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 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.

Hypoxia Inducible Factor-1α Is over Expressed in CLL B Cells Because of an Impaired Proteasome Pathway Associated with Defective Interaction with von Hippel-Landau Protein.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2115-2115 ◽  
Author(s):  
Yean K. Lee ◽  
Ann K. Strege ◽  
Nancy D. Bone ◽  
Linda E. Wellik ◽  
D. A. Chan ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Nuclear Extract ◽  
Degradation Pathway ◽  
Mrna Levels ◽  
Apoptosis Resistance ◽  
Vegf Mrna ◽  
Prolyl Hydroxylases

Abstract We have found that CLL B cells spontaneously secrete vascular endothelial growth factor (VEGF) and that a VEGF autocrine pathway can induce apoptosis resistance in these cells. Recently, we also found that hypoxia-inducible factor-1 alpha (HIF-1α) is highly expressed in CLL B cells. Since this protein is a potent transcription factor for the induction of VEGF, we were interested in further definition of HIF-1α regulation and its function in CLL B cells. CLL blood B cells overexpress HIF-1α protein but not mRNA for HIF-1α compared to normal blood and splenic B cells. Immunohistochemistry (IHC) showed that circulating blood CLL B cells and a subset of CLL marrow cells uniformly express HIF. Hypoxic conditions (i.e., 1% O2) did not increase the protein levels of HIF-1α nor mRNA for HIF-1α in CLL B cells, indicating that the high HIF-1α protein level is due to post-translation modification. Blockade of signaling pathways known to increase HIF-1α levels also did not alter the high levels of HIF-1α in CLL B cells. IHC and nuclear extraction assay demonstrated that HIF-1α was predominantly located in the CLL B cell nucleus. In addition, the nuclear extract when immunoprecipitated for HIF-1α was shown to be complexed with the co-activator p300, indicating that HIF-1α is transcriptionally active. Co-immunoprecipitation assay showed that HIF-1α from CLL B cells does not associate and form a complex with von Hippel-Landau protein tumor suppressor (pVHL), indicating that the proteasome dependent degradation pathway for HIF-1α protein in CLL B cells is dysfunctional. Using immunoblot or IHC methods, we were unable to detect pVHL protein in CLL B cells; however, we were able to use immunoprecipitation of CLL B cell lysates to demonstrate there is pVHL in CLL B cells. Prolyl hydroxylases (PHD 1, 2, and 3) are negative regulators for HIF-1α via hydroxylation of amino acid prolines in the oxygen degradation domain (ODD) which permits interaction with pVHL. RT-PCR results revealed that there is a subset of CLL patients who had ≥ 50% reduction of PHD 1 and 3 mRNA levels. However using a hydroxylation specific polyclonal antibody we found that HIF-1α from CLL B cells is indeed hydroxylated. Finally, silencing of HIF-1α by RNA interference in CLL B cells was associated with a selective decrease in VEGF mRNA levels but not VEGF-R1, Mcl-1 and prolyl hydroxylases (PHD 1–3) other downstream target genes of HIF-1α. These data show that the high endogenous HIF-1α levels in CLL B cells are due to a defect in HIF-1α degradation via the proteosomal pathway. We believe that this abnormality is linked to the autocrine VEGF pathway in CLL B cells and ultimately results in increases in their apoptotic resistance. Inhibition of HIF-1α levels may be of therapeutic benefit to CLL patients.

scholarly journals The bzd Gene Cluster, Coding for Anaerobic Benzoate Catabolism, in Azoarcus sp. Strain CIB

2004 ◽  
Vol 186 (17) ◽  
pp. 5762-5774 ◽  
Author(s):  
María J. López Barragán ◽  
Manuel Carmona ◽  
María T. Zamarro ◽  
Bärbel Thiele ◽  
Matthias Boll ◽  
...  
Keyword(s):  
Degradation Pathway ◽  
Ring Cleavage ◽  
Gene Products ◽  
Physiological Control ◽  
Significant Similarity ◽  
Thauera Aromatica ◽  
Benzoate Degradation ◽  
Coa Ligase ◽  
Transcriptional Units ◽  
Coa Reductase

ABSTRACT We report here that the bzd genes for anaerobic benzoate degradation in Azoarcus sp. strain CIB are organized as two transcriptional units, i.e., a benzoate-inducible catabolic operon, bzdNOPQMSTUVWXYZA, and a gene, bzdR, encoding a putative transcriptional regulator. The last gene of the catabolic operon, bzdA, has been expressed in Escherichia coli and encodes the benzoate-coenzyme A (CoA) ligase that catalyzes the first step in the benzoate degradation pathway. The BzdA enzyme is able to activate a wider range of aromatic compounds than that reported for other previously characterized benzoate-CoA ligases. The reduction of benzoyl-CoA to a nonaromatic cyclic intermediate is carried out by a benzoyl-CoA reductase (bzdNOPQ gene products) detected in Azoarcus sp. strain CIB extracts. The bzdW, bzdX, and bzdY gene products show significant similarity to the hydratase, dehydrogenase, and ring-cleavage hydrolase that act sequentially on the product of the benzoyl-CoA reductase in the benzoate catabolic pathway of Thauera aromatica. Benzoate-CoA ligase assays and transcriptional analyses based on lacZ-reporter fusions revealed that benzoate degradation in Azoarcus sp. strain CIB is subject to carbon catabolite repression by some organic acids, indicating the existence of a physiological control that connects the expression of the bzd genes to the metabolic status of the cell.

scholarly journals Pseudomonas putida Strain PCL1444, Selected for Efficient Root Colonization and Naphthalene Degradation, Effectively Utilizes Root Exudate Components

2002 ◽  
Vol 15 (7) ◽  
pp. 734-741 ◽  
Author(s):  
Irene Kuiper ◽  
Lev V. Kravchenko ◽  
Guido V. Bloemberg ◽  
Ben J. J. Lugtenberg
Keyword(s):  
Salicylic Acid ◽  
Succinic Acid ◽  
Pseudomonas Putida ◽  
Root Exudate ◽  
Carbon Sources ◽  
Degradation Pathway ◽  
Root Tip ◽  
Logarithmic Phase ◽  
Naphthalene Degradation ◽  
Catabolic Genes

Previously, we have described the selection of a plant-bacterium pair that is efficient in rhizoremediating naphthalene pollution in microcosm studies. After repeated selection for efficient root tip colonization upon inoculation of seeds of grass cv. Barmultra and for stable and efficient growth on naphthalene, Pseudomonas putida PCL1444 was selected as the most efficient colonizer of Barmultra roots. Here, we report the analysis of Barmultra root exudate composition and our subsequent tests of the growth rate of the bacterium and of the expression of the naphthalene degradation genes on individual exudate components. High performance liquid chromatography analysis of the organic acid and sugar root-exudate components revealed that glucose and fructose are the most abundant sugars, whereas succinic acid and citric acid are the most abundant organic acids. Tn5luxAB mutants of PCL1444 impaired in naphthalene degradation appeared to be impaired in genes homologous to genes of the upper naphthalene degradation pathway present in various Pseudomonas strains and to genes of the lower pathway genes for naphthalene degradation in P. stutzeri. Highest expression for both pathways involved in naphthalene degradation during growth in minimal medium with the carbon source to be tested was observed at the start of the logarithmic phase. Naphthalene did not induce the upper pathway, but a different pattern of expression was observed in the lower pathway reporter, probably due to the conversion of naphthalene to salicylic acid. Salicylic acid, which is described as an intermediate of the naphthalene degradation pathway in many Pseudomonas strains, did induce both pathways, resulting in an up to sixfold higher expression level at the start of the logarithmic phase. When expression levels during growth on the different carbon sources present in root exudate were compared, highest expression was observed on the two major root exudate components, glucose and succinic acid. These results show an excellent correlation between successful naphthalene rhizoremediation by the Barmultra-P. putida PCL1444 pair and both efficient utilization of the major exudate components for growth and high transcription of the naphthalene catabolic genes on the major exudate components. Therefore, we hypothesize that efficient root colonizing and naphthalene degradation is the result of the applied colonization enrichment procedure.

scholarly journals Role of the crc Gene in Catabolic Repression of the Pseudomonas putida GPo1 Alkane Degradation Pathway

2001 ◽  
Vol 183 (21) ◽  
pp. 6197-6206 ◽  
Author(s):  
Luis Yuste ◽  
Fernando Rojo
Keyword(s):  
Pseudomonas Putida ◽  
Transcription Initiation ◽  
Defined Medium ◽  
Degradation Pathway ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Rich Medium ◽  
Alkane Degradation ◽  
Catabolic Repression ◽  
Elevated Expression

ABSTRACT Expression of the alkane degradation pathway encoded in the OCT plasmid of Pseudomonas putida GPo1 is induced in the presence of alkanes by the AlkS regulator, and it is down-regulated by catabolic repression. The catabolic repression effect reduces the expression of the two AlkS-activated promoters of the pathway, named PalkB and PalkS2. The P. putida Crc protein participates in catabolic repression of some metabolic pathways for sugars and nitrogenated compounds. Here, we show that Crc has an important role in the catabolic repression exerted on the P. putida GPo1 alkane degradation pathway when cells grow exponentially in a rich medium. Interestingly, Crc plays little or no role on the catabolic repression exerted by some organic acids in a defined medium, which shows that these two types of catabolic repression can be genetically distinguished. Disruption of thecrc gene led to a six- to sevenfold increase in the levels of the mRNAs arising from the AlkS-activatedPalkB and PalkS2 promoters in cells growing exponentially in rich medium. This was not due to an increase in the half-lives of these mRNAs. Since AlkS activates the expression of its own gene and seems to be present in limiting amounts, the higher mRNA levels observed in the absence of Crc could arise from an increase in either transcription initiation or in the translation efficiency of the alkS mRNA. Both alternatives would lead to increased AlkS levels and hence to elevated expression of PalkBand PalkS2. High expression of alkS from a heterologous promoter eliminated catabolic repression. Our results indicate that catabolic repression in rich medium is directed to down-regulate the levels of the AlkS activator. Crc would thus modulate, directly or indirectly, the levels of AlkS.

The Pseudomonas putida Crc global regulator controls the hierarchical assimilation of amino acids in a complete medium: Evidence from proteomic and genomic analyses

PROTEOMICS ◽  
2009 ◽  
Vol 9 (11) ◽  
pp. 2910-2928 ◽  
Author(s):  
Renata Moreno ◽  
Montserrat Martínez-Gomariz ◽  
Luis Yuste ◽  
Concha Gil ◽  
Fernando Rojo
Keyword(s):  
Amino Acids ◽  
Pseudomonas Putida ◽  
Complete Medium ◽  
Global Regulator ◽  
Genomic Analyses

scholarly journals Global Regulation of Food Supply by Pseudomonas putida DOT-T1E

2010 ◽  
Vol 192 (8) ◽  
pp. 2169-2181 ◽  
Author(s):  
Craig Daniels ◽  
Patricia Godoy ◽  
Estrella Duque ◽  
M. Antonia Molina-Henares ◽  
Jesús de la Torre ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Carbon Sources ◽  
Nitrogen Sources ◽  
Nutrient Supply ◽  
Receptor Protein ◽  
Wild Type ◽  
Global Regulator ◽  
Growth And Survival ◽  
Complex Decision Making ◽  
Supply Optimization

ABSTRACT Pseudomonas putida DOT-T1E was used as a model to develop a “phenomics” platform to investigate the ability of P. putida to grow using different carbon, nitrogen, and sulfur sources and in the presence of stress molecules. Results for growth of wild-type DOT-T1E on 90 different carbon sources revealed the existence of a number of previously uncharted catabolic pathways for compounds such as salicylate, quinate, phenylethanol, gallate, and hexanoate, among others. Subsequent screening on the subset of compounds on which wild-type DOT-TIE could grow with four knockout strains in the global regulatory genes Δcrc, Δcrp, ΔcyoB, and ΔptsN allowed analysis of the global response to nutrient supply and stress. The data revealed that most global regulator mutants could grow in a wide variety of substrates, indicating that metabolic fluxes are physiologically balanced. It was found that the Crc mutant did not differ much from the wild-type regarding the use of carbon sources. However, certain pathways are under the preferential control of one global regulator, i.e., metabolism of succinate and d-fructose is influenced by CyoB, and l -arginine is influenced by PtsN. Other pathways can be influenced by more than one global regulator; i.e., l-valine catabolism can be influenced by CyoB and Crp (cyclic AMP receptor protein) while phenylethylamine is affected by Crp, CyoB, and PtsN. These results emphasize the cross talk required in order to ensure proper growth and survival. With respect to N sources, DOT-T1E can use a wide variety of inorganic and organic nitrogen sources. As with the carbon sources, more than one global regulator affected growth with some nitrogen sources; for instance, growth with nucleotides, dipeptides, d-amino acids, and ethanolamine is influenced by Crp, CyoB, and PtsN. A surprising finding was that the Crp mutant was unable to flourish on ammonium. Results for assayed sulfur sources revealed that CyoB controls multiple points in methionine/cysteine catabolism while PtsN and Crc are needed for N-acetyl-l-cysteamine utilization. Growth of global regulator mutants was also influenced by stressors of different types (antibiotics, oxidative agents, and metals). Overall and in combination with results for growth in the presence of various stressors, these phenomics assays provide multifaceted insights into the complex decision-making process involved in nutrient supply, optimization, and survival.

scholarly journals A Novel Gene, Encoding 6-Hydroxy-3-Succinoylpyridine Hydroxylase, Involved in Nicotine Degradation by Pseudomonas putida Strain S16

2008 ◽  
Vol 74 (5) ◽  
pp. 1567-1574 ◽  
Author(s):  
Hongzhi Tang ◽  
Shuning Wang ◽  
Lanying Ma ◽  
Xiangzhou Meng ◽  
Zixin Deng ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Gene Cluster ◽  
Wild Type Strain ◽  
Degradation Pathway ◽  
Wild Type ◽  
Pyrrolidine Ring ◽  
Gene Encoding ◽  
Resting Cell ◽  
Nicotine Degradation ◽  
Degradation Intermediates

ABSTRACT Previous research suggested that Pseudomonas spp. may attack the pyrrolidine ring of nicotine in a way similar to mammalian metabolism, resulting in the formation of pseudooxynicotine, the direct precursor of a potent tobacco-specific lung carcinogen. In addition, the subsequent intermediates, 6-hydroxy-3-succinoylpyridine (HSP) and 2,5-dihydroxypyridine (DHP) in the Pseudomonas nicotine degradation pathway are two important precursors for drug syntheses. However, there is little information on the molecular mechanism for nicotine degradation via the pyrrolidine pathway until now. In this study we cloned and sequenced a 4,879-bp gene cluster involved in nicotine degradation. Intermediates N-methylmyosmine, pseudooxynicotine, 3-succinoylpyridine, HSP, and DHP were identified from resting cell reactions of the transformant containing the gene cluster and shown to be identical to those of the pyrrolidine pathway reported in wild-type strain Pseudomonas putida S16. The gene for 6-hydroxy-3-succinoylpyridine hydroxylase (HSP hydroxylase) catalyzing HSP directly to DHP was cloned, sequenced, and expressed in Escherichia coli, and the purified HSP hydroxylase (38 kDa) is NADH dependent. DNA sequence analysis of this 936-bp fragment reveals that the deduced amino acid shows no similarity with any protein of known function.

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