Rationally rewiring the connectivity of the XylR/Pu regulatory node of the m-xylene degradation pathway in Pseudomonas putida

2016 ◽  
Vol 8 (4) ◽  
pp. 571-576
Author(s):  
Aitor de las Heras ◽  
Esteban Martínez-García ◽  
Maria Rosa Domingo-Sananes ◽  
Sofia Fraile ◽  
Víctor de Lorenzo
Keyword(s):  
Pseudomonas Putida ◽  
Degradation Pathway ◽  
Sigma 54 ◽  
Physiological Limit ◽  
Transcriptional Output

Rational rewiring of the components of the sigma-54 dependent promoterPuenables transcriptional output to reach its physiological limit.

scholarly journals Levels and Activity of the Pseudomonas putida Global Regulatory Protein Crc Vary According to Growth Conditions

2005 ◽  
Vol 187 (11) ◽  
pp. 3678-3686 ◽  
Author(s):  
Ana Ruiz-Manzano ◽  
Luis Yuste ◽  
Fernando Rojo
Keyword(s):  
Pseudomonas Putida ◽  
Regulatory Protein ◽  
Carbon Sources ◽  
Signal Transduction Pathway ◽  
Growth Conditions ◽  
Degradation Pathway ◽  
Complete Medium ◽  
Alkane Degradation ◽  
Mutant Proteins ◽  
Catalytic Sites

ABSTRACT The global regulatory protein Crc is involved in the repression of several catabolic pathways for sugars, hydrocarbons, and nitrogenated and aromatic compounds in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolite repression), therefore modulating carbon metabolism. We have analyzed whether the levels or the activity of Crc is regulated. Crc activity was followed by its ability to inhibit the induction by alkanes of the P. putida OCT plasmid alkane degradation pathway when cells grow in a complete medium, where the effect of Crc is very strong. The abundance of crc transcripts and the amounts of Crc protein were higher under repressing conditions than under nonrepressing conditions. The presence of crc on a high-copy-number plasmid considerably increased Crc levels, but this impaired its ability to inhibit the alkane degradation pathway. Crc shows similarity to a family of nucleases that have highly conserved residues at their catalytic sites. Mutation of the corresponding residues in Crc (Asp220 and His246) led to proteins that can inhibit induction of the alkane degradation pathway when present at normal or elevated levels in the cell. Repression by these mutant proteins occurred only under repressing conditions. These results suggest that both the amounts and the activity of Crc are modulated and support previous proposals that Crc may form part of a signal transduction pathway. Furthermore, the activity of the mutant proteins suggests that Crc is not a nuclease.

scholarly journals The Target for the Pseudomonas putida Crc Global Regulator in the Benzoate Degradation Pathway Is the BenR Transcriptional Regulator

2007 ◽  
Vol 190 (5) ◽  
pp. 1539-1545 ◽  
Author(s):  
Renata Moreno ◽  
Fernando Rojo
Keyword(s):  
Pseudomonas Putida ◽  
Target Genes ◽  
Carbon Sources ◽  
Degradation Pathway ◽  
Complete Medium ◽  
Mrna Levels ◽  
Global Regulator ◽  
Benzoate Degradation ◽  
Transcriptional Units ◽  
Degradation Intermediates

ABSTRACT Crc protein is a global regulator involved in catabolite repression control of several pathways for the assimilation of carbon sources in pseudomonads when other preferred substrates are present. In Pseudomonas putida cells growing exponentially in a complete medium containing benzoate, Crc strongly inhibits the expression of the benzoate degradation genes. These genes are organized into several transcriptional units. We show that Crc directly inhibits the expression of the peripheral genes that transform benzoate into catechol (the ben genes) but that its effect on genes corresponding to further steps of the pathway (the cat and pca genes of the central catechol and β-ketoadipate pathways) is indirect, since these genes are not induced because the degradation intermediates, which act as inducers, are not produced. Crc inhibits the translation of target genes by binding to mRNA. The expression of the ben, cat, and pca genes requires the BenR, CatR, and PcaR transcriptional activators, respectively. Crc significantly reduced benABCD mRNA levels but did not affect those of benR. Crc bound to the 5′ end of benR mRNA but not to equivalent regions of catR and pcaR mRNAs. A translational fusion of the benR and lacZ genes was sensitive to Crc, but a transcriptional fusion was not. We propose that Crc acts by reducing the translation of benR mRNA, decreasing BenR levels below those required for the full expression of the benABCD genes. This strategy provides great metabolic flexibility, allowing the hierarchical assimilation of different structurally related compounds that share a common central pathway by selectively regulating the entry of each substrate into the central pathway.

scholarly journals Expression of the Pseudomonas putida OCT Plasmid Alkane Degradation Pathway Is Modulated by Two Different Global Control Signals: Evidence from Continuous Cultures

2003 ◽  
Vol 185 (16) ◽  
pp. 4772-4778 ◽  
Author(s):  
M. Alejandro Dinamarca ◽  
Isabel Aranda-Olmedo ◽  
Antonio Puyet ◽  
Fernando Rojo
Keyword(s):  
Carbon Source ◽  
Pseudomonas Putida ◽  
Catabolite Repression ◽  
Degradation Pathway ◽  
Independent Effect ◽  
Physiological Status ◽  
Carbon Limitation ◽  
Alkane Degradation ◽  
Global Control ◽  
Ubiquinol Oxidase

ABSTRACT Expression of the genes of the alkane degradation pathway encoded in the Pseudomonas putida OCT plasmid are subject to negative and dominant global control depending on the carbon source used and on the physiological status of the cell. We investigated the signals responsible for this control in chemostat cultures under conditions of nutrient or oxygen limitation. Our results show that this global control is not related to the growth rate and responds to two different signals. One signal is the concentration of the carbon source that generates the repressing effect (true catabolite repression control). The second signal is influenced by the level of expression of the cytochome o ubiquinol oxidase, which in turn depends on factors such as oxygen availability or the carbon source used. Since under carbon limitation conditions the first signal is relieved but the second signal is not, we propose that modulation mediated by the cytochrome o ubiquinol oxidase is not classical catabolite repression control but rather a more general physiological control mechanism. The two signals have an additive, but independent, effect, inhibiting induction of the alkane degradation pathway.

Bio-production of high-purity propionate by engineering l-threonine degradation pathway in Pseudomonas putida

2020 ◽  
Vol 104 (12) ◽  
pp. 5303-5313 ◽  
Author(s):  
Chao Ma ◽  
Qingxuan Mu ◽  
Lei Wang ◽  
Yanan Shi ◽  
Lingfeng Zhu ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
High Purity ◽  
Degradation Pathway

scholarly journals Role of BkdR, a Transcriptional Activator of the SigL-Dependent Isoleucine and Valine Degradation Pathway inBacillus subtilis

1999 ◽  
Vol 181 (7) ◽  
pp. 2059-2066 ◽  
Author(s):  
Michel Debarbouille ◽  
Rozenn Gardan ◽  
Maryvonne Arnaud ◽  
George Rapoport
Keyword(s):  
Catalytic Domain ◽  
Regulatory Gene ◽  
Nitrogen Sources ◽  
Degradation Pathway ◽  
Sigma 54 ◽  
Nutritional Conditions ◽  
New Gene ◽  
Butyrate Kinase ◽  
Branched Chain

ABSTRACT A new gene, bkdR (formerly called yqiR), encoding a regulator with a central (catalytic) domain was found inBacillus subtilis. This gene controls the utilization of isoleucine and valine as sole nitrogen sources. Seven genes, previously called yqiS, yqiT, yqiU,yqiV, bfmBAA, bfmBAB, andbfmBB and now referred to as ptb,bcd, buk, lpd, bkdA1,bkdA2, and bkdB, are located downstream from the bkdR gene in B. subtilis. The products of these genes are similar to phosphate butyryl coenzyme A transferase, leucine dehydrogenase, butyrate kinase, and four components of the branched-chain keto acid dehydrogenase complex: E3 (dihydrolipoamide dehydrogenase), E1α (dehydrogenase), E1β (decarboxylase), and E2 (dihydrolipoamide acyltransferase). Isoleucine and valine utilization was abolished in bcd and bkdR null mutants ofB. subtilis. The seven genes appear to be organized as an operon, bkd, transcribed from a −12, −24 promoter. The expression of the bkd operon was induced by the presence of isoleucine or valine in the growth medium and depended upon the presence of the sigma factor SigL, a member of the sigma 54 family. Transcription of this operon was abolished in strains containing a null mutation in the regulatory gene bkdR. Deletion analysis showed that upstream activating sequences are involved in the expression of the bkd operon and are probably the target ofbkdR. Transcription of the bkd operon is also negatively controlled by CodY, a global regulator of gene expression in response to nutritional conditions.

scholarly journals Engineering of Quasi-Natural Pseudomonas putida Strains for Toluene Metabolism through anortho-Cleavage Degradation Pathway

1998 ◽  
Vol 64 (2) ◽  
pp. 748-751 ◽  
Author(s):  
Sven Panke ◽  
Juan M. Sánchez-Romero ◽  
Víctor de Lorenzo
Keyword(s):  
Carbon Source ◽  
Pseudomonas Putida ◽  
Single Gene ◽  
Regulatory Gene ◽  
Gene Cassette ◽  
Degradation Pathway ◽  
Ring Cleavage ◽  
Step Process ◽  
Natural Strain ◽  
Genetic Technique

ABSTRACT To construct a bacterial catalyst for bioconversion of toluene and several alkyl and chloro- and nitro-substituted derivatives into the corresponding benzoates, the upper TOL operon of plasmid pWW0 ofPseudomonas putida was fully reassembled as a single gene cassette along with its cognate regulatory gene, xylR. The corresponding DNA segment was then targeted to the chromosome of aP. putida strain by using a genetic technique that allows deletion of all recombinant tags inherited from previous cloning steps and leaves the otherwise natural strain bearing exclusively the DNA segment encoding the phenotype of interest. The resulting strains grew on toluene as the only carbon source through a two-step process: conversion of toluene into benzoate, mediated by the upper TOL enzymes, and further metabolism of benzoate through the housekeepingortho-ring cleavage pathway of the catechol intermediate.

Widening functional boundaries of the σ54promoter Pu of Pseudomonas putida by defeating extant physiological constraints

2015 ◽  
Vol 11 (3) ◽  
pp. 734-742 ◽  
Author(s):  
Aitor de las Heras ◽  
Esteban Martínez-García ◽  
Maria Rosa Domingo-Sananes ◽  
Víctor de Lorenzo
Keyword(s):  
Pseudomonas Putida ◽  
Transcriptional Regulator ◽  
Physiological Constraints ◽  
Sigma 54

The functional boundaries of thePupromoter can be expanded by overproduction of both sigma-54 and the transcriptional regulator XylR.

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