Assessment of nitrification process in a sequencing batch reactor: Modelling and genomic approach.

Nitrification of ammoniacal nitrogen (N-NH4+) to nitrate (N-NO3-) was investigated in a lab-scale sequencing batch reactor (SBR) to evaluate its efficiency. During the nitrification process the removal of N-NH4+ reached 96%, resulting in 73% formation of N-NO3-. A lineal correlation (r2 = 0.9978) was obtained between the concentration of volatile suspended solids (VSS) and the maximal N-NO3- concentration at the end of each batch cycle under stationary state. The bacterial taxons in the initial inoculum were identified, revealing a complex diverse community mainly in the two major bacterial phyla Proteobacteria and Actinobacteria. The FAPROTAX algorithm predicted the presence in the inoculum of taxa involved in relevant processes of the nitrogen metabolism, highlighting the bacterial genera Nitrospira and Nitrosomonas that are both involved in the nitrification process. A kinetic model was formulated for predicting and validating the transformation of N-NH4+, N-NO2- and N-NO3- and the removal of organic and inorganic carbon (TOC and IC, respectively). The results showed how the increase in biomass concentration slowed down the transformation to oxidised forms of nitrogen and increased denitrification in the settling and filling stages under free aeration conditions.

Influence of packing material on the biofiltration of butyric acid: A comparative study from a physico-chemical, olfactometric and microbiological perspective.

The influence of bed material on the odor removal performance of a biofilter was studied. A compost-wood biofilter and a wood biofilter were treated with a gaseous stream contaminated with butyric acid and comparatively evaluated at pilot scale using olfactometric, physico-chemical and microbiological approaches. The variables analyzed in both biofilters were correlated with specific families of their microbiota composition. In addition to a higher nutrients content (nitrogen and phosphorus), the compost-wood biofilter registered maximum values in number of aerobic microorganisms (3.6·108 CFU/g) and in aerobic microbiological activity (≈40 mg O2/g VS of cumulative oxygen demand at 20 h). This may explain the higher performance of this biofilter compared to the wood biofilter, withstanding odor loads of up to 1450 ouE/m2·s with odor removal efficiencies close to 100%. The analysis of the microbial community showed that Actinobacteria, particularly the mostly aerobic Microbacteriaceae family, might play an important role in butyric acid degradation and hence reduce odor impact. The multidisciplinary analysis carried out in this work could be a very useful strategy for the optimal design of biofiltration operations.

Biofiltration of butyric acid: Monitoring odor abatement and microbial communities.

The objective of this study is to evaluate comparatively the odor removal efficacy of two biofilters operated under different conditions and to identify taxonomically the microbial communities responsible for butyric acid degradation. Both biofiltration systems, which were filled with non-inoculated wood chips and exposed to gas streams containing butyric acid, were evaluated under different operational conditions (gas airflow and temperature) from the physical-chemical, microbiological and olfactometric points of view. The physical-chemical characterization showed the acidification of the packing material and the accumulation of butyric acid during the biofiltration process (<60 days). The removal efficacy was found to be 98-100% during the first 20 days of operation, even at high odor concentration. Changes in the operational temperature increased the odor load factor from 400 to 1400 ouE/m2·s, which led to the reduction of microbiota in the packing material, and a drastic drop of the odor removal efficacy. However, the progressive increase in gas airflow improved the biodegradation efficacy of butyric acid up to 88% with odor loadings as high as 33,000 ouE/m3, while a linear relationship between odor inlet load and removal capacity was also found. The analysis of the microbial community showed that Proteobacteria was the most abundant phylum along the biofiltration time (58-92%) and regardless of the operational conditions. Finally, principal component analysis applied to the physical-chemical and microbiological data set revealed significant differences between the two biofilters under study.

Signal-regulator interactions. Genetic analysis of the effector binding site of xylS, the benzoate-activated positive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon.

This study reports a genetic analysis of the interactions between a positive regulator of gene expression and its effector molecules. Transcription of the TOL plasmid meta-cleavage pathway operon is specifically stimulated by the XylS protein positive regulator either through activation of this regulator by benzoate effectors or through its hyperproduction. One xylS mutant that exhibits constitutive expression of the operon promoter has been characterized, together with six mutants encoding altered XylS proteins that recognize as effectors benzoate analogues that are non-effectors for the XylS wild-type protein. The changes in two mutant regulators are located at the N-terminal end of the protein, within a putative beta-pleated domain. These mutant proteins exhibit a markedly increased affinity for normal benzoate effectors, with K's values fivefold to 60-fold lower than those of the wild-type XylS protein. They are additionally activated by new effectors having certain substituents at position 2, 3 and 4 of the aromatic ring. Two other mutant proteins recognize new effectors having substituents at position 4 and 5 of the aromatic ring, and contain mutations at their C-terminal end within a putative alpha-helix-rich domain. Three other mutations, one of which leads to constitutive expression from Pm, each result in an amino acid change in the central region of the regulator. These findings suggest but do not prove that the effector binding pocket of the XylS protein may be composed of two or more non-contiguous segments of its primary structure. The XylS protein exhibits homology with the AraC protein of Escherichia coli, a protein that stimulates transcription from ara promoters when it is activated by arabinose or benzoate. Mutations influencing effector activation of the XylS protein characterized in this study are all located in regions exhibiting a high degree of homology with the corresponding aligned sequence of AraC protein.

Hydrogen peroxide activates the SoxRS regulon in vivo.

By multiplex reverse transcription-PCR, we demonstrate that the SoxRS response, which protects cells against superoxide toxicity, is triggered also by hydrogen peroxide. SoxR-dependent inductions of 7. 3-, 7.6-, 4.6-, 2.2-, and 2.6-fold were quantified for soxS, micF, sodA, inaA, and fpr transcripts, respectively. This finding suggests an extensive and tight connectivity between different regulatory pathways in the Escherichia coli response to oxidative stress.

The XylS/AraC family of regulators.

At least twenty-seven proteins belong to the XylS/AraC family of prokaryote transcriptional regulators. All members of this family except CelD and TetD are positive transcriptional factors. Three subgroups were distinguished within the family in accordance with the Needleman and Wunsch algorithm. Multiple alignment of these proteins revealed that they shared a high degree of sequence homology at their C-terminal end, where a characteristic conserved motif, whose consensus sequence is I-DIA--GF-S--YF--F---G-TPS--R (where - means any aminoacid), was found. Within the homologous C-terminal region, but outside the above consensus motif, a putative DNA-binding domain organized as a helix-turn-helix motif was located in all regulators. For regulators recognizing chemical signals, the non-homologous N-terminal region of these regulators is presumed to contain binding sites for activator molecules that confer specificity.

The Escherichia coli MelR transcription activator: production of a stable fragment containing the DNA-binding domain.

A set of nested deletions has been made in the Escherichia coli melR gene, encoding the MelR transcription activator protein. Expression of the resulting melR derivatives led to the production of nine MelR proteins with N-terminal deletions of different lengths. The properties of the shortened proteins have been studied both in vivo and in vitro. None of the truncated proteins activate transcription from the E.coli melAB promoter but three; MelR220, MelR183 and MelR173, inhibit activation of the melAB promoter by chromosomally-encoded full-length MelR. In gel retardation assays, both MelR183 and MelR173 clearly retard DNA fragments carrying the melAB promoter. MelR173 has been overproduced in a T7 expression system and shown to be stable in vivo for up to 2 h. DNAase I footprinting assays of partially purified protein show that it binds to the melAB promoter, protecting the same sites as the full-length protein. This fragment may be suitable for further structure/function studies of this class of transcription activator.

Interactions between the Escherichia coli MelR transcription activator protein and operator sequences at the melAB promoter.

The Escherichia coli MelR protein binds to two sites at the melAB promoter and activates transcription in response to melibiose. The binding of purified MelR to the melAB promoter and to a number of derivatives carrying point mutations has been studied. A melAB promoter fragment that is very weakly activated by the melR gene product has been made by creating multiple symmetrical changes in both MelR-binding sites. A library of random substitutions in MelR was screened to find mutants that were able to activate this mutant promoter. One of these mutants contained a substitution in a proposed helix-turn-helix region that most likely relaxes the sequence-specificity of MelR binding. The other mutants contain substitutions throughout the central part of MelR and appear to alter the conformation of MelR, such that activity is triggered at lower melibiose concentrations and by arabinose.

In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to oxidative stress.

Simultaneous expression of seven genes in Escherichia coli was measured by a reverse transcription-multiplex PCR fluorescence procedure. Genes studied were (i) oxyR (transcriptional regulator); (ii) katG, dps, gorA, and ahpCF (controlled by OxyR); (iii) sodA (controlled by SoxRS); and (iv) trxA (not related to OxyR or SoxRS). Except for trxA, transcription of all genes was activated during the course of growth of wild-type bacteria, though notable variations were observed with respect to both the time and extent of activation. Whereas oxyR, katG, dps, and gorA were activated during exponential growth, ahpCF and sodA were stimulated in stationary phase. Maximal induction ranged from 4.6- to 86.5-fold, for gorA and dps, respectively. Treatment with H2O2 stimulated expression of the genes (katG, dps, ahpCF, and gorA) previously identified as members of the OxyR regulon, except for oxyR itself. Induction by H2O2 was a remarkably rapid and reversible process that took place in an OxyR-dependent and sigmaS-independent manner. NaCl induced expression of the genes controlled by OxyR, including the oxyR locus. This transcriptional up-regulation was preserved in a strain with the DeltaoxyR::kan mutation, but it was abolished (ahpCF) or significantly reduced (oxyR and dps) in a strain with the rpoS::Tn10 mutation, potentially reflecting positive transcriptional regulation of the oxyR regulon by sigmaS. Expression of trxA was not increased either by H2O2 stress or by a shift to high-osmolarity conditions.

Identification of critical amino-terminal regions of XylS. The positive regulator encoded by the TOL plasmid.

The XylS protein is the positive regulator of the promoter controlling the meta-cleavage pathway (Pm) for catabolism of certain alkylbenzoates on the TOL plasmid of Pseudomonas. Transcription from Pm is mediated by XylS either in the presence of benzoate effectors or through XylS hyperproduction. Two regions of the NH2 terminus of XylS (residues 37-45) had been predicted to be involved in effector control of XylS transcriptional activation. Different methods were used to induce mutations in this region, including genetic selections (where Pm controlled a tetracycline resistance gene), bisulfite mutagenesis at a unique restriction site, and extensive oligonucleotide mutagenesis at residues 41 and 45. The mutants fell into four classes based on their phenotypes with respect to effector-mediated activation of a Pm-lacZ fusion: (a) effector profiles similar to wild type, (b) no Pm stimulation with benzoates, (c) altered effector specificity, and (d) higher basal Pm activities, in some cases including changes in effector specificity. In some mutants, higher basal Pm activity was apparently due to mutations that increased XylS stability. Substitutions at Arg-41 resulted in all four mutant phenotypes, indicating that this is a critical residue in XylS for effector stimulation of transcription activity.

XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.

The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to beta-galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229-->Ile) and Val274 (Asp274-->Val) substitutions over the N-terminal His41 (Arg41-->His) substitution, and the intraallelic dominance of Thr45 (Arg45-->Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88-->Leu) and Arg256 (Pro256-->Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Val288 (Asp288-->Val), and the double mutant was susceptible to activation by benzoates.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway.

The TOL plasmid upper pathway operon encodes enzymes involved in the catabolism of aromatic hydrocarbons such as toluene and xylenes. The regulator of the gene pathway, the XylR protein, exhibits a very broad effector specificity, being able to recognize as effectors not only pathway substrates but also a wide variety of mono- and disubstituted methyl-, ethyl-, and chlorotoluenes, benzyl alcohols, and p-chlorobenzaldehyde. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two upper pathway enzymes, exhibit very broad substrate specificities and transform unsubstituted substrates and m- and p-methyl-, m- and p-ethyl-, and m- and p-chloro-substituted benzyl alcohols and benzaldehydes, respectively, at a high rate. In contrast, toluene oxidase only oxidizes toluene, m- and p-xylene, m-ethyltoluene, and 1,2,4-trimethylbenzene [corrected], also at a high rate. A biological test showed that toluene oxidase attacks m- and p-chlorotoluene, albeit at a low rate. No evidence for the transformation of p-ethyltoluene by toluene oxidase has been found. Hence, toluene oxidase acts as the bottleneck step for the catabolism of p-ethyl- and m- and p-chlorotoluene through the TOL upper pathway. A mutant toluene oxidase able to transform p-ethyltoluene was isolated, and a mutant strain capable of fully degrading p-ethyltoluene was constructed with a modified TOL plasmid meta-cleavage pathway able to mineralize p-ethylbenzoate. By transfer of a TOL plasmid into Pseudomonas sp. strain B13, a clone able to slowly degrade m-chlorotoluene was also obtained.

Modulation of the function of the signal receptor domain of XylR, a member of a family of prokaryotic enhancer-like positive regulators.

The XylR protein controls expression from the Pseudomonas putida TOL plasmid upper pathway operon promoter (Pu) in response to aromatic effectors. XylR-dependent stimulation of transcription from a Pu::lacZ fusion shows different induction kinetics with different effectors. With toluene, activation followed a hyperbolic curve with an apparent K of 0.95 mM and a maximum beta-galactosidase activity of 2,550 Miller units. With o-nitrotoluene, in contrast, activation followed a sigmoidal curve with an apparent K of 0.55 mM and a Hill coefficient of 2.65. m-Nitrotoluene kept the XylR regulator in an inactive transcriptional form. Therefore, upon binding of an effector, the substituent on the aromatic ring leads to productive or unproductive XylR forms. The different transcriptional states of the XylR regulator are substantiated by XylR mutants. XylRE172K is a mutant regulator that is able to stimulate transcription from the Pu promoter in the presence of m-nitrotoluene; however, its response to m-aminotoluene was negligible, in contrast with the wild-type regulator. These results illustrate the importance of the electrostatic interactions in effector recognition and in the stabilization of productive and unproductive forms by the regulator upon aromatic binding. XylRD135N and XylRD135Q are mutant regulators that are able to stimulate transcription from Pu in the absence of effectors, whereas substitution of Glu for Asp135 in XylRD135E resulted in a mutant whose ability to recognize effectors was severely impaired. Therefore, the conformation of mutant XylRD135Q as well as XylRD135N seemed to mimic that of the wild-type regulator when effector binding occurred, whereas mutant XylRD135E seemed to be blocked in a conformation similar to that of wild-type XylR and XylRE172K upon binding to an inhibitor molecule such as m-nitrotoluene or m-aminotoluene.

In vivo construction of a hybrid pathway for metabolism of 4-nitrotoluene in Pseudomonas fluorescens.

Pseudomonas fluorescens 410PR grows on 4-nitrobenzoate but does not metabolize 4-nitrotoluene. The TOL pWW0 delta pm plasmid converts 4-nitrotoluene into 4-nitrobenzoate through its upper pathway, but it does not metabolize 4-nitrobenzoate. P. fluorescens 410PR(pWW0 delta pm) transconjugants were isolated and found to be able to grow on 4-nitrotoluene. This phenotype was stable after growth for at least 300 generations without any selective pressure. P. fluorescens 410PR(pWW0 delta pm) converted 4-nitrotoluene into 4-nitrobenzoate via 4-nitrobenzylalcohol and 4-nitrobenzaldehyde. 4-Nitrobenzoate was metabolized via 4-hydroxylaminobenzoate and finally yielded NH4+ and 3,4-dihydroxybenzoate, which was mineralized.

DNA binding and DNA bending by the MelR transcription activator protein from Escherichia coli.

The Escherichia coli melR gene encodes MelR protein which is a member of the AraC/XylS family of bacterial transcription activators. The function of MelR was investigated by making a targeted deletion in the melR gene of the Escherichia coli chromosome. MelR is a transcription activator essential for melibiose- dependent expression of the melAB operon which is needed for bacterial growth with melibiose as a carbon source. To investigate the interactions of MelR at the melAB promoter, both full length MelR and a shortened derivative, MelR173, containing the C-terminal DNA-binding domain, were purified as fusions to glutathione- S -transferase. Circular permutation studies show that both full-length MelR and MelR173 induce an apparent bend upon binding to target sites at the melAB promoter. Bound full-length MelR, but not MelR173, can oligomerise to form larger complexes that are likely to be involved in transcription activation.