1.3.99.1) comprising four subunits (SdhA, SdhBCD that are clustered [TIE 00016 through 0019]. Complex IV consists of cytochrome-coxidase/cytochrome aa3 genes. It is likely that under high oxygen tension the aa3-type oxidase is used, and that the cbb3-type oxidase is used under microaerophilic conditions [54]. The quinol oxidase bd is a high-affinity terminal oxidase for growth at low O 2 tension. Strikingly, the cbb3-type oxidase genes have a G + C content of 63%, a value higher than average GC content of the T1E and S12 strains. Z. Udaondo et al. / FEBS Letters 586 (2012) 2932–2938 2935 18733468, 2012, 18, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1016/j.febslet.2012.07.031 by Csic Organización Central Om (Oficialia Mayor) (Urici), Wiley Online Library on [09/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License In the S12 strain, proteomics analyses suggested differential expression of the cytochrome oubiquinol oxidase complex upon exposure to solvents, and showed that CyoB and CyoA exhibit a similar change in expression level in toluene adapted cells, while for the NADH dehydrogenase complex, only four subunits of the seven identified subunits were differentially expressed when cells grew in the presence of toluene. One of the most relevant features observed with a D cyoB mutant of T1E is that it exhibited a marked reduction in global metabolism suggesting that the limitation in energy generation associated with this terminal oxidase has a general effect on cell metabolism in the presence of toluene. For example, fatty acid biosynthesis is greatly limited, which agrees with previous observations that CyoB mutant cells produce membrane invaginations that affect cell membrane structure and consequently result in extreme sensitivity to solvent shocks [55]. Efflux pumps, especially those belonging to the RND family, are considered to be most important mechanism for solvent tolerance in Gram-negative bacteria [45]. RND transporters are proton-driven efflux systems comprising three proteins that form a multicomponent complex extending from the inner membrane to the outer membrane (Fig. 2)[56]. This molecular organization permits bacteria to extrude compounds via two possible pathways: from the periplasm to the external medium or from the cytoplasm to the external medium [57]. Members of the ABC-transporters (ATP Binding Cassette) have also been implicated in solvent tolerance [58]. Most Gram-negative bacteria encode several RND efflux pumps in their genomes (up to 20 in the highly solvent tolerant strain P. putida T1E). Survival analysis of P. putida T1E cultures after toluene shock (addition of a second phase of toluene) revealed that three RND efflux pumps, with different but overlapping substrate specificity, are directly involved in toluene resistance. We have generated a collection of these mutants in T1E and have tested their role in toluene tolerance. The presence of TtgGHI, encoded on the 133-kb pGRT1 plasmid, was found to be absolutely necessary to survive a 0.3% (v/v) toluene shock [14,62]. Mutants for two other pumps, namely TtgABC [59] and TtgDEF [33], were also isolated. The TtgABC pump is the main antibiotic extrusion pump, and is able to extrude ampicillin, chloramphenicol, tetracycline and flavonoids in addition to toluene and other solvents [43,60,61]. The TtgDEF (PP3425–PP3427) efflux pump has been shown to be involved in aromatic hydrocarbon detoxification [33]. In the S12 strain upon toluene exposure most RND transporter systems become downregulated except the efflux pumps SrpABC (highly similar to TtgGHI) and PP1272. The most prominent upregulation was observed for proteins of the SrpABC efflux system (>15-fold), which can be directly linked to the solvent tolerance of P. putida S12. The upregulation of PP1272 in the presence of toluene is interesting because this observation suggests that P. putida S12 also uses additional efflux systems for organic solvent transport. 4.4. Extracytoplasmic sigma factors (ECFs) and regulation of efflux pump expression P. putida encodes 20 ECFs. We have shown that one of these ECFs, known as ECF-Pp12 (PP3006), plays a role in tolerance to 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 PP3005 open reading frame is an inner membrane protein, whereas the PP3007 protein is periplasmic (G. Navarro-Avilés and J.L. Ramos, unpublished). A non-polar D 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 to stress imposed by heat, antibiotics, NaCl, paraquat, sodium dodecyl sulfate, H 2 O 2 and benzoate. In the D rpoT mutant background, expression of approximately 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 Fig. 2. (A) Model of an RND efflux pump based on the structure of its components. This model represents the possible assemblage of the TtgGHI proteins based on the Acr system [34]. (B) Gene organization of the efflux pumps involved in toluene tolerance in P. putida DOT-T1E (left panel) and response of the wild-type (black) and its isogenic mutants to a sudden solvent shock (right panel). Further details can be found in [42]. 2936 Z. Udaondo et al. / FEBS Letters 586 (2012) 2932–2938 18733468, 2012, 18, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1016/j.febslet.2012.07.031 by Csic Organización Central Om (Oficialia Mayor) (Urici), Wiley Online Library on [09/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 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 D rpoT strain expression of the toluene efflux pump ttgGHI genes is several fold lower than in the parental strain. Recently, extensive analyses of the regulation of the ttgABC,ttgDEF and ttgGHI operons in T1E and SrpABC in S12 have been carried out. TtgR, which belongs to the TetR family of regulators, is the specific transcriptional repressor of the ttgABC. The basal expression of this pump is increased in the presence of antibiotics, flavonoids and alcohols but it does not vary in the presence of aromatic compounds [1,63,64]. The TtgR operator is a 36 bp sequence located in the ttgR–ttgA intergenic region and overlaps with the - 10 and -35 regions of the ttgABC promoter, and the -10 region of the ttgR promoter. In the absence of effectors, the TtgR dimer is bound to its operator site repressing its own expression and that of the efflux pump. Effector binding to the protein–DNA complex induces the dissociation of TtgR, and allows transcription. The crystal structure of the TtgR protein in complex with effectors has been determined [65]. TtgR has a hydrophobic binding pocket, which explains TtgR’s ability to bind different ligands. Within this pocket two binding sites were identified: one that binds ligands with high affinity and the second that binds molecules with low affinity. TtgV belongs to the IclR family of transcriptional regulators and is the main regulator in the modulation of the expression of ttgDEF and ttgGHI (reviewed in [66]). The ttgDEF operon is silent in the absence of effectors, while a basal expression level of the ttgGHI operon has been reported [64,67]. Exposure of P. putida T1E to aromatic compounds (i.e. 4-nitrotoluene, benzonitrile, 1-naphthol), increased the transcription rate of ttgDEF and ttgGHI [67]. TtgV is a tetramer in solution and also when bound to its 42-bp target operator in the ttgDEF and ttgGHI intergenic regions. The repressorr binds to and masks the -10 region of each promoter [66,68]. It should be noted that the promoters of ttgG and ttgV partially overlap each other [67]. Each TtgV monomer has two domains, one comprising the HTH DNA binding domain at the amino terminal end, and an effector binding region at the central region and C terminus of the protein [69]. The two domains are bridged by a linker, whose role is to serve as a signal transmission element between the two domains that are physically disconnected [70]. Residues R98 and E102 within the linker, which bridge both domains, are critical for the correct transmission of the signal from the effector binding pocket to the DNA binding domain. Once the transmission is achieved TtgV modifies its structure and is released from the DNA, at this point the RNA polymerase is able to bind the promoter regions and initiate ttgG and ttgV transcription [67,68]. Although a TtgV/effector cocrystal structure has not been obtained, comparison of the apoTtgV structure with the structure of the TtgV–DNA complex showed a major re-arrangement in TtgV [69,70], which is consistent with a model proposed above. Regulation of the SrpABC efflux pump in P. putida S12 (homologous to the TtgGHI in T1E) exhibits great complexity. In this bacterium, two different regulators, SrpS (TtgV in T1E) and SrpR participate in control of the expression of the efflux pump operon [71,72]. In addition, two insertion elements, ISS12 and ISPpu21, can insert into srpS to block expression, thus derepressing expression of the efflux pump [72]. SrpS is a repressor of the efflux pump and SrpR is an antirepressor which binds to SrpS in such a way that it inhibits the SrpS–DNA interaction, thereby facilitating its release if prebound to the promoter region [72]. In short, solvent tolerant strains uses a multifactorial response that involves fine-tuning of lipid fluidity, activation of a general stress-response system, enhanced energy generation, and induction of specific efflux pumps that extrude solvents to the medium. Local and global transcriptional regulators participate in a complex network of metabolic functions, acting as the decision makers in the response to solvents. Acknowledgements Work in our laboratory was supported by Fondo Social Europeo and Fondos FEDER from the European Union through project BIO2010-17227 and Junta de Andalucía Proyecto de Excelencia CVI-3010. References [1] Daniels, C., Godoy, P., Duque, E., Molina-Henares, M.A., de la Torre, L., del Arco, J.M., Herrera, C., Segura, A., Guazzaroni, M.E., Ferrer, M. and Ramos, J.L. (2010) Global regulation of food supply by Pseudomonas putida DOT-T1E. J. Bacteriol. 192, 2169–2181. [2] Nelson, K.E., Weinel, C., Paulsen, I.T., Dodson, R.J., Hilbert, H., Martins dos Santos, V.A., Fouts, D.E., Gill, S.R., et al. (2002) Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ. Microbiol. 4, 799–808. [3] Silby, M., Winstanley, W.C., Godfrey, S.A., Levy, S.B. and Jackson, R.W. (2011) Pseudomonas genomes: diverse and adaptable. FEMS Microbiol. Rev. 35, 652– 680. [4] Wu, X., Monchy, S., Taghavi, S., Zhu, W., Ramos, J.L. and van der Lelie, D. (2011) Comparative genomics and functional analysis of niche-specific adaptation in Pseudomonas putida. FEMS Microbiol. Rev. 35, 299–323. [5] Ramos, J.L., Krell, T., Danield, C., Segura, A. and Duque, E. (2009) Responses of Pseudomonas to small toxic molecules by a mosaic of domains. Curr. Opin. Microbiol. 12, 215–220. [6] Nogales, J., Palsson, B.O. and Thiele, I. (2008) A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory. BMC Syst. Biol. 2, 79. [7] Puchalka, J., Oberhardt, M.A., Godinho, M., Bielecka, A., Regenhardt, D., Timmis, K.N., Papin, J.A. and Martins dos Santos, V.A.P. (2008) Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology. PLoS Comput. Biol. 4, e1000210. [8] Ussery, D.W., Kiil, K., Lagesen, K., Sicheritz-Ponten, T., Bohlin, J. and Wassenaar, T.M. (2009) The genus Burkholderia: analysis of 56 genomic sequences. Genome Dyn. 6, 140–157. [9] Ramos, J.L., Duque, E., Huertas, M.-J. and Haïdour, A. (1995) Isolation and expansión of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons. J. Bacteriol. 177, 3911–3916. [10] Mosqueda, G., Ramos-González, M.I. and Ramos, J.L. (1999) Toluene metabolism by the solvent-tolerant Pseudomonas putida DOT-T1 strain, and its role in solvent impermeabilization. Gene 232, 69–76. [11] Rojo, F. (2010) Carbon catabolite repression in Pseudomonas: Optimizing metabolic versatility and interactions with the environment. FEMS Microbiol. Rev. 34, 658–684. [12] Rojas, A., Duque, E., Mosqueda, G., Golden, G., Hurtado, A., Ramos, J.L. and Segura, A. (2001) Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E. J. Bacteriol. 183, 3967– 3973. [13] Ramos-González, M.I., Godoy, P., Alaminos, M., Ben-Bassat, A. and Ramos, J.L. (2001) Physiological characterization of Pseudomonas putida DOT-T1E tolerance to p-hydroxybenzoate. Appl. Environ. Microbiol. 67, 4338–4341. [14] Molina, L., Duque, E., Gómez, M.J., Krell, T., Lacal, J., García-Puente, A., García, V., Matilla, M.A., Ramos, J.L. and Segura, A. (2011) The pGRT1 plasmid of Pseudomonas putida DOT-T1E encodes functions relevant for survival under harsh conditons in the environment. Environ. Microbiol. 13, 2315–2327. [15] Hallin, P.F., Staerfeldt, H.H., Rotenberg, E., Binnewies, T.T., Benham, C.J. and Ussery, D.W.W. (2009) GeneWiz browser: an ineractive tool for visualizing. Sequenced chromosomes. Stand. Gen. Sci. 1, 204–215. [16] Zylstra, G.J., McCombie, W.R., Gibson, D.T. and Finette, B.A. (1988) Toluene degradation by Pseudomonas putida F1: genetic organization of the tod operon. Appl. Environ. Microbiol. 54, 1498–1503. [17] Gibson, D., Zylstra, G.J. and Chauhan, S. (1990) Pseudomonas: Biotransformations, Pathogenesis, and Evolving Biotechnology, ASM Press, Washington DC. pp. 121–132. [18] Lau, P.C., Wang, Y., Patel, A., et al. (1997) A bacterial basic region leucine zipper histidine kinase regulating toluene degradation. Proc. Natl. Acad. Sci. U S A 94, 1453–1458. [19] Lacal, J., Busch, A., Guazzaroni, M.E., Krell, T. and Ramos, J.L. (2006) The TodS/ TodT two-component regulatory system recognizes a wide range of effectors and works with DNA-bending proteins. Proc. Nat. Acad. Sci. U S A 103, 8191– 8196. Z. Udaondo et al. / FEBS Letters 586 (2012) 2932–2938 2937 18733468, 2012, 18, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1016/j.febslet.2012.07.031 by Csic Organización Central Om (Oficialia Mayor) (Urici), Wiley Online Library on [09/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License [20] Eaton, R.W. (1996) P-Cumate catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA carrying the cmt operon. J. Bacteriol. 178, 1351–1362. [21] Eaton, R.W. (1997) P-cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to pcumate. J. Bacteriol. 179, 3171–3180. [22] Spain, J.C. and Gibson, D.T. (1988) Oxidation of substituted phenols by Pseudomonas putida F1 and Pseudomonas sp. strain JS6. Appl. Environ. Microbiol. 54, 1399–1404. [23] Wackett, L.P. and Gibson, D.T. (1988) Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1. Appl. Environ. Microbiol. 54, 1703–1708. [24] Spain, J.C., Zylstra, G.J., Blake, C.K. and Gibson, D.T. (1989) Monohydroxylation of phenol and 2,5-dichlorophenol by toluene dioxygenase in Pseudomonas putida F1. Appl. Environ. Microbiol. 55, 2648–2652. [25] Busch, A., Lacal, J., Martos, A., Ramos, J.L. and Krell, T. (2007) Bacterial sensor kinase TodS interacts with agonistic and antagonistic signals. Proc. Nat. Acad. Sci. U S A 104, 13774–13779. [26] Segura, A., Rojas, A., Hurtado, A., Huertas, M.J. and Ramos, J.L. (2003) Comparative genomic analysis of solvent extrusion pumps in Pseudomonas strains exhibiting different degrees of solvent tolerance. Extremophiles 7, 371–376. [27] Volkers, R.J.M., De Jong, A.L., Hulst, A.G., Van Baar, B.L.M., De Bont, J.A.M. and Wery, J. (2006) Chemostat-based proteomic analysis of toluene-affected Pseudomonas putida S12. Environ. Microbiol. 8, 1674–1679. [28] Wijte, D., van Baar, B.L.M., Heck, A.J.R. and Altelaar, F.M. (2011) Probing the proteome response to toluene exposure in the solvent tolerant Pseudomonas putida S12. J. Proteome Res. 10, 394–403. [29] Van der Werf, M.J., Overkamp, K.M., Muilwijk, B., Koek, M.M., van der Werffvan der Vat, B.J.C., Jellema, R.H., Coulier, L. and Hankemeier, T. (2008) Comprehensive analysis of the metabolome of Pseudomonas putida S12 grown on different carbon sources. Mol. BioSyst. 4, 315–327. [30] Segura, A., Godoy, P., van Dillewijn, P., Hurtado, A., Arroyo, N., Santacruz, S. and Ramos, J.L. (2005) Proteomic analysis reveals the participation of energyand stress-related proteins in the response of Pseudomonas putida DOT-T1E to toluene. J. Bacteriol. 187, 5937–5945. [31] Ramos, J.L., Duque, E., Rodríguez-Herva, J.J., Godoy, P., Haïdour, A., Reyes, F. and Fernández-Barrero, A. (1997) Mechanisms for solvent tolerance in bacteria. J. Biol. Chem. 272, 3887–3890. [32] Bernal, P., Muñoz-Rojas, J., Hurtado, A., Ramos, J.L. and Segura, A. (2007) A Pseudomonas putida cardiolipin synthesis mutant exhibits increased sensitivity to drugs related to transport functionality. Environ. Microbiol. 9, 1135–1145. [33] Mosqueda, G. and Ramos, J.L. (2000) A set of genes encoding a second toluene efflux system in Pseudomonas putida DOT-T1E is linked to the tod genes for toluene metabolism. J. Bacteriol. 182, 937–943. [34] Kieboom, J., Dennis, J.J., Zylstra, G.J. and de Bont, J.A.M. (1998) Active efflux of organic solvents in Pseudomonas putida S12 is induced by solvents. J. Bacteriol. 180, 6769–6772. [35] Domínguez-Cuevas, P., Marín, P., Ramos, J.L. and Marqués, S. (2005) RNA polymerase holoenzymes can share a single transcription start site for the Pm promoter: Critical nucleotides in the 7 to 8 region are needed to select between RNA polymerase with r 38 or r 32 . J. Biol. Chem. 280, 41315–41323. [36] Sikkema, J., de Bont, J.A.M. and Poolman, B. (1995) Mechanisms of membrane toxicity of hydrocarbons. Microbiol. Mol. Biol. Rev. 59, 201–222. [37] Junker, F. and Ramos, J.L. (1999) Involvement of the cis/trans isomerase CtiT1 in solvent resistance in Pseudomonas putida DOT-T1E. J. Bacteriol. 181, 5693– 5700. [38] Heipieper, H.J. and De Bont, J.A.M. (1994) Adaptation of Pseudomonas putida S12 to ethanol and toluene at the level of fatty acid composition of membranes. Appl. Environ. Microbiol. 60, 4440–4444. [39] Heipieper, H.J. (2003) Enzymes in lipid modification. J. Lipid Sci. Technol. 105, 385. [40] Chang, Y.Y. and Cronan, J.E. (1999) Membrane cyclopropane fatty acid content is a major factor in acid resistance of Escherichia coli. Mol. Microbiol. 33, 249– 259. [41] Pini, C., Godoy, P., Bernal, P., Ramos, J.L. and Segura, A. (2011) Regulation of the cyclopropane synthase cfaB gene in Pseudomonas putida KT2440. FEMS Microbiol. Lett. 321, 107–114. [42] Pini, C.V., Bernal, P., Godoy, P., Ramos, J.L. and Segura, A. (2009) Cyclopropane fatty acids are involved in organic solvent tolerance but not in acid stress resistance in Pseudomonas putida DOT-T1E. Microbiol. Biotechnol. 2, 253– 261. [43] Duque, E., Rodríguez-Herva, J.-J., de la Torre, J., Domínguez-Cuevas, P., MúñozRojas, J. and Ramos, J.L. (2007) The RpoT regulon of Pseudomonas putida DOTT1E and its role in stress endurance against solvents. J. Bacteriol. 189, 207– 219. [44] Pinkart, H.C. and White, D.C. (1997) Phospholipid biosynthesis and solvent tolerance in Pseudomonas putida strains. J. Bacteriol. 179, 4219–4226. [45] Ramos, J.L., Duque, E., Gallegos, M.T., Godoy, P., Ramos-González, M.I., Rojas, A., Terán, W. and Segura, A. (2002) Mechanisms of solvent tolerance in gramnegative bacteria. Ann. Rev. Microbiol. 56, 743–768. [46] Rühl, J., Hein, E.-M., Hayen, H., Schmid, A. and Blank, L.M. (2011) The glycerophospholipid inventory of Pseudomonas putida is conserved between strains and enables growth condition-related alterations. Microbiol. Biotechnol. 5, 45–58. [47] Brynildsen, M.P. and Liao, J.C. (2009) An integrated network approach identifies the isobutanol response network of Escherichia coli. Mol. Syst. Biol. 5, 277. [48] Rutherford, B.J., Dahl, R.H., Price, R.E., Szmidt, H.L., Benke, P.I., Mukhopadhyay, A. and Keasling, J.D. (2010) Functional genomic study of exogenous n-butanol stress in Escherichia coli. Appl. Environ. Microbiol. 76, 1935–1945. [49] Akanuma, G., Ueki, M., Ishizuka, M., Ohnishi, Y. and Horinouchi, S. (2010) Control of aerial mycelium formation by the BldK oligopeptide ABC transporter in Streptomyces griseus. FEMS Microbiol. Lett. 315, 54–62. [50] Arnér, E.S.J. and Holmgren, A. (1989) Physiological functions of thioredoxin and thioredoxin and reductase. Eur. J. Biochem. 267, 6102–6109. [51] van Dillewijn, P., Couselo, J.L., Corredoria, E., Delgado, A., Witthich, R.M., Ballester, A. and Ramos, J.L. (2008) Bioremediation of 2,4,6-trinitrotoluene by bacterial nitroreductase expressing transgenic aspen. Environ. Sci. Tech. 42, 7405–7410. [52] Isken, S. and de Bont, J.A.M. (1996) Active efflux of toluene in a solventresistant bacterium. J. Bacteriol. 178, 6056–6058. [53] Del Castillo, T., Ramos, J.L., Rodríguez-Herva, J.J., Führer, T., Sauer, U. and Duque, E. (2007) Convergent peripheral pathways catalyze initial glucose catabolism in Pseudomonas putida: Genomic and flux analysis. J. Bacteriol. 189, 5142–5152. [54] Pitcher, R.S. and Watmough, N.J. (2004) The bacterial cytochrome cbb 3 oxidases. Biochim. Biophys. Acta 1655, 388–399. [55] Duque, E., García, V., de la Torre, J., Godoy, P., Bernal, P. and Ramos, J.L. (2004) Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida. Environ. Microbiol. 6, 1021–1031. [56] Nikaido, H. and Takatsuka, Y. (2009) Mechanisms of RND multidrug efflux pumps. Biochim. Biophys. Acta 1794, 769–781. [57] Murakami, S., Nakashima, R., Yamashita, E. and Yamaguchi, A. (2002) Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419, 587. [58] Kim, K., Lee, S., Lee, K. and Lim, D. (1998) Isolation and characterization of toluene-sensitive mutants from the toluene-resistant bacterium Pseudomonas putida GM73. J. Bacteriol. 180, 3692–3696. [59] Godoy, P., Molina-Henares, A.J., de la Torre, J., Duque, E. and Ramos, J.L. (2010) Characterization of the RND family of multidrug efflux pumps: in silico to in vivo confirmation of four functionally distinct subgroups. Microbiol. Biotechnol. 3, 691–700. [60] Terán, W., Felipe, A., Segura, A., Rojas, A., Ramos, J.L. and Gallegos, M.T. (2003) Antibiotic-dependent induction of Pseudomonas putida DOT-T1E TtgABC efflux pump is mediated by the drug binding repressor TtgR. Antimicrob. Agents Chemother. 47, 3067–3072. [61] Roca, A., Rodríguez-Herva, J.J., Duque, E. and Ramos, J.L. (2008) Physiological responses of Pseudomonas putida to formaldehyde during detoxification. Microbiol. Biotechnol. 1, 158–169. [62] Rodríguez-Herva, J.J., García, V., Hurtado, A., Segura, A. and Ramos, J.L. (2007) The ttgGHI solvent efflux pump operon of Pseudomonas putida DOT-T1E is located on a large self-transmissible plasmid. Environ. Microbiol. 9, 1550– 1561. [63] Duque, E., Segura, A., Mosqueda, G. and Ramos, J.L. (2001) Global and cognate regulators control the expression of the organic solvent efflux pumps TtgABC and TtgDEF of Pseudomonas putida. Mol. Microbiol. 39, 1100–1106. [64] Rojas, A., Duque, R., Schmid, S., Hurtado, S., Ramos, J.L. and Segura, A. (2004) Biotransformation in double-phase systems: physiological responses of Pseudomonas putida DOT-T1E to a double phase made of aliphatic alcohols and biosynthesis of substituted catechols. Appl. Environ. Microbiol. 70, 3637– 3643. [65] Alguel, Y., Meng, C., Terán, W., Krell, T., Ramos, J.L., Gallegos, M.T. and Zhang, X. (2007) Crystal structure of multidrug binding protein TtgR in complex with antibiotics and plant antimicrobials. J. Mol. Biol. 369, 829–840. [66] Fillet, S., Daniels, C., Pini, C., Krell, T., Duque, E., Bernal, E., Segura, A., Lu, D., Zhang, X. and Ramos, J.L. (2012) Transcriptional control of the main aromatic hydrocarbon efflux pump in Pseudomonas. Environ. Microbiol. Rep. 4, 158– 167. [67] Guazzaroni, M.E., Terán, W., Zhang, X., Gallegos, M.T. and Ramos, J.L. (2004) TtgV bound to a complex operator site represses transcription of the promoter for the multidrug and solvent extrusion TtgGHI pump. J. Bacteriol. 186, 2921– 2927. [68] Guazzaroni, M.E., Gallegos, M.T., Ramos, J.L. and Krell, T. (2007) Different modes of binding of monoand bioaromatic effectors to the transcriptional regulator TtgV. Role in differential derepression from its cognate operator. J. Biol. Chem. 282, 16308–16316. [69] Lu, D., Fillet, S., Meng, C., Alguel, Y., Kloppsteck, P., Bergeron, J., Krell, T., Gallegos, M.T., Ramos, J.L. and Zhang, X. (2010) Crystal structure of TtgV in complex with its DNA operator reveals a general model for cooperative DNA binding of tetrameric gene regulators. Genes Dev. 24, 2556–2575. [70] Fillet, S., Krell, T., Morel, B., Lu, D., Zhang, X. and Ramos, J.L. (2011) Intramolecular signal transmission in a tetrameric repressor of the IclR family. Proc. Nat. Acad. Sci. U S A 108, 15372–15377. [71] Wery, J., Hidayat, B., Kieboom, J. and de Bont, J.A.M. (2000) An insertion sequence prepares Pseudomonas putida S12 for severe solvent stress. J. Biol. Chem. 276, 5700–5706. [72] Sun, X. and Dennis, J.J. (2009) A novel insertion sequence derepresses efflux pump expression and preadapts Pseudomonas putida S12 for extreme solvent stress. J. Bacteriol. 191, 6773–6777. 2938 Z. Udaondo et al. / FEBS Letters 586 (2012) 2932–2938 18733468, 2012, 18, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1016/j.febslet.2012.07.031 by Csic Organización Central Om (Oficialia Mayor) (Urici), Wiley Online Library on [09/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License