Analysis of Pseudomonas gene products using lacIq/Ptrp-lac plasmids and transposons that confer conditional phenotypes

Lorenzo, Vı́ctor de; Eltis, Lindsay D.; Kessler, Birgit; Timmis, Kenneth N.

doi:10.1016/0378-1119(93)90533-9

Cited by 406 publications

(220 citation statements)

References 20 publications

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“…Overexpression of the nrdJ Region Products-The broad-host-range expression vector pVLT31 (20) was used to overproduce the proteins encoded in the nrdJ genomic region. To construct pETS131, the nrdJa region was amplified by PCR using primers OP1-VLT-up and OP2-J1-lw and using genomic DNA from the P. aeruginosa PAO1 strain.…”

Section: Methodsmentioning

confidence: 99%

Two Proteins Mediate Class II Ribonucleotide Reductase Activity in Pseudomonas aeruginosa

Torrents¹,

Poplawski²,

Sjöberg

2005

Journal of Biological Chemistry

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The opportunistic human pathogen Pseudomonas aeruginosa is one of a few microorganisms that code for three different classes (I, II, and III) of the enzyme ribonucleotide reductase (RNR). Class II RNR of P. aeruginosa differs from all hitherto known class II enzymes by being encoded by two consecutive open reading frames denoted nrdJa and nrdJb and separated by 16 bp. Split nrdJ genes were also found in the few other ␥-proteobacteria that code for a class II RNR. Interestingly, the two genes encoding the split nrdJ in P. aeruginosa were co-transcribed, and both proteins were expressed. Exponentially growing aerobic cultures were predominantly expressing the class I RNR (encoded by the nrdAB operon) compared with the class II RNR (encoded by the nrdJab operon). Upon entry to stationary phase, the relative amount of nrdJa transcript increased about 6 -7-fold concomitant with a 6-fold decrease in the relative amount of nrdA transcript. Hydroxyurea treatment known to knock out the activity of class I RNR caused strict growth inhibition of P. aeruginosa unless 5-deoxyadenosylcobalamin, a cofactor specifically required for activity of class II RNRs, was added to the rich medium. Rescue of the hydroxyureatreated cells in the presence of the vitamin B 12 cofactor strongly implies that P. aeruginosa produces a functionally active NrdJ protein. Biochemical studies showed for the first time that presence of both NrdJa and NrdJb subunits were absolutely essential for enzyme activity. Based on combined genetic and biochemical results, we suggest that the two-component class II RNR in P. aeruginosa is primarily used for DNA repair and/or possibly DNA replication at low oxygen tension.

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Section: Methodsmentioning

confidence: 99%

Two Proteins Mediate Class II Ribonucleotide Reductase Activity in Pseudomonas aeruginosa

Torrents¹,

Poplawski²,

Sjöberg

2005

Journal of Biological Chemistry

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“…The mobilizable plasmids encoding ptac-flhDC (pGY20) and ptac-yplAB (pDHS32) transcriptional fusions are derivatives of pVLT33 (13). Construction of pGY20 was described (9).…”

Section: Methodsmentioning

confidence: 99%

A new pathway for the secretion of virulence factors by bacteria: The flagellar export apparatus functions as a protein-secretion system

Young

Schmiel

Miller

1999

Proc. Natl. Acad. Sci. U.S.A.

354

312

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Biogenesis of the f lagellum, a motive organelle of many bacterial species, is best understood for members of the Enterobacteriaceae. The f lagellum is a heterooligomeric structure that protrudes from the surface of the cell. Its assembly initially involves the synthesis of a dedicated protein export apparatus that subsequently transports other f lagellar proteins by a type III mechanism from the cytoplasm to the outer surface of the cell, where oligomerization occurs. In this study, the f lagellum export apparatus was shown to function also as a secretion system for the transport of several extracellular proteins in the pathogenic bacterium Yersinia enterocolitica. One of the proteins exported by the f lagellar secretion system was the virulence-associated phospholipase, YplA. These results suggest type III protein secretion by the f lagellar system may be a general mechanism for the transport of proteins that inf luence bacterial-host interactions.

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“…Strains, Media and Growth-DHBD was hyperexpressed in P. putida KT2442 freshly transformed with pLEBD4, a broad host range expression plasmid containing the structural gene encoding the dioxygenase, as described previously (16). For DHBD expression, the strain was grown in Luria broth containing a potassium phosphate buffer described for Terrific Broth (17) 3 , 262.5 mM MgSO 4 , 10 mM CaCl 2 , and 0.1 mM thiamine.…”

Section: Methodsmentioning

confidence: 99%

Molecular Basis for the Stabilization and Inhibition of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase by t-Butanol

Vaillancourt

Han

Fortin

et al. 1998

Journal of Biological Chemistry

120

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The steady-state cleavage of catechols by 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD), the extradiol dioxygenase of the biphenyl biodegradation pathway, was investigated using a highly active, anaerobically purified preparation of enzyme. The kinetic data obtained using 2,3-dihydroxybiphenyl (DHB) fit a compulsory order ternary complex mechanism in which substrate inhibition occurs. The K m for dioxygen was 1280 ؎ 70 M, which is at least 2 orders of magnitude higher than that reported for catechol 2,3-dioxygenases. K m and K d for DHB were 22 ؎ 2 and 8 ؎ 1 M, respectively. DHBD was subject to reversible substrate inhibition and mechanism-based inactivation. In air-saturated buffer, the partition ratios of catecholic substrates substituted at C-3 were inversely related to their apparent specificity constants. Small organic molecules that stabilized DHBD most effectively also inhibited the cleavage reaction most strongly. The steady-state kinetic data and crystallographic results suggest that the stabilization and inhibition are due to specific interactions between the organic molecule and the active site of the enzyme. t-Butanol stabilized the enzyme and inhibited the cleavage of DHB in a mixed fashion, consistent with the distinct binding sites occupied by t-butanol in the crystal structures of the substrate-free form of the enzyme and the enzyme-DHB complex. In contrast, crystal structures of complexes with catechol and 3-methylcatechol revealed relationships between the binding of these smaller substrates and t-butanol that are consistent with the observed competitive inhibition.The microbial degradation of aromatic compounds constitutes an essential link in the global carbon cycle. The aerobic degradation of aromatic compounds such as toluene, naphthalene, and biphenyl generally proceeds via a catecholic catabolite with hydroxyl substituents on two adjacent carbon atoms. This catecholic compound is cleaved by a dioxygenase from one of two very different classes. Intradiol dioxygenases utilize non-heme ferric iron to cleave the aromatic nucleus ortho to (between) the hydroxyl substituents whereas extradiol dioxygenases utilize non-heme ferrous iron to cleave the aromatic nucleus meta (adjacent) to the hydroxyl substituents. The mechanism of intradiol dioxygenases is better understood due to their greater stability, favorable properties for spectroscopic examination, and the accessibility of catalytic intermediates (1, 2). Interest in extradiol dioxygenases is nonetheless considerable, not only because of their general metabolic significance and catalytic properties, but also because of the potential exploitation of these enzymes in the degradation of environmental pollutants such as polychlorinated biphenyls.2,3-Dihydroxybiphenyl 1,2-dioxygenase (DHBD) 1 is a component of the aerobic biphenyl degradation pathway of a number of microorganisms and cleaves 2,3-dihydroxybiphenyl (DHB) in an extradiol fashion as shown in Scheme 1. Crystallographic studies of DHBD from Burkholderia cepacia LB400 (3) and Pseudomonas...

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Analysis of Pseudomonas gene products using lacIq/Ptrp-lac plasmids and transposons that confer conditional phenotypes

Cited by 406 publications

References 20 publications

Two Proteins Mediate Class II Ribonucleotide Reductase Activity in Pseudomonas aeruginosa

Two Proteins Mediate Class II Ribonucleotide Reductase Activity in Pseudomonas aeruginosa

A new pathway for the secretion of virulence factors by bacteria: The flagellar export apparatus functions as a protein-secretion system

Molecular Basis for the Stabilization and Inhibition of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase by t-Butanol

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