Inferring the genetic network of m‐xylene metabolism through expression profiling of the xyl genes of Pseudomonas putida mt‐2

Velázquez, Francisco; Parro, Vı́ctor

doi:10.1111/j.1365-2958.2005.04787.x

Cited by 32 publications

(26 citation statements)

References 52 publications

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“…For comparison, succinate was used as an alternative carbon source since it is generally regarded as a good carbon source for KT2440 and has been used in several previous transcriptome studies (87,88) and proteomic studies (8,38,42,69,94). The operons required for carbazole catabolism, both on pCAR1 and on the chromosome, were strongly induced, and the regulatory gene antR was also upregulated during growth with carbazole.…”

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confidence: 99%

Transcriptome Analysis ofPseudomonas putidaKT2440 Harboring the Completely Sequenced IncP-7 Plasmid pCAR1

et al. 2007

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The IncP-7 plasmid pCAR1 of Pseudomonas resinovorans CA10 confers the ability to degrade carbazole upon transfer to the recipient strain P. putida KT2440. We designed a customized whole-genome oligonucleotide microarray to study the coordinated expression of pCAR1 and the chromosome in the transconjugant strain KT2440(pCAR1). First, the transcriptome of KT2440(pCAR1) during growth with carbazole as the sole carbon source was compared to that during growth with succinate. The carbazole catabolic car and ant operons were induced, along with the chromosomal cat and pca genes involved in the catechol branch of the ␤-ketoadipate pathway. Additionally, the regulatory gene antR encoding the AraC/XylS family transcriptional activator specific for car and ant operons was upregulated. The characterization of the antR promoter revealed that antR is transcribed from an RpoN-dependent promoter, suggesting that the successful expression of the carbazole catabolic operons depends on whether the chromosome contains the specific RpoN-dependent activator. Next, to analyze whether the horizontal transfer of a plasmid alters the transcription network of its host chromosome, we compared the chromosomal transcriptomes of KT2440(pCAR1) and KT2440 under the same growth conditions. Only subtle changes were caused by the transfer of pCAR1, except for the significant induction of the hypothetical gene PP3700, designated parI, which encodes a putative ParA-like ATPase with an N-terminal Xre-type DNA-binding motif. Further transcriptional analyses showed that the parI promoter was positively regulated by ParI itself and the pCAR1-encoded protein ParA.Many catabolic plasmids can be transferred horizontally between different bacteria and play an important role in the distribution of the ability to degrade and utilize recalcitrant chemical compounds (15,90). Several catabolic plasmids within members of the genus Pseudomonas have been identified and have been classified mostly into incompatibility groups IncP-1, IncP-2, IncP-7, and IncP-9. Recently, the complete genome sequences of several IncP-1, IncP-7, and IncP-9 catabolic plasmids were determined (16,28,44,45,47,76,77,83,86,93). The 199,035-bp catabolic plasmid pCAR1 was originally discovered in Pseudomonas resinovorans CA10, which is able to utilize carbazole as its sole source of carbon, nitrogen, and energy (53, 56), and was the first IncP-7 plasmid to be completely sequenced (45). pCAR1 carries the car and ant operons, which encode the upper and meta pathway enzymes and the anthranilate 1,2-dioxygenase, respectively (see Fig. 1A). The operons are transcribed from two identical anthranilate-inducible promoters, P ant , under the control of the AraC/XylS family activator AntR (85), and the constitutive promoter P carAa also originates the transcription of the car operon (50). Carbazole catabolism begins with the upper pathway to yield anthranilate and 2-hydroxypenta-2,4-dienoate, and then 2-hydroxypenta-2,4-dienoate is mineralized into pyruvate and acetyl coenzyme A (acetylCoA) by the met...

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confidence: 99%

Transcriptome Analysis ofPseudomonas putidaKT2440 Harboring the Completely Sequenced IncP-7 Plasmid pCAR1

et al. 2007

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“…The transcription originating at the Pm promoter leads to the formation of nine enzymes involved in the meta pathway. Thus, the benzoate yielded through the action of the enzymes synthesized in the upper operon is cis-dioxygenated in the meta pathway to form 3-methylcatechol, which is cleaved in meta and consequently channelled into the Krebs cycle (Velazquez et al, 2005). Similarly to the assumption made for the upper pathway, the conversion of benzoate to Krebs cycle intermediates is modelled as being exclusively controlled by a single rate-limiting enzyme.…”

Section: Ps Promotermentioning

confidence: 99%

Linking genes to microbial growth kinetics—An integrated biochemical systems engineering approach

Koutinas

Kiparissides

Silva‐Rocha

et al. 2011

Metabolic Engineering

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“…Membranes were blocked for 3 h at room temperature with 5% nonfat dry milk in phosphate-buffered saline. Blots were incubated at 4°C overnight with a 1/1,000 dilution of polyclonal antiserum against XylS (76). Blots were washed with phosphate-buffered saline solution and incubated with goat anti-rabbit immunoglobulin G (HϩL) conjugated with horseradish peroxidase (1:1,000 dilution) for 1 h (Caltag Laboratories).…”

Section: Methodsmentioning

confidence: 99%

Roles of Effectors in XylS-Dependent Transcription Activation: Intramolecular Domain Derepression and DNA Binding

et al. 2008

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XylS, an AraC family protein, activates transcription from the benzoate degradation pathway Pm promoter in the presence of a substrate effector such as 3-methylbenzoate (3MB). We developed a procedure to obtain XylS-enriched preparations which proved suitable to analyze its activation mechanism. XylS showed specific 3MB-independent binding to its target operator, which became strictly 3MB dependent in a dimerizationdefective mutant. We demonstrated that the N-terminal domain of the protein can make linker-independent interactions with the C-terminal domain and inhibit its capacity to bind DNA. Interactions are hampered in the presence of 3MB effector. We propose two independent roles for 3MB in XylS activation: in addition to its known influence favoring protein dimerization, the effector is able to modify XylS conformation to trigger N-terminal domain intramolecular derepression. We also show that activation by XylS involves RNA polymerase recruitment to the Pm promoter as demonstrated by chromatin immunoprecipitation assays. RNA polymerase switching in Pm transcription was reproduced in in vitro transcription assays. All 32 -, 38 -, and 70 -dependent RNA polymerases were able to carry out Pm transcription in a rigorous XylS-dependent manner, as demonstrated by the formation of open complexes only in the presence of the regulator.

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Inferring the genetic network of m‐xylene metabolism through expression profiling of the xyl genes of Pseudomonas putida mt‐2

Cited by 32 publications

References 52 publications

Transcriptome Analysis ofPseudomonas putidaKT2440 Harboring the Completely Sequenced IncP-7 Plasmid pCAR1

Transcriptome Analysis ofPseudomonas putidaKT2440 Harboring the Completely Sequenced IncP-7 Plasmid pCAR1

Linking genes to microbial growth kinetics—An integrated biochemical systems engineering approach

Roles of Effectors in XylS-Dependent Transcription Activation: Intramolecular Domain Derepression and DNA Binding

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