Cacao (Theobroma cacao L.) Response to Water Stress: Physiological Characterization and Antioxidant Gene Expression Profiling in Commercial Clones.

The increase in events associated with drought constraints plant growth and crop performance. Cacao (Theobroma cacao L.) is sensitive to water deficit stress (DS), which limits productivity. The aim of this research was to characterise the response of seven (CCN51, FEAR5, ICS1, ICS60, ICS95, EET8, and TSH565) commercially important cacao clones to severe and temporal water deficit stress. Ten-month-old cacao trees were submitted to two treatments: well-watered and water-stressed until the leaf water potential (Ψ leaf) reached values between -3.0 and -3.5 MPa. The effects of hydric stress on water relations, gas exchange, photochemical activity, membrane integrity and oxidative stress-related gene expression were evaluated. All clones showed decreases in Ψ leaf, but TSH565 had a higher capacity to maintain water homeostasis in leaves. An initial response phase consisted of stomatal closure, a general mechanism to limit water loss: as a consequence, the photosynthetic rate dropped by approximately 98% on average. In some clones, the photosynthetic rate reached negative values at the maximum stress level, evidencing photorespiration and was confirmed by increased intracellular CO2. A second and photosynthetically limited phase was characterized by a drop in PSII quantum efficiency, which affected all clones. On average, all clones were able to recover after 4 days of rewatering. Water deficit triggered oxidative stress at the early phase, as evidenced by the upregulation of oxidative stress markers and genes encoding ROS scavenging enzymes. The effects of water deficit stress on energy metabolism were deduced given the upregulation of fermentative enzyme-coding genes. Altogether, our results suggest that the EET8 clone was the highest performing under water deficit while the ICS-60 clone was more susceptible to water stress. Importantly, the activation of the antioxidant system and PSII repair mechanism seem to play key roles in the observed differences in tolerance to water deficit stress among clones.

De novo transcriptome analysis of white teak (Gmelina arborea Roxb) wood reveals critical genes involved in xylem development and secondary metabolism.

BACKGROUND: Gmelina arborea Roxb is a fast-growing tree species of commercial importance for tropical countries due to multiple industrial uses of its wood. Wood is primarily composed of thick secondary cell walls of xylem cells which imparts the strength to the wood. Identification of the genes involved in the secondary cell wall biosynthesis as well as their cognate regulators is crucial to understand how the production of wood occurs and serves as a starting point for developing breeding strategies to produce varieties with improved wood quality, better paper pulping or new potential uses such as biofuel production. In order to gain knowledge on the molecular mechanisms and gene regulation related with wood development in white teak, a de novo sequencing and transcriptome assembly approach was used employing secondary cell wall synthesizing cells from young white teak trees. RESULTS: For generation of transcriptome, RNA-seq reads were assembled into 110,992 transcripts and 49,364 genes were functionally annotated using plant databases; 5071 GO terms and 25,460 SSR markers were identified within xylem transcripts and 10,256 unigenes were assigned to KEGG database in 130 pathways. Among transcription factor families, C2H2, C3H, bLHLH and MYB were the most represented in xylem. Differential gene expression analysis using leaves as a reference was carried out and a total of 20,954 differentially expressed genes were identified including monolignol biosynthetic pathway genes. The differential expression of selected genes (4CL, COMT, CCoAOMT, CCR and NST1) was validated using qPCR. CONCLUSIONS: We report the very first de novo transcriptome of xylem-related genes in this tropical timber species of commercial importance and constitutes a valuable extension of the publicly available transcriptomic resource aimed at fostering both basic and breeding studies.

Transformation of TNT, 2,4-DNT, and PETN by Raoultella planticola M30b and Rhizobium radiobacter M109 and exploration of the associated enzymes.

The nitrated compounds 2,4-dinitrotoluene (2,4-DNT), 2,4,6-trinitrotoluene (TNT), and pentaerythritol tetranitrate (PETN) are toxic xenobiotics widely used in various industries. They often coexist as environmental contaminants. The aims of this study were to evaluate the transformation of 100 mg L-1 of TNT, 2,4-DNT, and PETN by Raoultella planticola M30b and Rhizobium radiobacter M109c and identify enzymes that may participate in the transformation. These strains were selected from 34 TNT transforming bacteria. Cupriavidus metallidurans DNT was used as a reference strain for comparison purposes. Strains DNT, M30b and M109c transformed 2,4-DNT (100%), TNT (100, 94.7 and 63.6%, respectively), and PETN (72.7, 69.3 and 90.7%, respectively). However, the presence of TNT negatively affects 2,4-DNT and PETN transformation (inhibition > 40%) in strains DNT and M109c and fully inhibited (100% inhibition) 2,4-DNT transformation in R. planticola M30b.Genomes of R. planticola M30b and R. radiobacter M109c were sequenced to identify genes related with 2,4-DNT, TNT or PETN transformation. None of the tested strains presented DNT oxygenase, which has been previously reported in the transformation of 2,4-DNT. Thus, unidentified novel enzymes in these strains are involved in 2,4-DNT transformation. Genes encoding enzymes homologous to the previously reported TNT and PETN-transforming enzymes were identified in both genomes. R. planticola M30b have homologous genes of PETN reductase and xenobiotic reductase B, while R. radiobacter M109c have homologous genes to GTN reductase and PnrA nitroreductase. The ability of these strains to transform explosive mixtures has a potentially biotechnological application in the bioremediation of contaminated environments.

Isolation, identification, and pathogenicity of Steinernema carpocapsae and its bacterial symbiont in Cauca-Colombia.

In Colombia, identification of entomopathogenic nematodes (EPN's) native species is of great importance for pest management programs. The aim of this study was to isolate and identify EPNs and their bacterial symbiont in the department of Cauca-Colombia and then evaluate the susceptibility of two Hass avocado (Persea americana) pests to the EPNs isolated. EPNs were isolated from soil samples by the insect baiting technique. Their bacterial symbiont was isolated from hemolymph of infected Galleria mellonella larvae. Both organisms were molecularly identified. Morphological, and biochemical characterization was done for the bacteria. Susceptibility of Epitrix cucumeris and Pandeleteius cinereus adults was evaluated by individually exposing adults to 50 infective juveniles. EPNs were allegedly detected at two sampled sites (natural forest and coffee cultivation) in 5.8% of the samples analyzed. However, only natural forest EPN's could be isolated and multiplied. The isolate was identified as Steinernema carpocapsae BPS and its bacterial symbiont as Xenorhabus nematophila BPS. Adults of both pests were susceptible to S. carpocapsae indicating this EPN potential for its management. The results of this study constitute the first record of S. carpocapsae in Colombia and the susceptibility of P. cinereus to this EPN.

Complexity in efflux pump control: cross-regulation by the paralogues TtgV and TtgT.

Pseudomonas putida DOT-T1E, known for its high tolerance to solvents, possesses three Resistance-Nodulation-Cell Division-type (RND) efflux pumps, namely TtgABC, TtgDEF and TtgGHI, which are involved in the active extrusion of solvents. Expression of the ttgABC and ttgGHI operons was previously shown to be regulated by the adjacently encoded repressors, TtgR and TtgV, respectively. Upstream of the third RND operon, ttgDEF, is located a putative regulator gene, ttgT. In this study, TtgT is shown to bind to the promoter region of the ttgDEF operon, and to be released from DNA in the presence of organic solvents. In vitro studies revealed that TtgV and TtgT bind the same operator sites in both the ttgDEF and the ttgGHI promoters. However, the affinity of TtgV for the ttgDEF operator was higher than that of TtgT, which, together with the fact that the ttgV promoter seems to be almost twice stronger than the ttgT promoter, explains why TtgV takes over in the regulation of the two efflux pump operons. The functional replacement of the cognate, chromosomally encoded TtgT by the plasmid-encoded paralogue TtgV illustrates a new mode of efflux pump regulation of which the physiological relevance is discussed.

Optimization of the palindromic order of the TtgR operator enhances binding cooperativity.

TtgR is the specific transcriptional repressor of the TtgABC efflux pump. TtgR and the TtgB efflux pump proteins possess multidrug-binding capacity, and their concerted action is responsible for the multidrug resistance phenotype of Pseudomonas putida DOT-T1E. TtgR binds to a pseudo-palindromic site that overlaps the ttgR/ttgA promoters. Dimethylsulfate footprint assays reveal a close interaction between TtgR and the central region of this operator. The results of analytical ultracentrifugation demonstrate that TtgR forms stable dimers in solution, and that two dimers bind to the operator. Microcalorimetric analysis of the binding of the two TtgR dimers to the cognate operator showed biphasic behavior, and an interaction model was developed for the cooperative binding of two TtgR dimers to their target operators. The binding of the two TtgR dimers to the operator was characterized by a Hill coefficient of 1.63+/-0.13 (k(D)=18.2(+/-6.3) microM, k(D)(')=0.91(+/-0.49) microM), indicating positive cooperativity. These data are in close agreement with the results of sedimentation equilibrium studies of TtgR-DNA complexes. A series of oligonucleotides were generated in which the imperfect palindrome of the TtgR operator was empirically optimized. Optimization of the palindrome did not significantly alter the binding of the initial TtgR dimer to the operator, but increased the cooperativity of binding and consequently the overall affinity. The minimal fragment for TtgR binding was a 30-mer DNA duplex, and analysis of its sequence revealed two partially overlapping inverted repeats co-existing within the large pseudo-palindrome operator. Based on the architecture of the operator, the thermodynamics of the process, and the TtgR-operator interactions we propose a model for the binding of TtgR to its target sequence.

Crystal structures of multidrug binding protein TtgR in complex with antibiotics and plant antimicrobials.

Antibiotic resistance is a widely spread phenomenon. One major mechanism that underlies antibiotic resistance in bacteria is the active extrusion of toxic compounds through the membrane-bound efflux pumps that are often regulated at the transcriptional level. TtgR represses the transcription of TtgABC, a key efflux pump in Pseudomonas putida, which is highly resistant to antibiotics, solvents and toxic plant secondary products. Previously we showed that TtgR is the only reported repressor that binds to different classes of natural antimicrobial compounds, which are also extruded by the efflux pump. We report here five high-resolution crystal structures of TtgR from the solvent-tolerant strain DOT-T1E, including TtgR in complex with common antibiotics and plant secondary metabolites. We provide structural basis for the unique ligand binding properties of TtgR. We identify two distinct and overlapping ligand binding sites; the first one is broader and consists of mainly hydrophobic residues, whereas the second one is deeper and contains more polar residues including Arg176, a unique residue present in the DOT-T1E strain but not in other Pseudomonas strains. Phloretin, a plant antimicrobial, can bind to both binding sites with distinct binding affinities and stoichiometries. Results on ligand binding properties of native and mutant TtgR proteins using isothermal titration calorimetry confirm the binding affinities and stoichiometries, and suggest a potential positive cooperativity between the two binding sites. The importance of Arg176 in phloretin binding was further confirmed by the reduced ability of phloretin in releasing the mutant TtgR from bound DNA compared to the native protein. The results presented here highlight the importance and versatility of regulatory systems in bacterial antibiotic resistance and open up new avenues for novel antimicrobial development.

Effector-repressor interactions, binding of a single effector molecule to the operator-bound TtgR homodimer mediates derepression.

The RND family transporter TtgABC and its cognate repressor TtgR from Pseudomonas putida DOT-T1E were both shown to possess multidrug recognition properties. Structurally unrelated molecules such as chloramphenicol, butyl paraben, 1,3-dihydroxynaphthalene, and several flavonoids are substrates of TtgABC and activate pump expression by binding to the TtgR-operator complex. Isothermal titration calorimetry was employed to determine the thermodynamic parameters for the binding of these molecules to TtgR. Dissociation constants were in the range from 1 to 150 microm, the binding stoichiometry was one effector molecule per dimer of TtgR, and the process was driven by favorable enthalpy changes. Although TtgR exhibits a large multidrug binding profile, the plant-derived compounds phloretin and quercetin were shown to bind with the highest affinity (K(D) of around 1 microm), in contrast to other effectors (chloramphenicol and aromatic solvents) for which exhibited a more reduced affinity. Structure-function studies of effectors indicate that the presence of aromatic rings as well as hydroxyl groups are determinants for TtgR binding. The binding of TtgR to its operator DNA does not alter the protein effector profile nor the effector binding stoichiometry. Moreover, we demonstrate here for the first time that the binding of a single effector molecule to the DNA-bound TtgR homodimer induces the dissociation of the repressor-operator complex. This provides important insight into the molecular mechanism of effector-mediated derepression.

The TetR family of transcriptional repressors.

We have developed a general profile for the proteins of the TetR family of repressors. The stretch that best defines the profile of this family is made up of 47 amino acid residues that correspond to the helix-turn-helix DNA binding motif and adjacent regions in the three-dimensional structures of TetR, QacR, CprB, and EthR, four family members for which the function and three-dimensional structure are known. We have detected a set of 2,353 nonredundant proteins belonging to this family by screening genome and protein databases with the TetR profile. Proteins of the TetR family have been found in 115 genera of gram-positive, alpha-, beta-, and gamma-proteobacteria, cyanobacteria, and archaea. The set of genes they regulate is known for 85 out of the 2,353 members of the family. These proteins are involved in the transcriptional control of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes, and pathogenicity. The regulatory network in which the family member is involved can be simple, as in TetR (i.e., TetR bound to the target operator represses tetA transcription and is released in the presence of tetracycline), or more complex, involving a series of regulatory cascades in which either the expression of the TetR family member is modulated by another regulator or the TetR family member triggers a cell response to react to environmental insults. Based on what has been learned from the cocrystals of TetR and QacR with their target operators and from their three-dimensional structures in the absence and in the presence of ligands, and based on multialignment analyses of the conserved stretch of 47 amino acids in the 2,353 TetR family members, two groups of residues have been identified. One group includes highly conserved positions involved in the proper orientation of the helix-turn-helix motif and hence seems to play a structural role. The other set of less conserved residues are involved in establishing contacts with the phosphate backbone and target bases in the operator. Information related to the TetR family of regulators has been updated in a database that can be accessed at www.bactregulators.org.

TtgV bound to a complex operator site represses transcription of the promoter for the multidrug and solvent extrusion TtgGHI pump.

The TtgGHI efflux pump of Pseudomonas putida extrudes a variety of antibiotics and solvents. We show that the ttgGHI operon is transcribed in vitro and in vivo from a single promoter and not from two overlapping promoters as previously proposed. The expression of this promoter is controlled by the TtgV repressor, whose operator expands through four helical turns that overlap the -10 region of the promoter. We also show that TtgV is released from its operator on binding of effectors such as aliphatic alcohols. Mutational analysis of the ttgGHI promoter revealed that substitutions at -13, -12, and -8 yielded promoters that were unable to drive transcription whereas certain mutations at -9, -11, and -6 to -3 increased expression in vivo. The cause of the increased expression was either a decrease in the affinity of the TtgV protein for its operator or an increase in the affinity of RNA polymerase for the mutant promoters.

Antibiotic-dependent induction of Pseudomonas putida DOT-T1E TtgABC efflux pump is mediated by the drug binding repressor TtgR.

Pseudomonas putida is well known for its metabolic capabilities, but recently, it has been shown to exhibit resistance to a wide range of antibiotics. In P. putida DOT-T1E, the TtgABC efflux pump, which has a broad substrate specificity, extrudes antibiotics such as ampicillin, carbenicillin, tetracycline, nalidixic acid, and chloramphenicol. We have analyzed the expression of the ttgABC efflux pump operon and its regulatory gene, ttgR, in response to several structurally unrelated antibiotics at the transcriptional level and investigated the role of the TtgR protein in this process. ttgABC and ttgR are expressed in vivo at a moderate basal level, which increases in the presence of hydrophobic antibiotics like chloramphenicol and tetracycline. In vitro experiments show that, in the absence of inducers, TtgR binds to a palindromic operator site which overlaps both ttgABC and ttgR promoters and dissociates from it in the presence of chloramphenicol and tetracycline. These results suggest that the TtgR repressor is able to bind to structurally different antibiotics, which allows induction of TtgABC multidrug efflux pump expression in response to these antimicrobial agents. This is the first case in which the expression of a drug transporter of the resistance-nodulation-division family has been shown to be regulated directly by antibiotics.

In vivo and in vitro evidence that TtgV is the specific regulator of the TtgGHI multidrug and solvent efflux pump of Pseudomonas putida.

The TtgGHI efflux pump of Pseudomonas putida DOT-T1E plays a key role in the innate and induced tolerance of this strain to aromatic hydrocarbons and antibiotics. The ttgGHI operon is expressed constitutively from two overlapping promoters in the absence of solvents and at a higher level in their presence, but not in response to antibiotics. Adjacent to the ttgGHI operon is the divergently transcribed ttgVW operon. In TtgV-deficient backgrounds, although not in a TtgW-deficient background, expression of the ttgGHI and ttgVW operons increased fourfold. This suggests that TtgV represses expression from the ttgG promoters and controls its own. TtgW plays no major role in the regulation of expression of these promoters. Primer extension revealed that the divergent ttgG and ttgV promoters overlap, and mobility shift assays indicated that TtgV binds to this region with high affinity. DNaseI footprint assays revealed that TtgV protected four DNA helical turns that include the -10 and -35 boxes of the ttgV and ttgG promoters.

Mechanisms of solvent tolerance in gram-negative bacteria.

Organic solvents can be toxic to microorganisms, depending on the inherent toxicity of the solvent and the intrinsic tolerance of the bacterial species and strains. The toxicity of a given solvent correlates with the logarithm of its partition coefficient in n-octanol and water (log Pow). Organic solvents with a log Pow between 1.5 and 4.0 are extremely toxic for microorganisms and other living cells because they partition preferentially in the cytoplasmic membrane, disorganizing its structure and impairing vital functions. Several possible mechanisms leading to solvent-tolerance in gram-negative bacteria have been proposed: (a) adaptive alterations of the membrane fatty acids and phospholipid headgroup composition, (b) formation of vesicles loaded with toxic compounds, and (c) energy-dependent active efflux pumps belonging to the resistance-nodulation-cell division (RND) family, which export toxic organic solvents to the external medium. In these mechanisms, changes in the phospholipid profile and extrusion of the solvents seem to be shared by different strains. The most significant changes in phospholipids are an increase in the melting temperature of the membranes by rapid cis-to-trans isomerization of unsaturated fatty acids and modifications in the phospholipid headgroups. Toluene efflux pumps are involved in solvent tolerance in several gram-negative strains, e.g., Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa. The AcrAB-TolC and AcrEF-TolC efflux pumps are important for n-hexane tolerance in E. coli. A number of P. putida strains have been isolated that tolerate toxic hydrocarbons such as toluene, styrene, and p-xylene. At least three efflux pumps (TtgABC, TtgDEF, and TtgGHI) are present in the most extensively characterized solvent-tolerant strain, P. putida DOT-T1E, and the number of efflux pumps has been found to correlate with the degree of solvent tolerance in different P. putida strains. The operation of these efflux pumps seems to be coupled to the proton motive force via the TonB system, although the intimate mechanism of energy transfer remains elusive. Specific and global regulators control the expression of the efflux pump operons of E. coli and P. putida at the transcriptional level.