Silvia Marqués

Transcriptional Tradeoff between Metabolic and Stress-response Programs in Pseudomonas putida KT2440 Cells Exposed to Toluene

When Pseudomonas putida KT2440 cells encounter toluene in the growth medium, they perceive it simultaneously as a potential nutrient to be metabolized, as a membrane-damaging toxic drug to be extruded, and as a macromolecule-disrupting agent from which to protect proteins. Each of these inputs requires a dedicated transcriptional response that involves a large number of genes. We used DNA array technology to decipher the interplay between these responses in P. putida KT2440 subjected to a short challenge (15 min) with toluene. We then compared the results with those in cells exposed to o-xylene (a non-biodegradable toluene counterpart) and 3-methylbenzoate (a specific substrate of the lower TOL pathway of the P. putida pWW0 plasmid). The resulting expression profiles suggest that the bulk of the available transcriptional machinery is reassigned to endure general stress, whereas only a small share of the available machinery is redirected to the degradation of the aromatic compounds. Specifically, both toluene and o-xylene induce the TOL pathways and a dedicated but not always productive metabolic program. Similarly, 3-methylbenzoate induces the expression not only of the lower meta pathway but also of the non-productive and potentially deleterious genes for the metabolism of (nonsubstituted) benzoate. In addition, toluene (and to a lesser extent o-xylene) inhibit motility functions as an unequivocal response to aromatic toxicity. We argue that toluene is sensed by P. putida KT2440 as a stressor rather than as a nutrient and that the inhibition by the aromatic compounds of many functions we tested is the tradeoff for activating stress tolerance genes at a minimal cost in terms of energy.

Transcriptional control of the Pseudomonas putida TOL plasmid catabolic pathways

TOL plasmid pWWO of Pseudomonas putida contains two operons that specify a pathway for the degradation of aromatic hydrocarbons. The upper pathway operon encodes the enzymes for the oxidation of toluene/xylenes to benzoate/toluates, and the meta‐cleavage pathway operon encodes the enzymes for the further oxidation of these compounds to Krebs cycle intermediates. Their expression is controlled by the gene products of two divergently transcribed regulatory genes, xylR and xylS. The XylR protein, which belongs to the NtrC family of regulators, is expressed from two tandem promoters and autoregulates its synthesis. XylR stimulates transcription from the xylS gene promoter (Ps) and the upper pathway operon promoter (Pu) in the presence of pathway substrates. Both promoters are a54 dependent, and Pu also requires the presence of integration host factor (IHF) for activation of transcription. Binding sites for XylR and IHF in the Pu promoter and for XylR in the Ps promoters have been defined. The XylS protein, which belongs to the AraC family of regulators, stimulates transcription from the mefa‐cleavage pathway operon promoter (Pm) in the presence of benzoates. The effector binding pocket and ONA‐binding region of XylS have been defined through the isolation of mutants that exhibit altered effector specificity and modified transcriptional patterns, respectively. Expression of the mefa‐cleavage pathway operon is also induced by xylene‐activated XylR protein via a cascade regulatory system in which this protein, in combination with σ;54, stimulates the expression from the xylS promoter. The increased concentration of XylS in turn leads to high level expression of the

Characterization of the anaerobic microbial community in oil-polluted subtidal sediments: aromatic biodegradation potential after the Prestige oil spill

The influence of massive crude oil contamination on the microbial population of coastal sediments was investigated in the Cíes Islands 18 and 53 months after the tanker Prestige sank off the NW coast of Spain. Communities were studied by means of culturable and non-culturable methods at three horizons in the sediment (2-5 cm, 12-15 cm and 25-30 cm) in an area heavily affected by the spill. Most probable number of aerobic hydrocarbon degraders was highest in the upper zone and decreased dramatically with depth. Aromatic oxidizing nitrate-reducing bacteria counts were slightly higher than aerobes in the oxidized layer, and also decreased considerably with depth. Iron-reducing bacteria were barely detectable. The highest counts were obtained for sulfate-reducing bacteria, which represented the most relevant fraction of aromatic oxidizers, being maximal at 12-15 cm depth. The community response to high pollution levels was characterized by an increase in culturable populations active towards crude oil components despite the strong decay in the total cell counts. Analysis of whole 16S rRNA gene libraries obtained from the two sampling times and different depths (1460 sequences in all) showed a predominance of Gamma- and Deltaproteobacteria, which was confirmed by fluorescent in situ hybridization. Desulfobacteraceae was the most abundant group among Deltaproteobacteria, followed by sequences affiliated with the order Myxococcales. All retrieved sequences of this order affiliated with a marine myxobacterial clade. Interestingly, sequences affiliated to the order Desulfarculales constituted half of the Deltaproteobacteria sequences retrieved from the heaviest contaminated sample. Principal coordinates analysis of 16S rRNA gene libraries suggested fluctuation in the community distribution with time. Changes in the abundance of certain groups such as Bacteroidetes contributed to these observed differences. Although predominance of certain metabolic types in each horizon could be delimited, a considerable overlap in the use of electron acceptors was observed, confirming that each selected zone could be influenced by more than one respiratory metabolism. Altogether, our results evidence the presence in these sediments of a microbial community with potential to respond against hydrocarbon contamination, consistent with the long pollution history of the site.

Integration of Signals through Crc and PtsN in Catabolite Repression of Pseudomonas putida TOL Plasmid pWW0

ABSTRACT Toluene degradation in Pseudomonas putida KT2440 pWW0 plasmid is subjected to catabolite repression. Pu and PS1 promoters of the pWW0 TOL plasmid are down-regulated in vivo during exponential growth in rich medium. In cells growing on minimal medium, yeast extract (YE) addition mimics exponential-phase rich medium repression of these promoters. We have constructed and tested mutants in a series of global regulators described in Pseudomonas. We describe that a mutant in crc (catabolite repression control) partially relieves YE repression. Macroarray experiments show that crc transcription is strongly increased in the presence of YE, inversely correlated with TOL pathway expression. On the other hand, we have found that induced levels of expression from Pu and PS in the presence of YE are partially derepressed in a ptsN mutant of P. putida. PtsN but not Crc seems to directly interfere with XylR activation at target promoters. The effect of the double mutation in ptsN and crc is not the sum of the effects of each independent mutation and suggests that both regulators are elements of a common regulatory pathway. Basal expression levels from these promoters in the absence of inducer are still XylR dependent and are also repressed in the presence of yeast extract. Neither crc nor ptsN could relieve this repression.

The XylS‐dependent Pm promoter is transcribed in vivo by RNA polymerase with σ32 or σ38 depending on the growth phase

The Pm promoter of the TOL plasmid of Pseudomonas putida is expressed at high level along the growth curve. This transcription is dependent on the positive regulator XylS activated by 3‐methylbenzoate. The sigma factor σ38 is required for expression in early stationary phase and thereafter. To test whether σ70 was involved in Pm transcription in exponential phase, we have followed mRNA synthesis in a rpoD thermosensitive strain. No difference in Pm transcription was found between the wild type and the thermosensitive strain at the restrictive temperature of 42°C, indicating that transcription was independent of the sigma factor σ70. However, basal levels of mRNA expression from Pm in this strain in exponential phase were more than twofold higher at 42°C, suggesting involvement of σ32 in Pm transcription. In a rpoH background, no expression of Pm took place in the exponential phase, whereas it increased during stationary phase, and in a rpoH rpoS double mutant no activity from the Pm promoter was detected along the growth curve. We have shown that the increase in the amount of σ32 factor necessary for transcription in exponential phase is provided through induction of the heat shock response by the presence of the effector 3‐methylbenzoate, which is also required for activation of the positive regulator XylS. We conclude that activation of Pm transcription is achieved through a switch between two stress‐responsive factors, σ32 in exponential phase and σ38 in stationary phase. In both cases, transcription is dependent on the activator XylS and presents the same transcription start point.

Response of sulfate-reducing bacteria to an artificial oil-spill in a coastal marine sediment

In situ mesocosm experiments using a calcareous sand flat from a coastal area of the island of Mallorca in the Mediterranean Sea were performed in order to study the response of sulfate-reducing bacteria (SRB) to controlled crude oil contamination, or heavy contamination with naphthalene. Changes in the microbial community caused by the contamination were monitored by a combination of comparative sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, cultivation approaches and metabolic activity rates. Our results showed that crude oil and naphthalene negatively influenced the total microbial community as the natural increase in cell numbers due to the seasonal dynamics was attenuated. However, both contaminants enhanced the sulfate reduction rates, as well as the culturability of SRB. Our results suggested the presence of autochthonous deltaproteobacterial SRBs that were able to degrade crude oil or polycyclic aromatic hydrocarbons such as naphthalene in anaerobic sediment layers.

Effect of oil refinery sludges on the growth and antioxidant system of alfalfa plants

The refining process in the petrochemical industry generates oil refinery sludges, a potentially contaminating waste product, with a high content of hydrocarbons and heavy metals. Faster degradation of hydrocarbons has been reported in vegetated soils than in non-vegetated soils, but the impact of these contaminants on the plants physiology and on their antioxidant system is not well known. In this study, the effect of the addition of petroleum sludge to soil on the physiological parameters, nutrient contents, and oxidative and antioxidant status in alfalfa was investigated. An inhibition of alfalfa growth and an induction of oxidative stress, as indicated by an increase in protein oxidation, were found. Also, the superoxide dismutase isoenzymes, peroxidase, and those enzymes involved in the ascorbate-glutathione cycle showed significant activity increases, parallel to an enhancement of total homoglutathione, allowing plants being tolerant to this situation. This information is necessary to establish successful and sustainable plant-based remediation strategies.

Role of sigma S in transcription from the positively controlled Pm promoter of the TOL plasmid of Pseudomonas putida.

Transcription from the TOL plasmid Pm promoter is dependent on the XylS regulator activated by benzoate effectors. We analysed transcription from Pm in several backgrounds with differing Escherichia coli alpha and sigma subunits of RNA polymerase. In different RpoA backgrounds, transcription from Pm was as high as in the wild‐type background throughout the growth curve. In the σs‐deficient background provided by E. coli RH90, high levels of transcription from Pm (XylS/3‐methylbenzoate dependent) were observed in the early logarithmic growth phase but not in the late logarithmic phase or early stationary phase. This contrasted with the results obtained in the isogenic σs‐proficient background, in which high levels of transcription were observed throughout the growth curve. XylS/3‐methylbenzoate‐dependent transcription from Pm in the late logarithmic growth phase in the RH90 background was restored by cloned rpoS. The transcription initiation point of Pm was the same regardless of the growth phase and the σs background. The requirement of (σs for stimulation of transcription from Pm in the late logarithmic and early stationary phase was overcome by using certain mutant Pm promoters, e.g. Pm5 (C‐47 → G, A‐44 → G), and the mutant regulator XylSG44S. It is suggested that the transcription from Pm involves the use of two sigma factors: σ70 during the early logarithmic phase and σs thereafter.

Activation of the toluene-responsive regulator XylR causes a transcriptional switch between sigma54 and sigma70 promoters at the divergent Pr/Ps region of the TOL plasmid.

The mechanism by which XylR, the toluene‐responsive activator of the σ54‐dependent Pu and Ps promoters of the Pseudomonas TOL plasmid pWW0, downregulates its own σ70 promoter Pr has been examined. An in vitro transcription system was developed in order to reproduce the repression of Pr observed in cells of P. putida (pWW0) both in the presence and in the absence of the XylR inducer, benzyl alcohol. DNA templates bearing the two σ70‐RNA polymerase (RNAP) binding sites of Pr, which overlap the upstream activating sequences (UAS) for XylR in the divergent σ54 promoter Ps, were transcribed in the presence of a constitutively active XylR variant deleted of its N‐terminal domain (XylRΔA). The addition of ATP, known to trigger multimerization of the regulator at the UAS, enhanced the repression of Pr by XylR. Furthermore, we observed activation of the divergent σ54 promoter Ps during Pr downregulation by XylRΔA. These results support the notion that activation of XylR by aromatic inducers in vivo triggers a transcriptional switch between Pr and PsSuch a switch is apparently caused by the ATP‐dependent multimerization and strong DNA binding of the protein required for activation of the σ54 promoter. This device could reset the level of XylR expression during activation of the σ54Pu and Ps promoters of the TOL plasmid.

Role of the ptsN Gene Product in Catabolite Repression of the Pseudomonas putida TOL Toluene Degradation Pathway in Chemostat Cultures

ABSTRACT The Pseudomonas putida KT2440 TOL upper pathway is repressed under nonlimiting conditions in cells growing in chemostat with succinate as a carbon source. We show that the ptsN gene product IIANtr participates in this repression. Crc, involved in yeast extract-dependent repression in batch cultures, did not influence expression when cells were growing in a chemostat with succinate at maximum rate.