Silvia Marqués

The xyl genes of Pseudomonas putida TOL plasmid that specify catabolism of toluene and xylenes are organized in four transcriptional units: the upper-operon xylUWCAMBN for conversion of toluene/xylenes into benzoate/alkylbenzoates; the meta-operon xylXYZLTEGFJQKIH, which encodes the enzymes for further conversion of these compounds into Krebs cycle intermediates; and xylS and xylR, which are involved in transcriptional control. The XylS and XylR proteins are members of the XylS/AraC and NtrC families, respectively, of transcriptional regulators. The xylS gene is constitutively expressed at a low level from the Ps2 promoter. The XylS protein is activated by interaction with alkylbenzoates, and this active form stimulates transcription from Pm by sigma70- or sigmaS-containing RNA polymerase (the meta loop). The xylR gene is also expressed constitutively. The XylR protein, which in the absence of effectors binds in a nonactive form to target DNA sequences, is activated by aromatic hydrocarbons and ATP; it subsequently undergoes multimerization and structural changes that result in stimulation of transcription from Pu of the upper operon. This latter process is assisted by the IHF protein and mediated by sigma54-containing RNA polymerase. Once activated, the XylR protein also stimulates transcription from the Ps1 promoter of xylS without interfering with expression from Ps2. This process is assisted by the HU protein and is mediated by sigma54-containing RNA polymerase. As a consequence of hyperexpression of the xylS gene, the XylS protein is hyperproduced and stimulates transcription from Pm even in the absence of effectors (the cascade loop). The two sigma54-dependent promoters are additionally subject to global (catabolite repression) control.

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. 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. Pseudomonas putida KT2440 is a soil microorganism characterized by its metabolic versatility, which enables the strain to degrade a wide variety of natural and man-made aromatic compounds (1Nelson K.E. Weinel C. Paulsen I.T. Dodson R.J. Hilbert H. Martins dos Santos V.A. Fouts D.E. Gill S.R. Pop M. Holmes M. Brinkac L. Beanan M. DeBoy R.T. Daugherty S. Kolonay J. Madupu R. Nelson W. White O. Peterson J. Khouri H. Hance I. Chris Lee P. Holtzapple E. Scanlan D. Tran K. Moazzez A. Utterback T. Rizzo M. Lee K. Kosack D. Moestl D. Wedler H. Lauber J. Stjepandic D. Hoheisel J. Straetz M. Heim S. Kiewitz C. Eisen J.A. Timmis K.N. Dusterhoft A. Tummler B. Fraser C.M. Environ. Microbiol. 2002; 4: 799-808Crossref PubMed Scopus (1058) Google Scholar, 2Jiménez J.I. Miñambres B. García J.L. Díaz E. Ramos J.L. Pseudomonas. 3. Kluwer Academic/Plenum Publishers, London2004: 425-462Google Scholar). This strain can use toluene and m- and p-xylene via the pWW0 TOL plasmidencoded pathways (3Nakazawa T. Environ. Microbiol. 2002; 4: 782-786Crossref PubMed Scopus (108) Google Scholar, 4Ramos J.L. Marqués S. Timmis K.N. Annu. Rev. Microbiol. 1997; 51: 341-373Crossref PubMed Scopus (271) Google Scholar). When the strain is confronted with these aromatic compounds, cells face an enticing paradox. On the one hand, these chemical species can be mineralized to yield carbon and energy for growth, allowing the strain to colonize niches refractory to other microbes. On the other hand, toluene and xylenes are toxic for the bacteria above a certain threshold, since they partition in the cell membrane and disorganize it by removing lipids and proteins, which eventually leads to cell death (5Sikkema J. de Bont J.A. Poolman B. Microbiol. Rev. 1995; 59: 201-222Crossref PubMed Google Scholar). The mechanism of toluene, m-xylene, and p-xylene metabolism to CO2 and H2Oin P. putida bearing pWW0 involves two sets of reactions and depends the of two to by certain aromatic compounds and to the expression of specific pathways in J.L. Marqués S. Timmis K.N. Annu. Rev. Microbiol. 1997; 51: 341-373Crossref PubMed Scopus (271) Google Scholar). of metabolic genes is not the only of P. putida cells to aromatic as these compounds also a tolerance response which involves a number of that are not J.L. E. P. A. W. A. Annu. Rev. Microbiol. 2002; PubMed Scopus Google Scholar, A. W. A. E. Ramos J.L. Ramos J.L. Pseudomonas. Kluwer Academic/Plenum Publishers, London2004: Google Scholar). a number of in the characterized the J.L. E. P. A. W. A. Annu. Rev. Microbiol. 2002; PubMed Scopus Google Scholar). to the used cell The of as in tolerance J. de Bont J.A. J. PubMed Google Scholar, K. Lee S. Lee K. D. J. PubMed Google Scholar, J.L. E. P. A. J. PubMed Google Scholar, Ramos J.L. J. PubMed Scopus Google Scholar, A. E. A. Ramos J.L. A. J. PubMed Scopus Google Scholar, A. A. W. A. Ramos J.L. J. PubMed Scopus Google and the in that and is also in Pseudomonas putida KT2440 E. A. Ramos J.L. Microbiol. PubMed Scopus Google Scholar, W. A. A. A. Ramos J.L. PubMed Scopus (108) Google Scholar). of the is the mechanism of involves other as membrane via to of the Ramos J.L. J. PubMed Google Scholar, A. J. PubMed Scopus Google the of a number of A. P. P. A. S. Ramos J.L. J. PubMed Scopus Google Scholar). of P. putida cells to and a response that is to a share of the available expression machinery of the cell A. P. P. A. S. Ramos J.L. J. PubMed Scopus Google Scholar, D. I. 4: PubMed Scopus Google Scholar). The at P. putida cells a transcriptional machinery are to inputs from that a to a large metabolic a toxic that a and tolerance and with the machinery to of the the strain is to not of the of technology I. de Rev. Microbiol. PubMed Scopus Google Scholar). In we used a DNA array to in the in expression of P. putida KT2440 in response to to aromatic toluene, a aromatic (a carbon and a a to induce the degradation pathway but which be C. Timmis K.N. Ramos J.L. J. PubMed Google only as a and a specific of the lower TOL pathway a with lower to the of compounds of two is to the of and to the the program. results that to aromatic compounds a and specific metabolic response at the of aromatic pathways is with a general stress response at by the of genes to to membrane stress, and of proteins. The interplay between the the of the of and the of in of these to a of available to the transcriptional machinery from functions and these suggest that the metabolic and interplay is not but in the use of the transcriptional machinery are in expression and putida KT2440 minimal C. Timmis K.N. Ramos J.L. J. PubMed Google with as a carbon of with P. putida KT2440 to a of and at and the of the at the exposed to a of the compounds, a that cell growth cell (15 by at in in and the cell in and at The transcriptional by an from the of P. putida KT2440 and a the between the of the R.J. E. PubMed Scopus Google Scholar). The resulting to P. putida KT2440 with pWW0 by DNA of P. putida DNA used in in L. I. R. J.I. J. E. Díaz E. Ramos J.L. de Environ. Microbiol. PubMed Scopus Google Scholar). of an array of in and with and The the in the P. putida KT2440 (1Nelson K.E. Weinel C. Paulsen I.T. Dodson R.J. Hilbert H. Martins dos Santos V.A. Fouts D.E. Gill S.R. Pop M. Holmes M. Brinkac L. Beanan M. DeBoy R.T. Daugherty S. Kolonay J. Madupu R. Nelson W. White O. Peterson J. Khouri H. Hance I. Chris Lee P. Holtzapple E. Scanlan D. Tran K. Moazzez A. Utterback T. Rizzo M. Lee K. Kosack D. Moestl D. Wedler H. Lauber J. Stjepandic D. Hoheisel J. Straetz M. Heim S. Kiewitz C. Eisen J.A. Timmis K.N. Dusterhoft A. Tummler B. Fraser C.M. Environ. Microbiol. 2002; 4: 799-808Crossref PubMed Scopus (1058) Google with the for the TOL pWW0 A. L. C.M. Environ. Microbiol. 2002; 4: PubMed Scopus Google and a of used genes and The are also with of for the and genes at as as at from cells with the J. T. 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TOL plasmid pWW0 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 metacleavage 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, xyIR and xyIS. The XyIR protein, which belongs to the NtrC family of regulators, is expressed from two tandem promoters and autoregulates its synthesis. XyIR stimulates transcription from the xyIS gene promoter (Ps) and the upper pathway operon promoter (Pu) in the presence of pathway substrates. Both promoters are sigma 54 dependent, and Pu also requires the presence of integration host factor (IHF) for activation of transcription. Binding sites for XyIR and IHF in the Pu promoter and for XyIR in the Ps promoters have been defined. The XyIS protein, which belongs to the AraC family of regulators, stimulates transcription from the meta-cleavage pathway operon promoter (Pm) in the presence of benzoates. The effector binding pocket and DNA-binding region of XyIS have been defined through the isolation of mutants that exhibit altered effector specificity and modified transcriptional patterns, respectively. Expression of the meta-cleavage pathway operon is also induced by xylene-activated XyIR protein via a cascade regulatory system in which this protein, in combination with sigma 54, stimulates the expression from the xyIS promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

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.