29 results on '"Haas, D"'
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2. Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa
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Serino, L, primary, Reimmann, C, additional, Visca, P, additional, Beyeler, M, additional, Chiesa, V D, additional, and Haas, D, additional
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- 1997
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3. Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities
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Schnider, U, primary, Keel, C, additional, Blumer, C, additional, Troxler, J, additional, Défago, G, additional, and Haas, D, additional
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- 1995
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4. arcD, the first gene of the arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa, encodes an arginine-ornithine exchanger
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Verhoogt, H J, primary, Smit, H, additional, Abee, T, additional, Gamper, M, additional, Driessen, A J, additional, Haas, D, additional, and Konings, W N, additional
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- 1992
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5. Anaerobic regulation of transcription initiation in the arcDABC operon of Pseudomonas aeruginosa
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Gamper, M, primary, Zimmermann, A, additional, and Haas, D, additional
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- 1991
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6. Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa
- Author
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Galimand, M, primary, Gamper, M, additional, Zimmermann, A, additional, and Haas, D, additional
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- 1991
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7. Regulation of enzyme synthesis in the arginine deiminase pathway of Pseudomonas aeruginosa
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Mercenier, A, Simon, J P, Vander Wauven, C, Haas, D, and Stalon, V
- Abstract
The three enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa strain PAO were induced strongly (50- to 100-fold) by a shift from aerobic growth conditions to very low oxygen tension. Arginine in the culture medium was not essential for induction, but increased the maximum enzyme levels twofold. The induction of the three enzymes arginine deiminase (EC 3.5.3.6), catabolic ornithine carbamoyltransferase (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.3) appeared to be coordinate. Catabolic ornithine carbamoyltransferase was studied in most detail. Nitrate and nitrite, which can replace oxygen as terminal electron acceptors in P. aeruginosa, partially prevented enzyme induction by low oxygen tension in the wild-type strain, but not in nar (nitrate reductase-negative) mutants. Glucose was found to exert catabolite repression of the deiminase pathway. Generally, conditions of stress, such as depletion of the carbon and energy source or the phosphate source, resulted in induced synthesis of catabolic ornithine carbamoyltransferase. The induction of the deiminase pathway is thought to mobilize intra- and extracellular reserves of arginine, which is used as a source of adenosine 5'-triphosphate in the absence of respiration.
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- 1980
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8. Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway
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Vander Wauven, C, Piérard, A, Kley-Raymann, M, and Haas, D
- Abstract
Pseudomonas aeruginosa PAO was able to grow in the absence of exogenous terminal electron acceptors, provided that the medium contained 30 to 40 mM L-arginine and 0.4% yeast extract. Under strictly anaerobic conditions (O2 at less than 1 ppm), growth could be measured as an increase in protein and proceeded in a non-exponential way; arginine was largely converted to ornithine but not entirely consumed at the end of growth. In the GasPak anaerobic jar (Becton Dickinson and Co.), the wild-type strain PAO1 grew on arginine-yeast extract medium in 3 to 5 days; mutants could be isolated that were unable to grow under these conditions. All mutants (except one) were defective in at least one of the three enzymes of the arginine deiminase pathway (arcA, arcB, and arcC mutants) or in a novel function that might be involved in anaerobic arginine uptake (arcD mutants). The mutations arcA (arginine deiminase), arcB (catabolic ornithine carbamoyltransferase), arcC (carbamate kinase), and arcD were highly cotransducible and mapped in the 17-min chromosome region. Some mutations in the arc cluster led to low, noninducible levels of all three arginine deiminase pathway enzymes and thus may affect control elements required for induction of the postulated arc operon. Two fluorescent pseudomonads (P. putida and P. fluorescens) and P. mendocina, as well as one PAO mutant, possessed an inducible arginine deiminase pathway and yet were unable to grow fermentatively on arginine. The ability to use arginine-derived ATP for growth may provide P. aeruginosa with a selective advantage when oxygen and nitrate are scarce.
- Published
- 1984
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9. Anabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa: nucleotide sequence and transcriptional control of the argF structural gene
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Itoh, Y, Soldati, L, Stalon, V, Falmagne, P, Terawaki, Y, Leisinger, T, and Haas, D
- Abstract
In Pseudomonas aeruginosa PAO the anabolic ornithine carbamoyltransferase (OTCase, EC 2.1.3.3) is the product of the argF gene and the only arginine biosynthetic enzyme whose synthesis is repressible by arginine. We have determined the complete nucleotide sequence of the argF gene including its promoter-control region. The deduced amino acid sequence of the anabolic OTCase consists of 305 residues (Mr 33,924), and this was confirmed by the N-terminal amino acid sequence, the total amino acid composition, and the subunit Mr of the purified enzyme. The native anabolic OTCase (Mr 110,000 to 125,000) was found to be a trimer by cross-linking experiments. P. aeruginosa also has a catabolic OTCase (the arcB gene product), which catalyzes the reverse reaction of the anabolic conversion. At the nucleotide sequence level, the P. aeruginosa argF gene had 52.4% identity with the arcB gene. The Escherichia coli argF and argI genes, which code for anabolic OTCase isoenzymes, had 47.3 and 44.9% identity, respectively, with the P. aeruginosa argF sequence. This suggests that these four genes have evolved from a common ancestral gene. The arcB gene appears to be more closely related to the E. coli argF gene than to the P. aeruginosa argF gene. Two transcripts (mRNA-1, mRNA-2) of the P. aeruginosa argF gene were identified by S1 mapping. The transcription initiation site for mRNA-1 was preceded by sequences having partial homology with the E. coli -35 and -10 consensus promoter sequences. No sequence similar to consensus promoters of enteric bacteria was found upstream of the 5' end of mRNA-2. E. coli carrying a P. aeruginosa argF+ recombinant plasmid produced mRNA-1 with low efficiency but no (or very little) mRNA-2. Arginine repressed argF transcription in P. aeruginosa. In the argF promoter region no sequence homologous to the "arg box" (arginine operator module) of E. coli was found. The mechanism of arginine repression in P. aeruginosa thus appears to be different from that in E. coli.
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- 1988
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10. Mutants of Escherichia coli "cryptic" for certain periplasmic enzymes: evidence for an alteration of the outer membrane
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Beacham, I R, Haas, D, and Yagil, E
- Abstract
Mutants in which the expression of periplasmic enzymes by whole cells is reduced (termed "cryptic") are also found to show greatly reduced uptake of labeled adenosine 5'-monophosphate (5'-AMP), providing a rapid assay for crypticity. The crypticity of 3'- and 5'-nucleotidase has been examined as a function of substrate concentration. The Km for 3'- or 5'-AMP increases in the cryptic mutants when whole cells are used as the enzyme source. The Vmax is not altered. Electrophoretic analysis of protein prepared from cell envelopes showed that three cryptic mutants have a polypeptide absent from the outer membrane and a relatively high proportion of a polypeptide in the inner membrane. Analysis of the molar ratios of constituent sugars of the lipopolysaccharides showed no differences between three cryptic mutants and the parent strain. One cryptic mutant (3--41), however, has altered sensitivity to phage T4. By selection for phage resistance, derivatives of the cryptic mutants that are deoxycholate sensitive have been obtained. These mutants are no longer cryptic. We suggest that cryptic mutants have an altered outer membrane, with decreased permeability to 3'- and 5'-AMP, as a result of an altered polypeptide.
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- 1977
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11. Mapping of the arginine deiminase gene in Pseudomonas aeruginosa
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Mercenier, A, primary, Stalon, V, additional, Simon, J P, additional, and Haas, D, additional
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- 1982
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12. Promoter recognition and activation by the global response regulator CbrB in Pseudomonas aeruginosa.
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Abdou L, Chou HT, Haas D, and Lu CD
- Subjects
- Binding Sites, DNA Mutational Analysis, DNA, Bacterial genetics, DNA, Bacterial metabolism, Electrophoretic Mobility Shift Assay, Integration Host Factors metabolism, Lipase metabolism, Protein Binding, RNA Polymerase Sigma 54 metabolism, Bacterial Proteins metabolism, Gene Expression Regulation, Bacterial, Promoter Regions, Genetic, Pseudomonas aeruginosa genetics, Transcription Factors metabolism, Transcriptional Activation
- Abstract
In Pseudomonas aeruginosa, the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein. Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and σ(54) RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA. As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.
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- 2011
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13. Identification of the biosynthetic gene cluster for the Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid.
- Author
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Lee X, Fox A, Sufrin J, Henry H, Majcherczyk P, Haas D, and Reimmann C
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- Gene Order, Genes, Bacterial, Mutagenesis, Site-Directed, Aminobutyrates metabolism, Anti-Bacterial Agents biosynthesis, Antimetabolites metabolism, Biosynthetic Pathways genetics, Multigene Family, Pseudomonas aeruginosa genetics, Pseudomonas aeruginosa metabolism
- Abstract
L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.
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- 2010
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14. Adaptation of aerobically growing Pseudomonas aeruginosa to copper starvation.
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Frangipani E, Slaveykova VI, Reimmann C, and Haas D
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- Aerobiosis, Artificial Gene Fusion, Bacterial Proteins genetics, Culture Media chemistry, Down-Regulation, Gene Deletion, Genes, Reporter, Glucose metabolism, Oligonucleotide Array Sequence Analysis, Pseudomonas aeruginosa growth & development, Pseudomonas aeruginosa metabolism, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins genetics, Up-Regulation, beta-Galactosidase biosynthesis, beta-Galactosidase genetics, Bacterial Proteins biosynthesis, Copper metabolism, Gene Expression Profiling, Pseudomonas aeruginosa physiology
- Abstract
Restricted bioavailability of copper in certain environments can interfere with cellular respiration because copper is an essential cofactor of most terminal oxidases. The global response of the metabolically versatile bacterium and opportunistic pathogen Pseudomonas aeruginosa to copper limitation was assessed under aerobic conditions. Expression of cioAB (encoding an alternative, copper-independent, cyanide-resistant ubiquinol oxidase) was upregulated, whereas numerous iron uptake functions (including the siderophores pyoverdine and pyochelin) were expressed at reduced levels, presumably reflecting a lower demand for iron by respiratory enzymes. Wild-type P. aeruginosa was able to grow aerobically in a defined glucose medium depleted of copper, whereas a cioAB mutant did not grow. Thus, P. aeruginosa relies on the CioAB enzyme to cope with severe copper deprivation. A quadruple cyo cco1 cco2 cox mutant, which was deleted for all known heme-copper terminal oxidases of P. aeruginosa, grew aerobically, albeit more slowly than did the wild type, indicating that the CioAB enzyme is capable of energy conservation. However, the expression of a cioA'-'lacZ fusion was less dependent on the copper status in the quadruple mutant than in the wild type, suggesting that copper availability might affect cioAB expression indirectly, via the function of the heme-copper oxidases.
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- 2008
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15. Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa.
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Kay E, Humair B, Dénervaud V, Riedel K, Spahr S, Eberl L, Valverde C, and Haas D
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- Biofilms, Gene Expression Regulation, Bacterial, Mutation, RNA, Bacterial genetics, Ribosomal Proteins metabolism, Bacterial Proteins metabolism, Pseudomonas aeruginosa metabolism, RNA, Bacterial metabolism
- Abstract
In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extracellular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.
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- 2006
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16. Quorum-sensing-negative (lasR) mutants of Pseudomonas aeruginosa avoid cell lysis and death.
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Heurlier K, Dénervaud V, Haenni M, Guy L, Krishnapillai V, and Haas D
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- Adenosine metabolism, Cell Division, Escherichia coli genetics, Genetic Complementation Test, N-Glycosyl Hydrolases genetics, N-Glycosyl Hydrolases metabolism, Plasmids genetics, Pseudomonas aeruginosa cytology, Pseudomonas aeruginosa growth & development, Restriction Mapping, Bacterial Proteins genetics, DNA-Binding Proteins genetics, Pseudomonas aeruginosa genetics, Trans-Activators genetics
- Abstract
In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37 degrees C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to > or = 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh(-) phenotype, and five of these mutants, which were functionally complemented by lasR(+), had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants.
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- 2005
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17. Posttranscriptional repression of GacS/GacA-controlled genes by the RNA-binding protein RsmE acting together with RsmA in the biocontrol strain Pseudomonas fluorescens CHA0.
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Reimmann C, Valverde C, Kay E, and Haas D
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- Amino Acid Sequence, Base Sequence, Genes, Regulator, Molecular Sequence Data, Transcription, Genetic, Bacterial Proteins physiology, Gene Expression Regulation, Bacterial, Pest Control, Biological, Pseudomonas fluorescens genetics, RNA-Binding Proteins physiology, Repressor Proteins physiology
- Abstract
In the plant-beneficial soil bacterium Pseudomonas fluorescens CHA0, the production of biocontrol factors (antifungal secondary metabolites and exoenzymes) is controlled at a posttranscriptional level by the GacS/GacA signal transduction pathway involving RNA-binding protein RsmA as a key regulatory element. This protein is assumed to bind to the ribosome-binding site of target mRNAs and to block their translation. RsmA-mediated repression is relieved at the end of exponential growth by two GacS/GacA-controlled regulatory RNAs RsmY and RsmZ, which bind and sequester the RsmA protein. A gene (rsmE) encoding a 64-amino-acid RsmA homolog was identified and characterized in strain CHA0. Overexpression of rsmE strongly reduced the expression of target genes (hcnA, for a hydrogen cyanide synthase subunit; aprA, for the main exoprotease; and phlA, for a component of 2,4-diacetylphloroglucinol biosynthesis). Single null mutations in either rsmA or rsmE resulted in a slight increase in the expression of hcnA, aprA, and phlA. By contrast, an rsmA rsmE double mutation led to strongly increased and advanced expression of these target genes and completely suppressed a gacS mutation. Both the RsmE and RsmA levels increased with increasing cell population densities in strain CHA0; however, the amount of RsmA showed less variability during growth. Expression of rsmE was controlled positively by GacA and negatively by RsmA and RsmE. Mobility shift assays demonstrated specific binding of RsmE to RsmY and RsmZ RNAs. The transcription and stability of both regulatory RNAs were strongly reduced in the rsmA rsmE double mutant. In conclusion, RsmA and RsmE together account for maximal repression in the GacS/GacA cascade of strain CHA0.
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- 2005
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18. PchC thioesterase optimizes nonribosomal biosynthesis of the peptide siderophore pyochelin in Pseudomonas aeruginosa.
- Author
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Reimmann C, Patel HM, Walsh CT, and Haas D
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- Aminobutyrates pharmacology, Cysteine pharmacology, Bacterial Proteins physiology, Phenols metabolism, Pseudomonas aeruginosa metabolism, Thiazoles
- Abstract
In Pseudomonas aeruginosa, the antibiotic dihydroaeruginoate (Dha) and the siderophore pyochelin are produced from salicylate and cysteine by a thiotemplate mechanism involving the peptide synthetases PchE and PchF. A thioesterase encoded by the pchC gene was found to be necessary for maximal production of both Dha and pyochelin, but it was not required for Dha release from PchE and could not replace the thioesterase function specified by the C-terminal domain of PchF. In vitro, 2-aminobutyrate, a cysteine analog, was adenylated by purified PchE and PchF proteins. In vivo, this analog strongly interfered with Dha and pyochelin formation in a pchC deletion mutant but affected production of these metabolites only slightly in the wild type. Exogenously supplied cysteine overcame the negative effect of a pchC mutation to a large extent, whereas addition of salicylate did not. These data are in agreement with a role for PchC as an editing enzyme that removes wrongly charged molecules from the peptidyl carrier protein domains of PchE and PchF.
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- 2004
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19. Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1.
- Author
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Heurlier K, Williams F, Heeb S, Dormond C, Pessi G, Singer D, Cámara M, Williams P, and Haas D
- Subjects
- Amino Acid Sequence, Bacterial Proteins antagonists & inhibitors, Base Sequence, Cloning, Molecular, Molecular Sequence Data, Pseudomonas aeruginosa genetics, RNA-Binding Proteins antagonists & inhibitors, Repressor Proteins antagonists & inhibitors, Bacterial Proteins physiology, Gene Expression Regulation, Bacterial, Glycolipids biosynthesis, Lipase biosynthesis, Pseudomonas aeruginosa metabolism, RNA, Bacterial physiology, RNA-Binding Proteins physiology, Repressor Proteins physiology
- Abstract
In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules. RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P. aeruginosa. An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P. aeruginosa. A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P. aeruginosa. Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation. Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.
- Published
- 2004
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20. Negative control of quorum sensing by RpoN (sigma54) in Pseudomonas aeruginosa PAO1.
- Author
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Heurlier K, Dénervaud V, Pessi G, Reimmann C, and Haas D
- Subjects
- 4-Butyrolactone metabolism, Bacterial Proteins genetics, Cell Division genetics, Cyclic AMP Receptor Protein genetics, Cyclic AMP Receptor Protein metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, DNA-Directed RNA Polymerases genetics, Gene Expression Regulation, Bacterial, Genetic Complementation Test, Ligases, Mutagenesis, Site-Directed, RNA Polymerase Sigma 54, Repressor Proteins genetics, Repressor Proteins metabolism, Sigma Factor genetics, Trans-Activators genetics, Trans-Activators metabolism, Transcription Factors genetics, Transcription Factors metabolism, 4-Butyrolactone analogs & derivatives, Bacterial Proteins metabolism, DNA-Directed RNA Polymerases metabolism, Pseudomonas aeruginosa physiology, Sigma Factor metabolism
- Abstract
In Pseudomonas aeruginosa PAO1, the expression of several virulence factors such as elastase, rhamnolipids, and hydrogen cyanide depends on quorum-sensing regulation, which involves the lasRI and rhlRI systems controlled by N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, respectively, as signal molecules. In rpoN mutants lacking the transcription factor sigma(54), the expression of the lasR and lasI genes was elevated at low cell densities, whereas expression of the rhlR and rhlI genes was markedly enhanced throughout growth by comparison with the wild type and the complemented mutant strains. As a consequence, the rpoN mutants had elevated levels of both signal molecules and overexpressed the biosynthetic genes for elastase, rhamnolipids, and hydrogen cyanide. The quorum-sensing regulatory protein QscR was not involved in the negative control exerted by RpoN. By contrast, in an rpoN mutant, the expression of the gacA global regulatory gene was significantly increased during the entire growth cycle, whereas another global regulatory gene, vfr, was downregulated at high cell densities. In conclusion, it appears that GacA levels play an important role, probably indirectly, in the RpoN-dependent modulation of the quorum-sensing machinery of P. aeruginosa.
- Published
- 2003
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21. Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0.
- Author
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Heeb S, Blumer C, and Haas D
- Subjects
- 4-Butyrolactone metabolism, Amino Acid Sequence, Bacterial Proteins physiology, Base Sequence, Chromosome Mapping, Chromosomes, Bacterial, DNA, Bacterial, Exopeptidases genetics, Genes, Bacterial, Molecular Sequence Data, Multienzyme Complexes genetics, Mutagenesis, Nucleic Acid Conformation, Oxidoreductases genetics, Oxidoreductases Acting on CH-NH2 Group Donors, Pseudomonas fluorescens genetics, RNA-Binding Proteins genetics, RNA-Binding Proteins physiology, Regulatory Sequences, Nucleic Acid, Repressor Proteins genetics, Repressor Proteins physiology, Sigma Factor metabolism, Transcription Factors genetics, Transcription Factors metabolism, 4-Butyrolactone analogs & derivatives, Bacterial Proteins genetics, Bacterial Proteins metabolism, Genes, Regulator, Pseudomonas fluorescens metabolism, RNA, Bacterial genetics, RNA-Binding Proteins metabolism, Repressor Proteins metabolism
- Abstract
In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified from P. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end. Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated to N-acyl-homoserine lactones. A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression. Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease). Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal. Overexpression of rsmA had a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.
- Published
- 2002
- Full Text
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22. The global posttranscriptional regulator RsmA modulates production of virulence determinants and N-acylhomoserine lactones in Pseudomonas aeruginosa.
- Author
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Pessi G, Williams F, Hindle Z, Heurlier K, Holden MT, Cámara M, Haas D, and Williams P
- Subjects
- 4-Butyrolactone analysis, Bacterial Proteins metabolism, Bacterial Proteins physiology, Cloning, Molecular, Hydrogen Cyanide metabolism, Molecular Sequence Data, Mutation, Protein Biosynthesis, Pseudomonas aeruginosa metabolism, Pseudomonas aeruginosa pathogenicity, Pyocyanine metabolism, Repressor Proteins physiology, 4-Butyrolactone analogs & derivatives, 4-Butyrolactone metabolism, Bacterial Proteins genetics, Pseudomonas aeruginosa genetics, RNA-Binding Proteins, Repressor Proteins genetics
- Abstract
Posttranscriptional control is known to contribute to the regulation of secondary metabolism and virulence determinants in certain gram-negative bacteria. Here we report the isolation of a Pseudomonas aeruginosa gene which encodes a global translational regulatory protein, RsmA (regulator of secondary metabolites). Overexpression of rsmA resulted in a substantial reduction in the levels of extracellular products, including protease, elastase, and staphylolytic (LasA protease) activity as well as the PA-IL lectin, hydrogen cyanide (HCN), and the phenazine pigment pyocyanin. While inactivation of rsmA in P. aeruginosa had only minor effects on the extracellular enzymes and the PA-IL lectin, the production of HCN and pyocyanin was enhanced during the exponential phase. The influence of RsmA on N-acylhomoserine lactone-mediated quorum sensing was determined by assaying the levels of N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL) and N-butanoylhomoserine lactone (C4-HSL) produced by the rsmA mutant and the rsmA-overexpressing strain. RsmA exerted a negative effect on the synthesis of both 3-oxo-C12-HSL and C4-HSL, which was confirmed by using lasI and rhlI translational fusions. These data also highlighted the temporal expression control of the lasI gene, which was induced much earlier and to a higher level during the exponential growth phase in an rsmA mutant. To investigate whether RsmA modulates HCN production solely via quorum-sensing control, hcn translational fusions were employed to monitor the regulation of the cyanide biosynthesis genes (hcnABC). RsmA was shown to exert an additional negative effect on cyanogenesis posttranscriptionally by acting on a region surrounding the hcnA ribosome-binding site. This suggests that, in P. aeruginosa, RsmA functions as a pleiotropic posttranscriptional regulator of secondary metabolites directly and also indirectly by modulating the quorum-sensing circuitry.
- Published
- 2001
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23. Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa.
- Author
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Reimmann C, Patel HM, Serino L, Barone M, Walsh CT, and Haas D
- Subjects
- ATP-Binding Cassette Transporters genetics, Bacterial Proteins, Chorismic Acid metabolism, Gene Expression Regulation, Bacterial, Genes, Bacterial, Molecular Sequence Data, Oxidoreductases Acting on CH-NH Group Donors genetics, Repressor Proteins, Salicylates metabolism, Thiazoles metabolism, Iron Chelating Agents metabolism, Oxidoreductases Acting on CH-NH Group Donors metabolism, Phenols metabolism, Pseudomonas aeruginosa metabolism, Siderophores metabolism
- Abstract
The biosynthetic genes pchDCBA and pchEF, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene on the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downstream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the Yersinia pestis and Y. enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG abolished pyochelin formation, whereas mutations in pchH and pchI did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG together with the E. coli entD gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P. aeruginosa.
- Published
- 2001
- Full Text
- View/download PDF
24. Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa.
- Author
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Pessi G and Haas D
- Subjects
- Amino Acid Sequence, Anaerobiosis, Bacterial Proteins genetics, Bacterial Proteins metabolism, Base Sequence, DNA Mutational Analysis, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Molecular Sequence Data, Oxidoreductases Acting on CH-NH2 Group Donors, Promoter Regions, Genetic genetics, Pseudomonas aeruginosa growth & development, Pseudomonas aeruginosa metabolism, Signal Transduction, Trans-Activators genetics, Trans-Activators metabolism, Transcription Factors genetics, Transcription Factors metabolism, Gene Expression Regulation, Bacterial, Hydrogen Cyanide metabolism, Multienzyme Complexes genetics, Multienzyme Complexes metabolism, Oxidoreductases genetics, Oxidoreductases metabolism, Pseudomonas aeruginosa genetics, Transcription, Genetic
- Abstract
Virulence factors of Pseudomonas aeruginosa include hydrogen cyanide (HCN). This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase. The hcnABC genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of P. aeruginosa PAO1. The hcnA promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR. Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp. Their function was confirmed by transcriptional lacZ fusions. The promoter sequence displayed an FNR/ANR box at -42.5 bp upstream of T2 and a lux box centered around -42.5 bp upstream of T1. Expression of the hcn genes was completely abolished when this lux box was deleted or inactivated by two point mutations in conserved nucleotides. The lux box was recognized by both LasR [activated by N-(oxododecanoyl)-homoserine lactone] and RhlR (activated by N-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective P. aeruginosa mutants and in the N-acyl-homoserine lactone-negative heterologous host P. fluorescens CHA0. A second, less conserved lux box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression. Without LasR and RhlR, ANR could not activate the hcn promoter. Together, these data indicate that expression of the hcn promoter from T1 can occur under quorum-sensing control alone. Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR.
- Published
- 2000
- Full Text
- View/download PDF
25. Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin.
- Author
-
Schnider-Keel U, Seematter A, Maurhofer M, Blumer C, Duffy B, Gigot-Bonnefoy C, Reimmann C, Notz R, Défago G, Haas D, and Keel C
- Subjects
- Anti-Bacterial Agents pharmacology, Cloning, Molecular, DNA Transposable Elements genetics, DNA, Bacterial chemistry, DNA, Bacterial genetics, Fungicides, Industrial metabolism, Fusaric Acid pharmacology, Fusarium chemistry, Fusarium metabolism, Gene Expression Regulation, Bacterial drug effects, Genes, Bacterial genetics, Molecular Sequence Data, Mutation, Phenols, Phloroglucinol analogs & derivatives, Phloroglucinol metabolism, Plant Diseases microbiology, Pseudomonas fluorescens genetics, Pseudomonas fluorescens isolation & purification, Pyrroles, Salicylates pharmacology, Sequence Analysis, DNA, Anti-Bacterial Agents metabolism, Pseudomonas fluorescens metabolism, Salicylates metabolism
- Abstract
The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.
- Published
- 2000
- Full Text
- View/download PDF
26. Target joining of duplicated insertion sequence IS21 is assisted by IstB protein in vitro.
- Author
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Schmid S, Berger B, and Haas D
- Subjects
- Artificial Gene Fusion, Escherichia coli genetics, Replicon, Bacterial Proteins physiology, Bacteriophage lambda genetics, DNA Transposable Elements, Tandem Repeat Sequences
- Abstract
Tandemly repeated insertion sequence IS21, located on a suicide plasmid, promoted replicon fusion with bacteriophage lambda in vitro in the presence of ATP. This reaction was catalyzed in a cell extract containing the 45-kDa IstA protein (cointegrase) and the 30-kDa IstB helper protein of IS21 after both proteins had been overproduced in Escherichia coli. Without IstB, replicon fusion was inefficient and did not produce the 4-bp target duplications typical of IS21.
- Published
- 1999
- Full Text
- View/download PDF
27. The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa.
- Author
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Lu CD, Winteler H, Abdelal A, and Haas D
- Subjects
- Adenosine Triphosphate biosynthesis, Anaerobiosis, Antiporters biosynthesis, Arginine pharmacology, Bacterial Proteins biosynthesis, Base Sequence, Binding Sites, Gene Expression Regulation, Bacterial, Models, Genetic, Molecular Sequence Data, Protein Binding, Pseudomonas aeruginosa drug effects, Amino Acid Transport Systems, Antiporters genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, DNA-Binding Proteins, Promoter Regions, Genetic, Pseudomonas aeruginosa genetics, Repressor Proteins metabolism, Trans-Activators, Transcription Factors metabolism
- Abstract
Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcD transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at -73.5 bp in the arcD promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5'-TGACGC-3', which deviated in only 1 base from the common sequence motif 5'-TGTCGC-3' found in other ArgR binding sites of P. aeruginosa. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the arc operon, suggesting that ArgR alone does not activate the arcD promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of arc transcription.
- Published
- 1999
- Full Text
- View/download PDF
28. Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0.
- Author
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Laville J, Blumer C, Von Schroetter C, Gaia V, Défago G, Keel C, and Haas D
- Subjects
- Aerobiosis, Amino Acid Sequence, Anaerobiosis, Bacterial Proteins genetics, Base Sequence, Chromosome Mapping, Cloning, Molecular, DNA Primers genetics, DNA, Bacterial genetics, Escherichia coli genetics, Gene Expression Regulation, Bacterial, Iron-Sulfur Proteins genetics, Molecular Sequence Data, Mutation, Oxidoreductases Acting on CH-NH2 Group Donors, Pest Control, Biological, Plant Diseases microbiology, Plants, Toxic, Promoter Regions, Genetic, Pseudomonas aeruginosa genetics, Sequence Homology, Amino Acid, Nicotiana microbiology, Transcription Factors genetics, Bacterial Proteins metabolism, DNA-Binding Proteins, Escherichia coli Proteins, Genes, Bacterial, Multienzyme Complexes genetics, Multigene Family, Oxidoreductases genetics, Pseudomonas fluorescens genetics, Pseudomonas fluorescens metabolism, Trans-Activators, Transcription Factors metabolism
- Abstract
The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC ... ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.
- Published
- 1998
- Full Text
- View/download PDF
29. Expression of the argA gene carried by a defective lambda bacteriophage of Escherichia coli.
- Author
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Leisinger T, Haas D, and Kelker N
- Subjects
- Arginine metabolism, Arginine pharmacology, Enzyme Induction, Escherichia coli metabolism, Floxuridine pharmacology, Genes, Regulator, Hot Temperature, Mutation, Transduction, Genetic, Virus Replication, Coliphages growth & development, Escherichia coli enzymology, Genes, Glutamate Synthase biosynthesis, Transaminases biosynthesis
- Abstract
Evidence is presented that the increase in specific activity of N-acetylglutamate synthase observed upon heat induction of Escherichia coli (lambdadargA) is primarily due to a gene dosage effect.
- Published
- 1976
- Full Text
- View/download PDF
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