scholarly journals DL-α-Monofluoromethylputrescine is a potent irreversible inhibitor of Escherichia coli ornithine decarboxylase

1982 ◽  
Vol 204 (3) ◽  
pp. 771-775 ◽  
Author(s):  
A Kallio ◽  
P P McCann ◽  
P Bey
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Ornithine Decarboxylase ◽  
Half Life ◽  
Arginine Decarboxylase ◽  
Biosynthetic Enzyme ◽  
Irreversible Inhibitor ◽  
E Coli

DL-alpha-Monofluoromethylputrescine (compound R.M.I. 71864) is an enzyme-activated irreversible inhibitor of the biosynthetic enzyme ornithine decarboxylase from Escherichia coli. This compound, however, has much less effect in vitro on ornithine decarboxylase obtained from Pseudomonas aeruginosa. These findings are in contrast with those previously found with the substrate analogue DL-alpha-difluoromethylornithine (compound R.M.I. 71782). The K1 of the DL-alpha-monofluoromethylputrescine for the E. coli ornithine decarboxylase is 110 microM, and the half-life (t1/2) calculated for an infinite concentration of inhibitor is 2.1 min. When DL-alpha-monofluoromethylputrescine is used in combination with DL-alpha-difluoromethylarginine (R.M.I. 71897), an irreversible inhibitor of arginine decarboxylase, in vivo in E. coli, both decarboxylase activities are inhibited (greater than 95%) but putrescine levels are only decreased to about one-third of control values and spermidine levels are slightly increased.

scholarly journals Difluoromethylornithine irreversibly inactivates ornithine decarboxylase of Pseudomonas aeruginosa, but does not inhibit the enzymes of Escherichia coli

1981 ◽  
Vol 200 (1) ◽  
pp. 69-75 ◽  
Author(s):  
A Kallio ◽  
P P McCann
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Ornithine Decarboxylase ◽  
Arginine Decarboxylase ◽  
Decarboxylase Activity ◽  
Dependent Manner ◽  
Irreversible Inhibitor ◽  
E Coli ◽  
Ornithine Decarboxylase Activity

DL-alpha-Difluoromethylornithine, an enzyme-activated irreversible inhibitor of eukaryotic ornithine decarboxylase and consequently of putrescine biosynthesis, inhibited ornithine decarboxylase in enzyme extracts from Pseudomonas aeruginosa in a time-dependent manner t1/2 1 min, and also effectively blocked the enzyme activity in situ in the cell. Difluoromethylornithine, however, had no effect on the activity of ornithine decarboxylase assayed in enzyme extracts from either Escherichia coli or Klebsiella pneumoniae. However, the presence of the inhibitor in cell cultures did partially lower ornithine decarboxylase activity intracellularly in E. coli. Any decrease in the intracellular ornithine decarboxylase activity observed in E. coli and Pseudomonas was accompanied by a concomitant increase in arginine decarboxylase activity, arguing for a co-ordinated control of putrescine biosynthesis in these cells.

Biosynthetic ornithine and arginine decarboxylases: correlation of rates of synthesis with activities in Escherichia coli during exponential growth and following nutritional shift-up

1982 ◽  
Vol 28 (8) ◽  
pp. 945-950
Author(s):  
Stephen M. Boyle ◽  
Kazuo Adachi
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Ornithine Decarboxylase ◽  
Biological Activities ◽  
Decarboxylase Activity ◽  
Biosynthetic Enzyme ◽  
Guanosine Tetraphosphate ◽  
E Coli ◽  
K 12

Whether guanosine tetraphosphate (ppGpp) has a role in the regulation of the putrescine biosynthetic enzyme, ornithine decarboxylase, in Escherichia coli is controversial. Different laboratories have reported either direct or indirect correlations between ppGpp levels and ornithine decarboxylase activity using different in vivo conditions. In this report, using conditions in vivo to modulate ppGpp levels, experiments are described which bear on the controversy. The rates of synthesis and biological activities of the biosynthetic ornithine and arginine decarboxylases (ODC and ADC) were measured in E. coli K-12 during experimental growth and during nutritional shift-up. There were good correlations between changes in their respective activities and the rates of synthesis of these enzymes during steady state or shift-up. ODC activity or rate of synthesis changed directly in concert with ppGpp levels, while ADC activity or rate of synthesis changed inversely with ppGpp levels. These observations support the contention that ppGpp does not inhibit ODC activity.

scholarly journals The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 2838-2844 ◽  
Author(s):  
Nicoletta Castiglione ◽  
Serena Rinaldo ◽  
Giorgio Giardina ◽  
Francesca Cutruzzolà
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Consensus Sequence ◽  
Signal Molecules ◽  
Reporter System ◽  
Nucleotide Substitutions ◽  
E Coli

Pseudomonas aeruginosa is a well-known pathogen in chronic respiratory diseases such as cystic fibrosis. Infectivity of P. aeruginosa is related to the ability to grow under oxygen-limited conditions using the anaerobic metabolism of denitrification, in which nitrate is reduced to dinitrogen via nitric oxide (NO). Denitrification is activated by a cascade of redox-sensitive transcription factors, among which is the DNR regulator, sensitive to nitrogen oxides. To gain further insight into the mechanism of NO-sensing by DNR, we have developed an Escherichia coli-based reporter system to investigate different aspects of DNR activity. In E. coli DNR responds to NO, as shown by its ability to transactivate the P. aeruginosa norCB promoter. The direct binding of DNR to the target DNA is required, since mutations in the helix–turn–helix domain of DNR and specific nucleotide substitutions in the consensus sequence of the norCB promoter abolish the transcriptional activity. Using an E. coli strain deficient in haem biosynthesis, we have also confirmed that haem is required in vivo for the NO-dependent DNR activity, in agreement with the property of DNR to bind haem in vitro. Finally, we have shown, we believe for the first time, that DNR is able to discriminate in vivo between different diatomic signal molecules, NO and CO, both ligands of the reduced haem iron in vitro, suggesting that DNR responds specifically to NO.

scholarly journals dl-α-difluoromethyl[3,4-3H]arginine metabolism in tobacco and mammalian cells. Inhibition of ornithine decarboxylase activity after arginase-mediated hydrolysis of dl-α-difluoromethylarginine to dl-α-difluoromethylornithine

1988 ◽  
Vol 255 (1) ◽  
pp. 197-202 ◽  
Author(s):  
R D Slocum ◽  
A J Bitonti ◽  
P P McCann ◽  
R P Feirer
Keyword(s):  
Ornithine Decarboxylase ◽  
Mammalian Cells ◽  
Selective Inhibition ◽  
Arginine Decarboxylase ◽  
Decarboxylase Activity ◽  
Irreversible Inhibitor ◽  
Hydrolysis Of ◽  
Analogous Mechanism

DL-alpha-Difluoromethylarginine (DFMA) is an enzyme-activated irreversible inhibitor of arginine decarboxylase (ADC) in vitro. DFMA has also been shown to inhibit ADC activities in a variety of plants and bacteria in vivo. However, we questioned the specificity of this inhibitor for ADC in tobacco ovary tissues, since ornithine decarboxylase (ODC) activity was strongly inhibited as well. We now show that [3,4-3H]DFMA is metabolized to DL-alpha-difluoromethyl[3,4-3H]ornithine [(3,4-3H]DFMO), the analogous mechanism-based inhibitor of ODC, by tobacco tissues in vivo. Both tobacco and mammalian (mouse, bovine) arginases (EC 3.5.3.1) hydrolyse DFMA to DFMO in vitro, suggesting a role for this enzyme in mediating the indirect inhibition of ODC by DFMA in tobacco. These results suggest that DFMA may have other effects, in addition to the inhibition of ADC, in tissues containing high arginase activities. The recent development of potent agmatine-based ADC inhibitors should permit selective inhibition of ADC, rather than ODC, in such tissues, since agmatine is not a substrate for arginase.

scholarly journals Pseudomonas aeruginosa MutL protein functions in Escherichia coli

2005 ◽  
Vol 388 (3) ◽  
pp. 879-887 ◽  
Author(s):  
Daniela K. JACQUELÍN ◽  
Adrián FILIBERTI ◽  
Carlos E. ARGARAÑA ◽  
José L. BARRA
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Repair System ◽  
E Coli ◽  
New Findings

Escherichia coli MutS, MutL and MutH proteins act sequentially in the MMRS (mismatch repair system). MutH directs the repair system to the newly synthesized strand due to its transient lack of Dam (DNA-adenine methylase) methylation. Although Pseudomonas aeruginosa does not have the corresponding E. coli MutH and Dam homologues, and consequently the MMRS seems to work differently, we show that the mutL gene from P. aeruginosa is capable of complementing a MutL-deficient strain of E. coli. MutL from P. aeruginosa has conserved 21 out of the 22 amino acids known to affect functioning of E. coli MutL. We showed, using protein affinity chromatography, that the C-terminal regions of P. aeruginosa and E. coli MutL are capable of specifically interacting with E. coli MutH and retaining the E. coli MutH. Although, the amino acid sequences of the C-terminal regions of these two proteins are only 18% identical, they are 88% identical in the predicted secondary structure. Finally, by analysing (E. coli–P. aeruginosa) chimaeric MutL proteins, we show that the N-terminal regions of E. coli and P. aeruginosa MutL proteins function similarly, in vivo and in vitro. These new findings support the hypothesis that a large surface, rather than a single amino acid, constitutes the MutL surface for interaction with MutH, and that the N- and C-terminal regions of MutL are involved in such interactions.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections

2005 ◽  
Vol 49 (6) ◽  
pp. 2343-2351 ◽  
Author(s):  
Patricia Komp Lindgren ◽  
Linda L. Marcusson ◽  
Dorthe Sandvang ◽  
Niels Frimodt-Møller ◽  
Diarmaid Hughes
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Infection Model ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Tract Infections ◽  
Subsequent Selection

ABSTRACT Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

scholarly journals The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters

1993 ◽  
Vol 296 (3) ◽  
pp. 851-857 ◽  
Author(s):  
T Belyaeva ◽  
L Griffiths ◽  
S Minchin ◽  
J Cole ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Growth Conditions ◽  
Upstream Region ◽  
E Coli ◽  
Run Off ◽  
A Site ◽  
Specific Sequences

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

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